Neuroinflammation

Neuroinflammation

Neuroinflammation

Edited by

Alireza Minagar

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier 32 Jamestown Road London NW1 7BY 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA First edition 2011 Copyright © 2011 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangement with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-384913-7 For information on all Elsevier publications visit our website at elsevierdirect.com This book has been manufactured using Print On Demand technology. Each copy is produced to order and is limited to black ink. The online version of this book will show color figures where appropriate.

Preface

During the past two decades the scientific community has witnessed many major achievements and technical advances in our knowledge and understanding of the fundamental molecular mechanisms underlying various neuroinflammatory disorders affecting the human nervous system. The major players in the pathogenesis of these complex and intriguing neuroinflammatory and neurodegenerative disorders include activated immune cells, endothelial cells, immune cells resident within the central nervous system (CNS), and effects of several recently identified immunomodulators, cytokines and chemokines, which initiate and sustain the underlying pathologic processes of these often enigmatic disorders. Scientists around the world, through innumerable collaborative studies, have partially determined the diverse roles of these players in the course of neuroinflammation and are now applying this wealth of information toward the development of more potent and specific and less dangerous therapies for these difficult-to-treat diseases. The main objective of this book, entitled Neuroinflammation, is to provide interested readers with the most up-to-date and detailed reviews of current scientific concepts of neuroinflammation, with extensive updates on the most recent concepts on the pathogenesis of these CNS diseases. The core emphasis of this series of reviews on basic and clinical features of neuroinflammation will be of interest to a broad continuum of both basic scientific researchers and clinical scientists. Neuroinflammation is a rapidly expanding field, and our collection on this topic represents an educational tool that can assist students, scientists, and clinicians around the planet to better understand, diagnose, and treat these complex diseases. I very much appreciate the scholastic efforts of several wonderful contributors to this book, who made Neuroinflammation a reality and provided us with their excellent chapters on various topics. I also appreciate the efforts of Mr. Paul Prasad Chandramohan and the hardworking staff at Elsevier’s publishing production team, who provided us with their support and expertise during the production of this book. I hope that my colleagues will find this book to be a useful resource in their continuous research into the fundamental concepts of neuroinflammation. Alireza Minagar, MD, FAAN

Contributors

J. Steven Alexander  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Peter O. Behan  Division of Clinical Neuroscience, Faculty of Medicine, University of Glasgow, Glasgow, Scotland, UK; School of Life Sciences, Glasgow Caledonian University, Glasgow, Scotland, UK Aimee Borazanci  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Isabel Bosca  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK; Neurology Department, La Fe University Hospital, Valencia, Spain Hermine Brunner  Division of Rheumatology, Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio (HB) Abhijit Chaudhuri  Department of Neurology, Queen’s Hospital, Romford, Essex, England, UK Tiffany Chang  Department of Neurology, Tulane University School of Medicine, New Orleans, LA, USA Andrew L. Chesson Jr.  Sleep Medicine Program, Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Xi Chen  Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA Paweł Cies´ lik  Department of Internal, Autoimmune and Metabolic Diseases, Medical University of Silesia, Katowice, Poland Randall J. Cohrs  Department of Neurology, University of Colorado School of Medicine, Aurora, CO, USA Natalie Cornay  Louisiana State University School of Medicine, Shreveport, LA, USA Robin Davis  Louisiana State University School of Medicine, Shreveport, LA, USA Francesco Deleo  Division of Epilepsy, Clinic and Experimental Neurophysiology, IRCCS Foundation Neurological Institute C. Besta, Milano, Italy

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Contributors

Gabriele Di Comite  Department of Immunology, National Institute of Neuroscience, Tokyo, Japan Ludovico D’Incerti  Department of Neuroradiology, Neurological Institute C. Besta, Milano, Italy

IRCCS

Foundation

Donard S. Dwyer  Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, USA; Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, USA Masoud Etemadifar  Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran; Department of Neurology, Isfahan University of Medical Sciences, Isfahan, Iran Clare Fraser  The National Hospital for Neurology and Neurosurgery, London, UK; Moorfields Eye Hospital, London, UK; St Thomas’ Hospital, London, UK Don Gilden  Department of Neurology, University of Colorado School of Medicine, Aurora, CO, USA; Department of Microbiology, University of Colorado School of Medicine, Aurora, CO, USA Gavin Giovannoni  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK Eduardo Gonzalez-Toledo  Department of Radiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA; Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA D. Neil Granger  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Viktoria Gudi  Department of Neurology, Hannover Medical School, Hannover, Germany Meghan Harris  Department of Neurology, Louisiana State University School of Medicine, Shreveport, LA, USA Antoni Hrycek  Department of Internal, Autoimmune and Metabolic Diseases, Medical University of Silesia, Katowice, Poland Stephen Jaffe  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Edward Johnson  Louisiana State University School of Medicine, Shreveport, LA, USA Roger E. Kelley  Department of Neurology, Tulane University School of Medicine, New Orleans, LA, USA

Contributors

xvii

Marisa Klein-Gitelman  Division of Rheumatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL (MKG) Cesar Liendo  Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA Alexandra Lopez-Soriano  Department of Neurology, Buffalo Neuroimaging Analysis Center, State University of New York, Buffalo, NY, USA Amy E. Lovett-Racke  Department of Neurology, Ohio State University Medical Center, Columbus, OH, USA; Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Medical Center, Columbus, OH, USA Amir-Hadi Maghzi  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK; Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran; Isfahan Neuroscience Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Monica Marta  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK Ravi Mahalingam  Department of Neurology, University of Colorado School of Medicine, Aurora, CO, USA Nicholas E. Martinez  Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA Rae Ann Maxwell  Medical Science Liaison, Biogen Idec Neurology, US Medical Affairs Jeanie McGee  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA David E. McCarty  Sleep Medicine Program, Department of Neurology, Louisiana State University Helath Sciences Center, Shreveport, LA, USA Ute-Christiane Meier  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK Mandana Mohyeddin Bonab  Department of Immunology and the Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran Alireza Minagar  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

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Contributors

Mutsumi Nagai  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Maria A. Nagel  Department of Neurology, University of Colorado School of Medicine, Aurora, CO, USA Behrouz Nikbin  Department of Immunology and the Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran Eileen M. O’Connor  Medical Science Liaison, Biogen Idec Neurology, US Medical Affairs Seiichi Omura  Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA Magdalena Olszanecka-Glinianowicz  Department of Pathophysiology, Medical University of Silesia, Katowice, Poland Parrin Patterson  Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, USA Gordon T. Plant  The National Hospital for Neurology and Neurosurgery, London, UK; Moorfields Eye Hospital, London, UK; St Thomas’ Hospital, London, UK Refik Pul  Department of Neurology, Hannover Medical School, Hannover, Germany Brain Rubin  Department of Neurology, Louisiana State University School of Medicine, Shreveport, LA, USA Amy C. Rauchway  Department of Neurology and Psychiatry, Saint Louis University School of Medicine, St. Louis, MO, USA Michael K. Racke  Department of Neurology, Ohio State University Medical Center, Columbus, OH, USA; Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Medical Center, Columbus, OH, USA; Department of Neuroscience, Ohio State University Medical Center, Columbus, OH, USA Mohammad-Reza Savoj  Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran Fumitaka Sato  Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA Robert N. Schwendimann  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Contributors

xix

Yadollah Shakiba  Department of Immunology and the Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran Thomas Skripuletz  Department of Neurology, Hannover Medical School, Hannover, Germany Karen Small  Louisiana State University School of Medicine, Shreveport, LA, USA Martin Stangel  Department of Neurology, Hannover Medical School, Hannover, Germany Malú G. Tansey  Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA Corinna Trebst  Department of Neurology, Hannover Medical School, Hannover, Germany Ikuo Tsunoda  Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA Elke Voss  Department of Neurology, Hannover Medical School, Hannover, Germany Yuhong Yang  Department of Neurology, Ohio State University Medical Center, Columbus, OH, USA; Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Medical Center, Columbus, OH, USA Robert Zivadinov  Department of Neurology, Buffalo Neuroimaging Analysis Center, State University of New York, Buffalo, NY, USA; Department of Neurology, The Jacobs Neurological Institute, State University of New York, Buffalo, NY, USA

1 Multiple Sclerosis: Pathophysiology, Clinical Features, Diagnosis, and Management

Amir-Hadi Maghzi1,2,3, Aimee Borazanci4, Jeanie McGee4, J. Steven Alexander5, Eduardo Gonzalez-Toledo4,6, Alireza Minagar 4 1 

Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran Isfahan Neuroscience Research Center, Isfahan University of Medical Sciences, Isfahan, Iran 3  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK 4  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 5  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 6  Department of Radiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 2 

Introduction Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the central nervous system (CNS), which largely affects young adults with certain genetic backgrounds, often following exposure to several as yet unidentified environmental antigen(s) [1,2]. It is believed that the interactions between environmental and genetic influences are required to trigger the massive immune response against putative CNS antigens (e.g., myelin proteins that surround axons). This progressive inflammatory process affects both gray and white matters of the brain and spinal cord and ultimately causes neurodegeneration and axonal loss, with resultant permanent disability. Inflammatory demyelination in MS slows impulse conduction or leads to complete cessation of nerve impulse transmission. Axonal loss and neurodegeneration are the fundamental mechanisms underlying brain atrophy and permanent loss of motor function. The lesions of MS can affect any region of the neuroaxis; therefore, the anatomic location of MS lesions plays a significant role in determining clinical symptoms. Based on the clinical disease pattern, four types of MS are recognized: relapsing– remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00001-0 © 2011 Elsevier Inc. All rights reserved.

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MS (PPMS), and progressive relapsing MS (PRMS) [3]. Interestingly, it appears that these four different forms of MS have dissimilar underlying neuropathologies, which in turn indicates that MS may represent a heterogeneous group of related diseases. The clinical course of RRMS is characterized by clear disease relapses with development of new neurologic deficits or worsening of older symptoms that last more than 24 h; each relapse is typically separated from the last attack by at least 1 month of stability. Patients with relapse of MS, either with treatment with corticosteroids or spontaneously, may return to their baseline neurologic status or may recover partially, with residual neurologic deficits. Usually, during the interval between relapses there is no clinical disease progression. Clinically, RRMS is the most common form of MS; more than 85% of patients initially present with this form. SPMS is recognized by an initial RRMS that progresses to SPMS within 10–15 years. During this phase of MS, the underlying inflammatory cascade and clinical relapses decrease in severity, while the neurodegenerative process builds to become the dominant pathology. In certain ways, SPMS may be regarded as a long-term product of RRMS. Up to 15% of MS patients initially present with PPMS, which is characterized by a relentless progression with no obvious relapses; patients occasionally exhibit transient minor improvement. PRMS is characterized by progressive and devastating attacks of the disease from the beginning with acute relapses, with or without recovery. Importantly, the intervals between relapses are marked by continuous disease progression. This form of MS is the least common clinical form. The most common form of MS, RRMS, begins with a single unifocal or multifocal demyelinating attack (known as clinically isolated syndrome [CIS]), with a complete or partial resolution of the attack. This form of MS with its dominant neuroinflammatory sequelae is clinically recognized by relapses and development of new lesions on magnetic resonance imaging (MRI) studied by CNS neuroimaging. Of course, during this process, the neurodegenerative arm of MS continuously proceeds with progressive axonal and neuronal loss to the point that the patient’s capacity to sustain any new attacks without suffering additional disability decreases. Within a few years of the onset of RRMS, the underlying neuropathology of MS involves more neurodegeneration with fewer clinical relapses, more clinical deterioration, and accumulating disability. Patients with PPMS have a progressive course from the beginning without significant evidence of inflammatory lesions on CNS neuroimaging and with no proven therapeutic response to immunomodulatory medications as compared to RRMS.

Epidemiology The diverse worldwide epidemiology of MS provides clues to the genetic and environmental risk factors for MS. The observations from migrant studies showing that migration from high- to low-risk areas before puberty provides some protection against developing MS, and vice versa, highlight the importance of environmental factors in MS [4]. The highest incidence and prevalence of MS are more likely to be observed at the highest latitudes in both the northern and southern hemispheres.

Multiple Sclerosis: Pathophysiology, Clinical Features, Diagnosis, and Management

3

In addition, the prevalence and incidence of MS are shown to be associated with the distance from the equator [5]. This has been mostly linked to the effects of sunlight exposure and vitamin D, leading to the formulation of a hypothesis that vitamin D deficiency may enhance the risk of MS; this hypothesis was later strengthened by further immunologic studies [6]. In addition, similarities between the epidemiology of MS and primary Epstein–Barr virus (EBV) infection (infectious mononucleosis [IM]) have been well documented, and several studies have shown that the risk of MS is elevated after IM, which has led to a growing body of evidence linking EBV to MS [7]. MS is also observed more commonly among smokers, those of higher socioeconomic class, and those with low dietary vitamin D intake [8–10]. The epidemiology of MS has been changing during recent decades. Generally, there has been an increase in the prevalence and incidence of MS worldwide, especially in previously low-risk regions such as the Middle East [5,11]. This has been partly attributed to the better diagnosis of MS secondary to enhanced diagnostic criteria, an increase in the number of neurologists, more availability of disease-modifying drugs, enhanced awareness, and more widespread use of MRI. However, these factors cannot fully explain the increase in the prevalence of MS. It is plausible that changes in lifestyle and environmental exposures have also contributed to the increase in the prevalence and incidence of MS. More evidence comes from studies that have documented an increase in the sex ratio of MS during recent decades in different parts of the world [5,12,13]. Since all these changes have occurred during a short period, they are more likely due to environmental changes rather than genetic ones. For instance, in Western countries, where smoking has been recognized as a risk factor for MS, the increase in the sex ratio of MS has been attributed to the growing number of female smokers, while in an Iranian study this was linked to an increase in the vitamin D deficiency among the young female population [12,14]. Epidemiologic investigations have shown that individuals of Western European ancestry have a higher susceptibility to MS. On the contrary, there are ethnic populations such as Hutterites and the Natives of western Canada who appear to be resistant to the disease despite living in relatively high-risk regions for MS. First-degree relatives of MS patients have a 20 times higher incidence of MS than the general population. Studies on monozygotic twins show that the concordance rate is 30% compared with rates of less than 5% in dizygotic twins [15]. Genetically unrelated family members living in the same environment have a risk of MS that is no higher than the background population. All these observations point toward a genetic component for MS. To date the most significant genetic component discovered remains the HLA-DRB1*15 [15].

Pathophysiology The exact cause of MS remains unknown, but evidence indicates that its pathophysiology includes two key and interconnected components: neuroinflammation and neurodegeneration [1,16]. The inflammatory component of the pathophysiology of MS includes abnormally excessive activation of the immune system against

Neuroinflammation

Th2 cytokines (IL-4, 5, –6, –13) FOXP3 CD4+/ CD25+ T-regs Tr1/IL-10 TGF-β/Th3

EBV

APC (DC) Putative Ag

HSV-6

Inflammation

Antiinflammatory

4

Environment trauma

T-cells

Adherens tight junctions

Th1 cytokines (TNF-a, IFN-g, IL–12, –15, –17) HLA-DR CD71 CD80/B7-1 CCR-5/CXCR3

BBB disturbances

T-cells (CD4+, CD8+) B-cells, monocytes

VE-cadherin Occludin claudins

Adhesion

ICAM-1, VCAM-1/α4β1 MAdCAM-1/α4β7

Transmigration MS injury

Demyelination

(loss of oligodendrocytes)

(B-cells)

IgG/Oligoclonal bands

Myelin Ags MBP MOG

Neurodegeneration

Figure 1.1  Proposed MS pathogenesis. After exposure to environmental antigen(s) (e.g., EBV or HSV-6), myelin-sensitized autoreactive leukocytes are activated via binding of the T-cell receptor to the putative antigen(s), which is conveyed to them by antigen-presenting cells (such as dendritic cells) (trimolecular complexes). The activated leukocytes (T cells, B cells, and macrophages) cross the BBB (transmigration) through the disrupted cerebral endothelial tight and adherens junctions (by disintegrating junctional complexes containing VE-cadherin, occludin, claudin, and junctional adhesion molecules). The activated leukocytes also secrete a number of pro- and anti-inflammatory cytokines that play regulatory roles in polarization of the peripheral environment toward inflammatory or anti-inflammatory mechanisms. Once these cells gain access to the CNS environment, they identify more autoantigens and generate and release more cytokines and autoantibodies, causing loss of myelin/oligodendrocyte complex as well as neurodegeneration.

CNS antigen(s), which leads to interactions between autoreactive leukocytes and the inflamed cerebral endothelium, disintegration of the blood–brain barrier (BBB), and penetration of these activated leukocytes into the CNS parenchyma [1,17–19] (Figure 1.1). The early events that trigger these exuberant immune responses and activation of leukocytes against self-antigens remain hard to pin down, but viral and bacterial antigens are relevant and probable stimuli triggering the original MS pathophysiology [7,20]. Exposure to or infection with a number of viruses, such as hepatitis B and EBV, has been proposed as the activating factor for the T lymphocytes that are sensitized against viral proteins that share similar structural motifs with CNS proteins such as myelin basic protein (MBP); this is the so-called “molecular mimicry hypothesis.”

Multiple Sclerosis: Pathophysiology, Clinical Features, Diagnosis, and Management

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Pathophysiology of MS involves both the innate and acquired immune systems. One hypothesis about the pathophysiology of MS is that the initial event begins in the peripheral circulation with activation of immune cells outside the CNS such as dendritic cells [18,19]. Numerous scientific studies on experimental MS in mice (experimental autoimmune encephalomyelitis [EAE], the closest animal model of MS) have revealed that autoreactive CNS-antigen(s)-directed T lymphocytes (CD4, CD8) play significant roles in the development of CNS demyelinating lesions. At some point in the early stages of the MS development, T lymphocytes become sensitized against several suspected CNS antigens, including MBP, proteolipid protein, and myelin oligodendrocyte glycoprotein [21], and activate a massive immune response that leads to their migration across the BBB, leading to its dysregulation. Autoreactive T lymphocytes and monocytes interact with inflamed cerebral endothelial cells through rolling and firm binding in the cerebrovascular space. This binding process is the most significant component of the leukocyte–endothelial interaction and commits the leukocytes to migrate across postcapillary venules into the CNS environment [17]. Loss of the BBB endothelial integrity layer is associated with disassembly and destruction of endothelial tight junctions and junctional proteins such as occludin and VE-cadherin [22] as well as claudins, which facilitate movement of the leukocytes. Once autoreactive T lymphocytes and monocytes breach the CNS at the perivenular areas, the immune cascade escalates and more varied CNS antigens become identified as potential immune targets for T cells, a diversification of antigenic specificity over the course of the disease (the episode spreading concept) [23]. The pro-inflammatory Th1 lymphocytes express high levels of activation markers (HLA-DR and CD71), co-stimulatory molecules (CD80/ B7-1), and Th1-cell chemokine receptors (CCR5 and CXCR3). These cells produce high levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-12, IL-15, and IL-17. The Th2 lymphocytes, which switch the environment toward anti-inflammatory or protective mode, secrete cytokines such as IL-4, IL5, IL-6, and IL-13 [24]. Other cells involved in reducing the inflammatory response include various kinds of CD4 regulatory cells such as FOXP3 CD4CD25 Tregs, the IL-10–generating Tr1 cells, and the transforming growth factor β–generating Th3 cells. However, regardless of their Th polarization, the movement of immune cells into the CNS parenchyma can disturb BBB integrity. During the past decade, neuroimmunologists have focused on the role of emerging cytokines such as IL-12, IL-27, and IL-23 in the pathogenesis of MS. Members of the IL-12 family proteins are involved in regulation of T-lymphocyte responses and may be important in the pathophysiology of MS [25]. IL-17, a potent inflammatory cytokine, promotes CNS inflammation by disrupting the BBB, allowing greater permeation of autoreactive peripheral CD4 T cells into the CNS [26]. Recently, Alexander et al. [27] published the results of a 1-year prospective study of serum levels of IL-12p40, IL-17, and IL-23 prior to and at 3-month intervals after treatment with IFN-β1b. The investigators reported that continuous treatment with IFN-β1b reduced serum levels of IL-12p40 and IL-23 and showed a trend for decreasing IL-17. The investigators concluded that early treatment of MS with IFN-β1b may stabilize the clinical course of MS by decreasing levels of these inflammatory cytokines.

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Neuroinflammation

The humoral arm of the immune system also is significantly involved in the pathophysiology of MS. Under normal circumstances, B lymphocytes do not cross the BBB. However, during the course of MS and along the massive immune cascade, B lymphocytes become activated, produce antibodies, and cross the BBB. In 1942, Kabat et al. [28] reported the presence of the oligoclonal bands (evidence of intrathecal antibody production) that were not present in the concurrent serum. While it is known that oligoclonal bands are present in the cerebrospinal fluid (CSF) of patients with other inflammatory and infectious etiologies, their presence in the appropriate setting strongly supports a diagnosis of MS. Increased intrathecal synthesis of immunoglobulins is reflected by evidence of several factors and events in the CSF of MS patients, including the presence of oligoclonal bands, an elevated IgG index, B-cell clonal expansion somatic hypermutation [29,30], and B-cell receptor revision (editing) in clonally expanded B cells [31]. These all provide strong support for roles of activated B cells in the massive immune response that occurs during the pathogenesis of MS. Neuropathologic studies of CNS tissues from MS patients show an underlying “pathologic heterogeneity.” A milestone Mayo Clinic neuropathologic study of a large group of active MS white matter lesions suggested four patterns of MS neuropathologies based on the location of the plaques, the extent and pattern of damage and loss of oligodendrocytes, the pattern of myelin protein loss, and evidence of complement activation along with deposition of immunoglobulins [32]. Pattern I lesions reveal strong involvement of T lymphocytes and macrophages with sharp lesion edges and survival of oligodendrocytes and remyelination. In these lesions the expression of all myelin proteins, such as MBP, PLP, myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein (MOG), is decreased. Pattern II lesions demonstrate antibody complement–associated demyelination with plaques showing sharply defined borders. These lesions also show the presence of macrophages and T lymphocytes as well as surviving oligodendrocytes and remyelination. Pattern III plaques demonstrate hypoxiainduced distal oligodendrogliopathy with fuzzy borders and apoptosis of oligodendrocytes. There is no evidence of activity of humoral immune response in these lesions. Pattern IV lesions reveal sharply bordered plaques that also contain macrophages and T lymphocytes with no evidence of immunoglobulin or activated complement deposits within them. The proposed mechanism for the formation of these lesions is a primary oligodendrogliopathy (primary oligodendrocyte degeneration). This form of neuropathology is uncommon among MS patients and is limited to patients with PPMS.

Clinical Manifestations Lesions of MS affect many areas of the brain and spinal cord and may damage many aspects of the CNS functions. Clinically, MS may vary from benign MS to more aggressive and progressive forms of MS such as SPMS and PRMS. Fatigue is the most common complaint among MS patients. Fatigue, either mental or physical, affects all aspects of the MS patient’s life profoundly and results in reduced mental or physical activities. In many cases the fatigue compounds the coexisting depression and intensifies the symptoms.

Multiple Sclerosis: Pathophysiology, Clinical Features, Diagnosis, and Management

7

Clinical manifestations of MS stem from disturbances of the sensorimotor, bowel and bladder, sexual, brain stem and optic nerve, and neuropsychiatric functions. MS lesions demonstrate a predilection for certain areas of the neuroaxis, such as brain stem, periventricular area, cerebellum, optic nerve, and spinal cord, which in turn leads to the development of neuroanatomic-based symptoms. For example, involvement of the medial longitudinal fasciculus at the peri-aqueductal location causes internuclear ophthalmoplegia, which manifests with impairment of the conjugate lateral gaze with limitation of the adduction of the affected eye, along with compensatory nystagmus of the abducted eye. Patients with internuclear ophthalmoplegia complain of double vision, while convergence remains intact. Other abnormalities of the brain stem in the context of MS include impairments of extraocular motility, which manifest with horizontal or vertical gaze paresis, one-and-a-half syndrome, weakness of the extraocular muscles (cranial nerves 3, 4, or 6), or skew deviation. Facial paresis of the central or peripheral type manifests in MS and originates from demyelination of the facial nerve within the brain stem. Facial myokymia is an undulating, wavelike twitching that starts in the orbicularis oculi and occasionally presents in MS. Patients with MS frequently manifest dysarthria and dysphagia [33]. One particular form of speech impairment, scanning speech, is typical for MS patients. Hearing loss is uncommon in MS, but many MS patients frequently complain of vertigo. Many MS patients develop optic neuritis, either as the initial manifestation or as a part of their disease process. Optic neuritis usually begins with subacute visual loss in one or both eyes. As the disease progresses the scotoma becomes larger and the patient develops disturbances of color perception and contrast sensitivity. Patients with optic neuritis often complain of retro-orbital pain that is deteriorated with eye movement. Neuro-ophthalmologic evaluation may reveal a normal optic disc (if the neuritis is retrobulbar) or papillitis as well as the presence of relative afferent pupillary defect. Visual acuity is decreased and the patient suffers from visuospatial deficits. Motor symptoms in MS mainly originate from involvement of the corticospinal tract, which clinically translates into heaviness, stiffness, weakness, pain, or paralysis that can cause hemiparesis or paraparesis or paraplegia. In the course of MS, the lower extremities are more commonly affected than the upper extremities. Other manifestations of corticospinal tract involvement include the presence of hyperactive deep tendon reflexes, spasticity of the affected extremities, and the presence of the Babinski response. Corticospinal tract demyelination and neurodegeneration result from lesions within deep hemispheric white matter, basis pontis, cerebral peduncles, medullary pyramids, internal capsule, or spinal cord. Sensory symptoms of MS include tingling, burning, a pins-and-needles sensation, or complete loss of sensation. Frequently, MS patients complain of abnormal feeling occurring in a band-like distribution around the chest or abdomen. The sensory complaints in MS may stem from demyelination of the posterior columns (gracilis and cuneatus fascicule) or spinothalamic tracts. Lhermitte’s sign consists of a sudden electric-like sensation traveling down the spine or the extremities for a brief period. Apart from these sensory complaints, MS is potentially a painful disease and patients may develop trigeminal or glossopharyngeal neuralgia. Patients who suffer from

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these neuralgias report frequent, severe, short-lasting lancinating pain that affects their face or head and neck area. In addition, patients with MS have more generalized headaches and migraines than healthy individuals. Cerebellar involvement in MS stems from both vermian and hemispheric lesions and clinically manifests with gait ataxia, dysmetria upon performance of the finger-to-nose and heel-to-shin test, and inability to do tandem gait. Limb ataxia and intention tremor may be present in up to 50% of MS patients. Up to 65% of MS patients suffer from neuropsychiatric abnormalities. A decline in language skills, memory, and intellectual function is commonly observed in MS patients. Neuropsychological assessment of MS patients reveals mild to moderate slowing of thinking process, poor recent memory, word-finding difficulties, slow information processing, and difficulty with concentration [34–37]. The pattern of cognitive impairment in MS is similar to the pattern of other neurologic disorders that involve subcortical structures, such as HIV-related encephalopathy and Parkinson’s disease. Therefore, the presence of aphasia and neglect in MS patients is unusual. Mood disorders, particularly depression and bipolar mood disorder, are common among MS patients. These patients also frequently demonstrate euphoria, which indicates their inability to inhibit emotional expression. A number of abnormal involuntary movements may present in the context of MS. Some patients may develop rubral tremor, chorea, segmental myoclonus, and dystonia. Autonomic disturbances, including impairment of bowel and bladder dysfunction, and abnormal sweating, with unusual coldness or discoloration of the legs or feet, may be present in MS. Any cognitive or physical deficits in MS can be stereotypically and reversibly enhanced with exposure to heat, prolonged exercise, or infection (Uthoff’s phenomenon).

Neuroimaging: A Concise Review The use of various neuroimaging procedures, particularly different MR techniques, for the diagnosis, management, and follow-up of MS patients has fundamentally changed our view and understanding about the nature and pathophysiology of MS. The role of MRI in the world of MS is so significant that without an abnormal brain MRI, a diagnosis of MS must be reconsidered. MRI of the brain and spinal cord is the only objective tool that provides clinicians with a solid view about the natural history, disease activity, disease burden, severity of brain and spinal cord atrophy, and any therapeutic response to the administration of disease-modifying agents and immunosuppressants. Application of MRI has even changed the diagnostic criteria for MS and allows for the development of new T2-weighted lesions 1 month or more after the last MRI to fulfill the criteria for dissemination in time (Table 1.1) as well as development of one or more than one lesions in each of more than two characteristic sites for MS lesions to meet the criteria for dissemination in space [38]. As we learn more about MS, we also learn more about other diseases that imitate MS clinically. Therefore, MRI serves as an accurate diagnostic procedure to exclude MS imitators.

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Table 1.1  MRI Criteria for Dissemination in Space and Dissemination in Time for Patients with MS

Dissemination in Space McDonald [46]

Three or more of the following: Nine T2-weighted or one Gd-enhancing lesions; three or more periventricular lesions; one juxtacortical lesion; one or more posterior fossa lesions (*One spinal cord lesion can replace brain lesion.)

McDonald [47]

Three or more of the following: Nine T2-weighted lesions or one Gd-enhancing lesions; three or more periventricular lesions; one or more juxtacortical lesions; one or more posterior fossa lesions or spinal cord lesions (*A spinal cord lesion can replace an infratentorial lesion. **Any number of spinal cord lesions can be included in the total lesion count.)

New criteria

>1 lesion in each of >2 characteristic locations; periventricular, juxtacortical, posterior fossa, and spinal cord

Dissemination in Time McDonald [46]

Detection of Gd enhancement 3 or more months after CIS; a new T2-weighted lesion with reference to a previous scan 3 or more months after onset of CIS

McDonald [47]

A Gd-enhancing lesion 3 or more months after CIS; a new T2-weighted lesion with reference to a baseline scan obtained 30 days or more after onset of CIS

New criteria

A new T2-weighted lesion on follow-up MRI irrespective of timing of baseline scan

Gd, gadolinium; CIS, clinically isolated syndrome. Source: Adapted with permission from Lancet Neurology.

The other role of MRI is in follow-up and assessing a patient’s response to therapy. Currently, the treatments for MS approved by the US Food and Drug Administration (FDA) are expensive, and it is necessary to objectively document a patient’s favorable response or lack of response to treatment. Serial MRI of the patient’s brain and spinal cord, before and after treatment with disease-modifying agents, enables clinicians to assess and measure the quantity and the volume of MS lesions on various MR sequences; this in turn translates into a deeper understanding of the extent and severity of the underlying disease process. In addition, since MS is a “whole brain disease,” MRI shows us the extent of involvement of both white and gray matters and particularly demonstrates the severity of brain atrophy, which is a long-term indicator of disability in MS. At present, performing sophisticated MR techniques is the principal component of clinical trials for MS. The standard protocol for neuroimaging of MS patients proposed by the Consortium of MS Centers is presented in Table 1.2 [39]. The most informative MR sequences for neuroimaging of MS on routine MR

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Table 1.2  The Consortium of MS Centers Standardized Brain and Spinal Cord MRI Protocol Brain MRI Sequence

Diagnostic Scan for CIS

New Baseline or Comment Follow-Up Scan in Definite MS

Three-plane scout

Recommended Recommended

Axial sections through the subcallosal line (joins the undersurface of the rostrum and splenium of the corpus callosum)

Sagittal FLAIR

Recommended Recommended

Useful for corpus callosum lesions

Axial fast spin– or turbo spin–echo PD/T2

Recommended Recommended

TE1 < 30 ms TE2 < 80 ms Useful for infratentorial lesions missed by FLAIR

Axial FLAIR

Recommended Recommended

Useful for most white matter lesions, including juxtacortical

Axial pre-contrast T1

Optional

Optional

Useful for T1 black hole assessment

Three-dimensional T1

Optional

Optional

Useful for brain volume measures

Axial post-contrast T1

Recommended Optional

Minimum of 5 min delay using a standard dose

Spinal cord MRI sequence

Spinal cord imaging following contrast-enhanced brain MRI (no further contrast is needed) sequence

Three-plane localizer pre-contrast sagittal T1

Three-plane localizer postcontrast sagittal T1

Sagittal fast spin–echo PD/T2

Sagittal fast spin–echo PD/T2

Axial fast spin–echo PD/ T2 through lesions

Post-contrast axial T1-weighted through lesions

Three-dimensional T1 (optional)

Axial fast spin–echo PD/T2 through lesions

Post-contrast sagittal T1

Three-dimensional T1 (optional)

Post-contrast axial T1 through lesions Slice thickness should be <3 mm with no interslice gap (contiguous) on a >1.0  T closed MRI scanner. FLAIR, fluidattenuated inversion recovery; MS, multiple sclerosis; PD, proton density. Source: Adapted with permission from Am J Neuroradiol [39].

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Figure 1.2  (A) MRI. Axial view, FLAIR sequence. Plaque in infratentorial location, right cerebellar. (B) MRI. Axial view, FLAIR sequence. Multiple demyelinating plaques in characteristic periventricular location following the periependymal veins (Dawson’s fingers). (C) MRI. Transverse slice, FLAIR sequence. (D) MRI. MT sequence, same level as (C). Compare the lesser number of plaques on the MT sequence. The lower magnetization value indicates lipoprotein loss. In this patient the volume for plaques was 23.5 cc on FLAIR and 4.2 cc on MT, indicating 18% of plaques have lipoprotein breakdown.

machines include sagittal fluid-attenuated inversion recovery (FLAIR), axial fast spin– or turbo spin–echo proton density (PD/T2-weighted), axial FLAIR, and axial pre- and post-contrast (gadolinium[Gd]) T1-weighted images. The FLAIR technique is a unique procedure currently used for the detection of white matter lesions, which appear as hyperintense signals on this sequence (Figure 1.2A–D). Typical MS lesions on FLAIR sequence are hyperintense periventricular lesions as well as corpus callosum lesions (Dawson’s fingers). Application of T2-weighted sequence is a classic approach to visualize MS disease burden and lesion formation over time [40]. However, the specificity of this MR technique is low since different pathophysiologic events such as edema, demyelination, remyelination, inflammation, Wallerian degeneration, and axonal loss all present as hyperintense lesions on this sequence. T2-weighted images can be obtained by applying a number of spin echo– or fast spin echo–based methods. T2-weighted

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Figure 1.3  (A) MRI. T2-weighted axial view of brain showing MS plaques. T2 values were calculated: for a normal value of 95 ms in brain parenchyma, the plaques measure from 124 to 211 ms. (B) Post-contrast axial T1-weighted view showing enhancing plaque corresponding to one of the plaques appearing on the T2-weighted image.

lesions of MS are observed in the periventricular white matter, corpus callosum, and juxtacortical and infratentorial areas, including brain stem, cerebellum, and spinal cord (Figure 1.3A). The T2-weighted lesions may be multifocal or as the underlying disease process advances they may become confluent. With time, the number and volume of T2-weighted lesions increase. However, with further disease progression with progression of central atrophy, the total brain volume as well as white matter volume is decreased, which translates into an overall decrease in the volume of T2-weighted lesions. Therefore, the T2-weighted lesion load may not demonstrate an accurate correlation with overall disability. Another MR technique to study MS is T1-weighted images obtained pre- and post-contrast infusion. Gd, the contrast material, is infused intravenously. The development of Gd-enhancing lesions on post-contrast T1-weighted images usually occurs at the early stages of lesion formation and may indicate BBB leakage (Figure 1.3B). The duration of the GD-enhancing T1-weighted lesions is short; they usually disappear within 2–6 weeks [41]. In addition, most of these lesions are clinically silent; therefore, MRI is 5–10 times more sensitive to MS disease activity than clinical observation alone [42]. Contrast-enhancing lesions may enhance homogeneously or present as ring-enhancing lesions. However, some of the contrast-enhancing T1-weighted lesions do not show a complete ring and manifest as “open ring”– enhancing lesions, in which case the opening of the ring points toward the cortex. The presence of open ring–enhancing lesions strongly supports a diagnosis of MS. In general, the ring-enhancing lesions are typically larger than nonring-enhancing lesions, show a shorter period of enhancement, and show a lower diffusion and magnetization transfer (MT) ratio. Therefore, these lesions may indicate the evolution of these lesions into T1-weighted black holes and may be associated with a

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Figure 1.4  (A) MR spectroscopy of the MS plaque. Echo time [TE]30 ms. Note the presence of lactate, indicating anaerobic metabolism, decrease in NAA (low neurons/axons), slight increase in choline (increased membrane metabolism, increased metabolic turnover), increased myoinositol (glial marker). (B) MR axial post-contrast T1-weighted enhancing hyperacute plaque over the left central region. (C) Axial apparent diffusion coefficient (ADC) map. (D) Axial diffusion-weighted image (DWI). Note hyperintensity on DWI and hypointensity on ADC, indicating restricted diffusion, seen in hyperacute plaques. With time the values increase and there will be no restriction.

stronger chance for brain atrophy. While the presence of contrast enhancement to a certain degree reflects inflammatory disease activity and disruption of the BBB, these lesions do not correlate potently with disability on longitudinal studies. The contrast-enhancing lesions are better indicators of active inflammatory stage, they point toward upcoming clinical relapses, and they are sensitive to treatment with corticosteroids and other immunomodulatory medications. A subgroup of T2-weighted lesions, which is also observable on T1-weighted images represents areas of hypointensity. Some of these areas may represent a temporary stage in the development of new MS lesions and may be associated with the inflammatory infiltrate in the newly developing lesions. However, a certain number of these lesions turn into persistent T1-weighted hypointensities also known as T1 black holes. T1 black holes are believed to show severe tissue loss with axonal loss, and the load of persistent T1-weighted black holes demonstrates a strong correlation with chronic disability [43]. T1-weighted black holes have been reported in both cerebral white matter and the spinal cord [44]. Another novel MRI technique is proton magnetic spectroscopy (1H-MRS), which allows determination of the chemical components and metabolite alterations within MS lesions as well as normal-appearing white matter (Figure 1.4). Assessment of MS

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lesions using 1H-MRS allows insight into the biomolecules such as N-acetylaspartate (NAA), choline, creatine, myoinositol, glutamate, glutamine, lipids, and lactate. Each one of these metabolites appears at a unique site in the 1H-MRS spectrum (expressed at parts per million [ppm]). NAA is a specific marker of neuronal and axonal integrity and presents at 2.02 ppm. In active inflammatory lesions of MS there is a reduction of NAA peak along with increases in the choline, lactate, and lipid peaks. An increase in choline peak indicates increased cell membrane turnover marker, and its increase may be associated with infiltration of immune cells, demyelination, remyelination, and gliosis. Myoinositol is proposed to be a marker of glial proliferation.

Figure 1.5  (A) MRI. Axial diffusion-weighted image showing multiple hyperintense lesions, corresponding to MS plaques. Apparent diffusion coefficient (ADC) values (as n  10-3 mm2/s) were elevated compared to normal values (0.70  10-3 mm2/s). (B) MRI. Sagittal T1-weighted image with Gd contrast. MS plaques are perpendicular to ependyma (Dawson’s fingers).

Figure 1.6  (A) MRI. MT. MT ratio of a plaque is 33%, or 77% of normal. (B) Same patient 7 months after; plaque MT ratio is now 20%, or 44% of normal.

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Spinal cord lesions, which may be detected in up to 90% of patients with clinically definite MS, account for significant disability in these patients. The cervical cord is more frequently affected than the thoracic cord, and involvement of the spinal cord without cerebral involvement occurs in only 2% of MS patients. MS lesions affecting the spinal cord generally do not extend beyond two vertebral segments, and extensive longitudinal lesions, particularly when present acutely, should raise the diagnosis of neuromyelitis optica [45]. Many of the spinal cord lesions are clinically silent. Spinal cord lesions are better imaged on short tau inversion recovery (STIR) sequences. Other advanced MR procedures currently employed to better understand the pathogenesis of MS consist of diffusion-weighted MR (Figure 1.5), MT ratio (Figure 1.6), functional, perfusion MR imaging, and tractography.

Diagnosis The original diagnostic criteria proposed by Poser et al. (1983) [64] required clinical documentation of dissemination in time and space based on the physical examination. These criteria were modified to include MRI abnormalities [46,47]. The latest diagnostic criteria for MS are presented in Table 1.3.

Variants of MS Variants of MS include neuromyelitis optica (also known as Devic’s disease, which is discussed in a separate chapter), Marburg’s variant, Balo’s concentric sclerosis, and Schilder’s disease. Marburg’s variant of MS is a fulminant disorder with a rapidly progressive course, which is associated with severe axonal loss and causes the patient’s death. Balo’s concentric sclerosis is an uncommon form of MS that neuropathologically manifests with concentric rings of alternating demyelinated and undemyelinated areas. This form of MS, which is more common among Asians, presents as an acute condition and may result in severe disability or death. Unlike typical cases of MS, patients with Balo’s concentric sclerosis present with more cortical impairments such as seizures, aphasia, and cognitive decline. Schilder’s disease is a rare and progressive demyelinating disorder that affects children. Demyelinating lesions of Schilder’s disease are usually large, confluent, and extensive. Patients present with seizures, aphasia, weakness, speech impairment, personality changes, and poor concentration.

Differential Diagnosis A number of neurologic and nonneurologic diseases imitate MS and may present with nonspecific white matter lesions on brain or spinal cord MRI. In these cases, attention

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Table 1.3  Revised McDonald MS Diagnostic Criteria Clinical Presentation

Additional Requirements to Diagnose MS

2 or more attacksa

Noneb

2 or more objective clinical lesions 2 or more attacksa objective clinical evidence of 1 lesion

Dissemination in space, demonstrated by: MRIc or a positive CSFd and 2 or more MRI lesions consistent with MS or a further clinical attacka indicating a different location

1 attacka 2 or more objective clinical lesions

Dissemination in time, demonstrated by: MRIe or second clinical attacka

1 attacka objective clinical evidence of 1 lesion (monosymptomatic presentation; CIS)

Dissemination in space, demonstrated by: MRIc or positive CSFd and 2 or more MRI lesions consistent with MS and Dissemination in time, demonstrated by: MRIe or second clinical attacka

If criteria indicated are fulfilled, the diagnosis is multiple sclerosis (MS); if the criteria are not completely met, the diagnosis is “possible MS”; if the criteria are fully explored and not met, the diagnosis is “not MS.” a No additional tests are required; however, if tests (magnetic resonance imaging [MRI], cerebrospinal fluid [CSF]) are undertaken and are negative, extreme caution should be taken before making a diagnosis of MS. Alternative diagnoses must be considered. There must be no better explanation for the clinical picture. b MRI demonstration of space dissemination. c Positive CSF determined by oligoclonal bands detected by established methods (preferably isoelectric focusing) different from any such bands in serum or by a raised IgG index. d MRI demonstration of time dissemination. e Abnormal visual evoked potential of the type seen in MS (delay with a well-preserved waveform). Source: From [47], Ann Neurology 2005. With permission from John Wiley and Sons, Inc.

to the details of the clinical history and neurologic examination as well as other laboratory tests usually assists the clinician to differentiate these diseases from MS. These imitators of MS include neurosarcoidosis, neurosyphilis, leukodystrophies, acute disseminated encephalomyelitis, ischemic demyelination, migraine, systemic lupus erythematosus, B12 deficiency, HIV encephalitis, HTLV-1–associated myelopathy, mitochondrial encephalopathy, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, ischemic stroke, fat embolism, and Behçet’s disease.

Management Currently, MS remains an incurable disease. Treatments for MS can be classified into three major categories: treatment for acute attacks, use of disease-modifying agents, and immunosuppressive agents.

Treatment of Acute Relapses Patients with MS frequently experience relapses; they may resolve completely or leave the patient with clinically detectable residual neurologic deficits. In addition,

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once the patient experiences a number of relapses, he or she accumulates significant functional disability. Multiple clinical observations have demonstrated that MS patients who are treated with corticosteroids or adrenocorticotropic hormone (ACTh) recover faster than untreated patients. Due to the powerful anti-inflammatory and immunosuppressive properties of corticosteroids and ACTh, neurologists commonly use these agents to treat acute relapses of MS. These medications decrease edema and inflammation as well as the number of contrast-enhancing lesions on T1-weighted brain MR images. Clinically, patients improve and usually recover more rapidly. One of these corticosteroid medications is methylprednisolone (Solu-Medrol), which is infused intravenously 1 g/day for 5 days, followed by abrupt withdrawal or a tapering regimen. ACTh is not commonly used for treatment of acute relapses of MS.

Disease-Modifying Agents Currently, there are six FDA-approved disease-modifying agents for treatment of MS: (1) low-dose, low-frequency interferon-β1a (Avonex); (2) high-dose, highfrequency interferon-β1a (Rebif); (3) high-frequency interferon-β1b; (4) glatiramer acetate (GA), also known as copolymer 1, Cop-1, or Copaxone; (5) natalizumab (Tysabri), a monoclonal antibody against the cellular adhesion molecule a4-integrin; and (6) mitoxantrone (Table 1.4).

Beta-Interferons Beta-interferons decrease the number of contrast (Gd)-enhancing lesions on brain MRI, reduce severe relapses by 50%, and delay disease progression. The mechanisms of the beneficial effects of beta-interferons in the management of MS patients are only partially known. Proposed mechanisms of action for these agents include reduction of antigen presentation [48], decreasing the expression of co-stimulatory molecules on the dendritic and other cells [49,50], suppressing the proliferation of pro-inflammatory Th1 lymphocytes and upregulation of production of IL-10 [51], shifting of the cytokine environment from pro-inflammatory to anti-inflammatory [52,53], preventing transendothelial migration of autoreactive T lymphocytes by decreasing the production of matrix metalloproteinases [54,55], and restoring BBB integrity by upregulating the expression of occludin and VE-cadherin [22]. IFN-β1b also decreases serum levels of IL-12 and IL-23 [27]. Adverse effects of IFN-β1b include injection site reactions. The flu-like syndrome that occurs within hours of the injection usually resolves within 24 h. However, the severity of the flu-like syndrome may decrease with more injections over several weeks. Various techniques can be used to reduce the severity and duration of the flu-like symptoms. One recommendation is to inject IFN-β1b at bedtime and to use a titration schedule to increase the dose slowly, starting at 25% of the recommended dose and progressing to the full dose within 4–6 weeks. Patients should be instructed to take noncorticosteroid anti-inflammatory agents such as ibuprofen or acetaminophen prior to and after the injection. In severe cases, use of low-dose corticosteroids (prednisone 10 mg prior

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Table 1.4  Therapeutic Drugs Approved by the FDA for Treatment of MS Drug

Dosage

Route of Side Effects Administration

Safety Blood Work

Avonex (INF-β1a)

30 μg weekly Intramuscular

Flu-like syndrome

LFT/CBC Redness, necrosis

Rebif (INF-β1a)

8.0 million Subcutaneous units 3 times weekly

Flu-like syndrome

LFT/CBC Redness, necrosis

Betaseron (INF-β1b)

44 μg

Subcutaneous

Flu-like syndrome

LFT/CBC Redness, necrosis

Glatiramer acetate (Copaxone)

20 mg

Subcutaneous

None

None

Tysabri 300 mg once Intravenous (natalizumab) every 28 days

None A number of cases of progressive multifocal leukoencephalopathy have been reported in association with it

Mitoxantrone 12 mg/m2 Intravenous body surface once every 3 months, not to exceed 140 mg in lifetime

Cardiomyopathy, opportunistic infections, acute myeloid leukemia

Skin Reaction

Redness, lipodystrophy None

LFT/CBC Skin necrosis upon accidental skin exposure

LFT, liver function tests; CBC, complete blood count.

to injection) is recommended. Uncommonly, depression and suicidal ideation occur in patients being treated with IFN-β1b. The most common laboratory abnormalities associated with IFN-β1b are lymphopenia and elevated liver enzymes. Injection site reactions are common and typically occur when patients inject themselves at the same place frequently. This reaction ranges from redness of the skin to skin necrosis. Skin necrosis usually requires discontinuation of the IFN-β1b.

Glatiramer Acetate GA, which is administered 20 mg/day subcutaneously, reduces relapses by one third. GA is a synthetic random polymer of four amino acids (with a molecular weight of 6.4 kDa) that exist in the MBP. It is currently used for treatment of patients with RRMS. The four amino acids are tyrosine, glutamate, alanine, and lysine. Previous experiments demonstrated that GA was an effective therapy for preventing and

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ameliorating experimental allergic encephalomyelitis [56]. GA was approved in 1997 by the FDA for treatment of patients with RRMS. The mechanisms of the beneficial effects of GA on MS patients are not completely understood. Based on the proposed mechanism of action of GA, this medication induces a Th2/anti-inflammatory response in up to 50% of treated patients. The Th2 T cells in turn induce immunosuppressive type 2 monocytes and microglia, which also promote a switch to a Th2 environment. Currently, GA is not indicated for treatment of patients with SPMS and PPMS. Two recent head-to-head clinical trials comparing the clinical efficacy of IFN-β1a and IFN-β1b did not establish any difference between the efficacy of the beta-interferons and GA [57,58].

Natalizumab Natalizumab (Tysabri) is a humanized monoclonal antibody that targets the α4 chain of α4β1and α4β7 integrins. The α4β1 integrin very late antigen-4 (VLA-4) is expressed on all leukocytes except neutrophils and binds to the vascular cell adhesion molecule-4 (VCAM-4), which is expressed by the cerebral inflamed endothelium [59]. Natalizumab blocks the binding of the VLA-4 and VCAM-1, which in turn either blocks or significantly reduces transendothelial migration of the activated leukocytes into the CNS. The results of clinical trials of natalizumab in MS patients indicate that administration of this monoclonal antibody reduced the annual relapse rate by 68% and decreased the rate of disability progression by 42%, the number of brain contrastenhancing T1-weighted lesions by 92%, and the number of T2-weighted lesions by 83% [60,61]. Natalizumab is infused intravenously 300 mg once every 28 days. Its major adverse event is development of progressive multifocal leukoencephalopathy. Other monoclonal antibodies under clinical investigation for treatment of MS include daclizumab, rituximab, and alemtuzumab.

Mitoxantrone Mitoxantrone (Novantrone), an antineoplastic agent with profound immunosuppressive effects, is chemically related to the anthracyclines such as doxorubicin and acts as a potent immunosuppressive agent for treatment of MS. Mechanisms of action of mitoxantrone include intercalation with the DNA molecule, which in turn causes single- and double-stranded disruptions and suppresses DNA repair via inhibition of topoisomerase II. Mitoxantrone potently inhibits proliferation of B and T lymphocytes as well as macrophages. Other cells, such as antigen-presenting cells, are also killed and migration of the activated leukocytes is suppressed. Other modes of action for mitoxantrone include lowering the secretion of IFN-γ, TNF-α, and IL-2 [62]. Administration of mitoxantrone causes apoptosis of B and T lymphocytes [63]. Mitoxantrone is administered 12 mg/m2 every 3 months. It significantly reduces clinical relapses, disease progression, and MRI lesions in MS patients. Mitoxantrone has three significant side effects: severe leukopenia, acute myelogenous leukemia, and cardiac toxicity. In addition, patients treated with mitoxantrone are always at high risk for opportunistic infections.

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Prognosis MS is an unpredictable disease and in many cases, even with standard treatment, patients progress toward irreversible disability. Prognostic factors that indicate a better outcome include female gender, younger age at disease onset, initial disease presentation with optic neuritis or sensory symptoms, the presence of little residual disability after each relapse, a prolonged interval between the demyelinating attacks, a lower lesion load on the baseline brain scan at the onset, a progressive course from the beginning of the disease, poor recovery from relapses, and cerebellar or motor deficits.

Conclusion MS remains a very complicated and incurable disease with many as yet unrecognized features. Ongoing basic science and clinical research into the pathophysiology of MS has altered our view of the fundamental mechanisms of disease process and had led to the discovery of disease-modifying agents. With introduction of these agents, the natural course of MS has changed forever and our clinical practice has moved toward more rapid diagnosis and earlier treatment of these patients. It is hoped that more effective therapies with fewer side effects will translate into a better quality of life for MS patients. The authors hope this chapter will encourage readers to continue research into the nature of this enigmatic disease.

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[10] Zivadinov R, Weinstock-Guttman B, Hashmi K, Abdelrahman N, Stosic M, Dwyer M, et al. Smoking is associated with increased lesion volumes and brain atrophy in multiple sclerosis. Neurology 2009;73:504–10. [11] Saadatnia M, Etemadifar M, Maghzi AH. Multiple sclerosis in Isfahan, Iran. Int Rev Neurobiol 2007;79:357–75. [12] Maghzi AH, Ghazavi H, Ahsan M, Etemadifar M, Mousavi S, Khorvash F, et  al. Increasing female preponderance of multiple sclerosis in Isfahan, Iran: a populationbased study. Mult Scler 2010;16(3):359–61. [13] Orton SM, Herrera BM, Yee IM, Valdar W, Ramagopalan SV, Sadovnick AD, et al. Sex ratio of multiple sclerosis in Canada: a longitudinal study. Lancet Neurol 2006;5:932–6. [14] Alonso A, Hernán MA. Temporal trends in the incidence of multiple sclerosis: a syste­ matic review. Neurology 2008;71(2):129–35. [15] Dyment DA, Ebers GC, Sadovnick AD. Genetics of multiple sclerosis. Lancet Neurol 2004;3:104–10. [16] Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mörk S, Bö L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998;338:278–85. [17] Minagar A, Alexander JS. Blood–brain barrier disruption in multiple sclerosis. Mult Scler 2003;9(6):540–9. [18] Bar-Or A. The immunology of multiple sclerosis. Semin Neurol 2008;28:29–45. [19] Bar-Or A. Immunology of multiple sclerosis. Neurol Clin 2005;23:149–75. [20] Lovett-Racke AE, Racke MK. Epstein–Barr virus and multiple sclerosis. Arch Neurol 2006;63:810–1. [21] Bernard CC, Johns TG, Slavin A, Ichikawa M, Ewing C, Liu J, et al. Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis. J Mol Med 1997;75:77–88. [22] Minagar A, Ostanin D, Long AC, Jennings M, Kelley RE, Sasaki M, et al. Serum from patients with multiple sclerosis downregulates occludin and VE-cadherin expression in cultured endothelial cells. Mult Scler 2003;9:235–8. [23] Davies S, Nicholson T, Laura M, Giovannoni G, Altmann DM. Spread of T lymphocyte immune responses to myelin epitopes with duration of multiple sclerosis. J Neuropathol Exp Neurol 2005;64:371–7. [24] Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4 T cells. Annu Rev Immunol 1994;12:635–73. [25] Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 2003;19:641–4. [26] Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, et  al. Human Th17 lymphocytes promote blood–brain barrier disruption and central nervous system inflammation. Nat Med 2007;13:1173–5. [27] Alexander JS, Harris MK, Wells SR, Mills G, Chalamidas K, Ganta VC, et  al. Alterations in serum MMP-8, MMP-9, IL-12p40 and IL-23 in multiple sclerosis patients treated with interferon-beta1b. Mult Scler 2010;16:801–9. [28] Kabat EA, Moore DH, Landow H. An electrophoretic study of the protein components in cerebrospinal fluid and their relationship to the serum proteins. J Clin Invest 1942;21:571–7. [29] Owens GP, Ritchie AM, Burgoon MP, Williamson RA, Corboy JR, Gilden DH. Singlecell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid. J Immunol 2003;171:2725–33. [30] Qin Y, Duquette P, Zhang Y, Talbot P, Poole R, Antel J. Clonal expansion and somatic hypermutation of V(H) genes of B cells from cerebrospinal fluid in multiple sclerosis. J Clin Invest 1998;102(5):1045–50. [31] Monson NL, Brezinschek HP, Brezinschek RI, Mobley A, Vaughan GK, Frohman EM, et  al. Receptor revision and atypical mutational characteristics in clonally expanded

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[51] Zang Y, Hong J, Robinson R, Li S, Rivera VM, Zhang JZ. Immune regulatory properties and interactions of copolymer-I and beta-interferon 1a in multiple sclerosis. J Neuroimmunol 2003;137:144–53. [52] Ozenci V, Kouwenhoven M, Huang YM, Kivisäkk P, Link H. Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-alpha)- and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-beta) treatment. Clin Exp Immunol 2000;120:147–53. [53] Ozenci V, Kouwenhoven M, Teleshova N, Pashenkov M, Fredrikson S, Link H. Multiple sclerosis: pro- and anti-inflammatory cytokines and metalloproteinases are affected differentially by treatment with IFN-beta. J Neuroimmunol 2000;108(1–2):236–43. [54] Prat A, Al-Asmi A, Duquette P, Antel JP. Lymphocyte migration and multiple sclerosis: relation with disease course and therapy. Ann Neurol 1999;46(2):253–6. [55] Yushchenko M, Mäder M, Elitok E, Bitsch A, Dressel A, Tumani H, et al. Interferonbeta-1 b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis. J Neurol 2003;250:1224–8. [56] Teitelbaum D, Fridkis-Hareli M, Arnon R, Sela M. Copolymer 1 inhibits chronic relapsing experimental allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice and interferes with PLP-specific T cell responses. J Neuroimmunol 1996;64:209–17. [57] Mikol DD, Barkhof F, Chang P, REGARD study group, et al. Comparison of subcutaneous interferon beta-1a with glatiramer acetate in patients with relapsing multiple sclerosis (the REbif vs Glatiramer Acetate in Relapsing MS Disease [REGARD] study): a multicentre, randomised, parallel, open-label trial. Lancet Neurol 2008;7:903–914. [58] Connor P, Filippi M, Arnason B, BEYOND Study Group, et al. 250 microg or 500 microg interferon beta-1b versus 20 mg glatiramer acetate in relapsing–remitting multiple sclerosis: a prospective, randomised, multicentre study. Lancet Neurol 2009;8:889–897. [59] Sheremata WA, Minagar A, Alexander JS, Vollmer T. The role of alpha-4 integrin in the aetiology of multiple sclerosis: current knowledge and therapeutic implications. CNS Drugs 2005;19:909–22. [60] Polman CH, O'Connor PW, Havrdova E, Hutchinson M, Kappos L, Miller DH, et  al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 2006;354:899–910. [61] Polman CH, O’Connor PW, Havrdova E, AFFIRM Investigators, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 2006;354:899–910. [62] Fidler JM, DeJoy SQ, Gibbons Jr JJ. Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function. J Immunol 1986;137:727–32. [63] Bellosillo B, Piqué M, Barragán M, Castaño E, Villamor N, Colomer D. Aspirin and salicylate induce apoptosis and activation of caspases in B-cell chronic lymphocytic leukemia cells. Blood 1998;92:1406–14. [64] Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et  al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol. 1983;13:227–31.

2 Epstein–Barr Virus and

Multiple Sclerosis: Wrong Place, Wrong Time?

Amir-Hadi Maghzi1,2,3, Monica Marta1, Isabel Bosca1,4, Mohammad-Reza Savoj2, Masoud Etemadifar2, Gavin Giovannoni1, Ute-Christiane Meier1 1 

Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK 2  Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran 3  Isfahan Neuroscience Research Center, Isfahan University of Medical Sciences, Isfahan, Iran 4  Neurology Department, La Fe University Hospital, Valencia, Spain

Epstein–Barr Virus Epstein–Barr virus (EBV) was first isolated from Burkitt’s lymphoma in 1964 by Epstein and Barr. EBV as the causative agent for infectious mononucleosis (IM) was discovered in 1968, when a laboratory technician working on lymphoma samples was accidentally infected with EBV and developed IM. In 1970, the virus was found to infect and immortalize B cells. EBV is a gamma-herpes-4-virus and was the first herpes virus to be completely sequenced. Several diseases are associated with EBV, including Hodgkin’s and post-transplant lymphomas, oral hairy leukoplakia, and nasopharyngeal carcinomas [1–3]. However, although EBV has a population prevalence of more than 90% worldwide, only a few EBV-positive individuals suffer from diseases that are linked to EBV [1,2,4,5]. EBV has a double-stranded 172-kb DNA genome. Upon infection the genome circularizes and persists as an episome in infected cells. More than 70 open reading frames exist, which encode proteins expressed during lytic and latent EBV infection [1,3,5–7]. Lytic proteins encode viral proteins, which are necessary for the production of infectious virions, whereas latent proteins are needed to set up persistent infection by transforming and immortalizing host cells [1,3]. EBV also encodes non-translated RNAs (e.g., EBER1 and EBER2), which are almost ubiquitously Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00002-2 © 2011 Elsevier Inc. All rights reserved.

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expressed and highly abundant in EBV-infected cells (106–107 copies per infected cell nucleus). Four EBV latency programs have been described with distinct sets of expressed proteins and viral RNAs: (1) Latency 0: EBER1 and EBER2 but no expression of proteins; (2) Latency 1 (true latency program): EBER1 and EBER2, EBNA1; (3) Latency 2 (default program): EBER1 and EBER2 and expression of EBNA1, latent membrane protein (LMP)-1, LMP2A, LMP2B; and (4) Latency 3 (growth program): EBER1 and EBER2 and expression of EBNA-leader protein (LP), EBNA 2–6, LMP1, LMP2A, LMP2B [8]. Viral latency can be disrupted by a variety of cellular activators, inducing the switch from latent to lytic replication, mediated by the Zebra protein [9]. The major components of the lytic phase are the EBV DNA polymerase, BALF5, and early and late lytic proteins. Self-assembly of the EBV capsids requires viral capsid antigen (VCA), major capsid protein (BcLF1), and major surface membrane antigens (MA) gp350/220 [10]. Viral entry into B cells is mediated by gp350, which binds to CD21 (complement receptor) and possibly other receptors and triggers endocytosis. Entry seems to additionally require binding of MHC class II by gp42 to initiate fusion of viral and endosomal membranes to release viral genetic material into the cell. The role of most EBV proteins has been characterized mainly in vitro using lymphoblastoid cell lines (LCL). In brief, EBNA1 is critical for maintenance of the EBV genome during cell division. LMP1 is important for growth transformation. EBNA3 modulates the EBNA2 activity and may have some role in regulation of transcription. LMPs are transmembrane proteins and mimic an active receptor necessary for transformation of resting B cells [1,11].

EBV and Diseases More than 90% of people are infected with EBV and the prevalence is higher in less-developed countries. Infection in childhood is asymptomatic or presents with subtle signs and symptoms; however, about half of the individuals infected during/ after their late teens have the typical presentations of IM. EBV is usually transmitted via the oral route and replicates intensely in oropharyngeal epithelial cells. The virus enters its lytic program and infects local B cells. These B cells migrate to peripheral lymphoid tissue, but rare EBV B cells are also present in the bloodstream [1]. The virus has evolved strategies that render the host’s immune response unable to eliminate all infected cells, enabling the virus to set up persistent infection. The virus keeps immunologically silent in persistently infected B cells by turning off most genes, and only rarely undergoes reactivation. As the infection enters the latent phase, a few malignant diseases may emanate from chronic infection. Some of the neoplasms known to be caused by EBV are Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and posttransplant lymphoproliferative disorder (PTLD) [1,6,7,11–13].

Epstein–Barr Virus and Multiple Sclerosis: Wrong Place, Wrong Time?

27

Different types of malignancies are related to different virus programs [1,6]. For instance, Burkitt’s lymphoma is known to be associated with the Latency 1 programme, nasopharyngeal carcinoma and Hodgkin’s lymphoma with Latency 2 programme, and lymphoproliferative diseases in immunocompromised patients with Latency 3 programme, whereas hairy oral leukoplakia is related to the lytic program [1]. Over the years several autoimmune diseases, such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), have been linked to EBV infection [5,14].

Multiple Sclerosis MS is an inflammatory autoimmune disease of the human central nervous system. Infectious agents are plausible candidates in triggering and perpetuating the disease. Several viruses are suggested as a trigger for MS, but consistency and strength of association render EBV the outstanding candidate [15].

Epidemiologic Evidence Linking EBV to MS There are similarities between the epidemiology of MS and IM [16]. Several studies have shown that individuals with a previous history of IM are at higher risk for acquiring MS. A meta-analysis calculated the combined relative risk of MS after IM to be 2.3 (95% CI 1.7–3.0; P < 108) [17]. Besides demonstrating a potential role for EBV in MS, these findings may also suggest that the timing of primary EBV infection is important in developing MS. Those who are infected during adulthood have a higher risk of developing MS than those infected during childhood [17]. In highrisk regions for MS, the first encounter with EBV occurs during or after puberty in many individuals [18]. A large Danish study on patients with IM observed a more than twofold increased risk of MS in the IM cohort, adding to the evidence of association between IM and MS; in addition, in their study the risk of getting MS was augmented for more than 30 years after IM [19].

EBV Serology and MS Several studies have compared the presence of common EBV antibodies between MS patients and healthy controls. A systematic review demonstrated that nearly all MS patients are infected with EBV, compared with only about 90% of healthy individuals [20]. This study reported an odds ratio of developing MS of 13.5 (95% CI 6.3–31.4) in EBV-positive individuals. A second systematic review confirmed the relation of EBV seropositivity and MS, and calculated an odds ratio of developing MS for seronegative individuals of 0.06 (95% CI 0.03–0.13) [21]. Moreover, another study confirmed that the risk of MS in EBV-negative individuals is very low; however, it

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considerably increases in the same individuals after infection with EBV [22]. A metaanalysis of 30 case-control studies found an association between MS and exposure to EBV by determining the anti-VCA IgG antibodies (OR5.5; 95% CI3.37–8.81; P<0.0001), anti-complex EBNA IgG (OR5.4; 95% CI2.94–9.76; P<0.0001), and anti-EBNA-1 IgG (OR12.1; 95% CI3.13–46.89; P<0.0001); however, no significant association was observed when studying anti-early antigen (EA) IgG (OR1.3; 95% CI0.68–2.35; P0.457) or EBV DNA in serum (OR1.8; 95% CI0.99– 3.36; P0.051) [23]. Longitudinal prospective studies in which blood samples were collected prior to the onset of MS showed that the risk of developing MS is strongly associated with increased levels of EBV antibody titers prior to disease onset, and the strongest association was found for anti-EBNA1 IgG [24–26]. There is evidence that EBV antibodies might be a marker for clinical and radiologic disease activity in MS. It has been shown that patients with clinical relapse, compared to patients with clinical remission, have evidence of peripheral EBV reactivation as documented by increased IgM and IgA responses to EBV EA, and detectable EBV DNA in serum [27]. Moreover, patients with anti-EBV EA antibodies have more gadolinium (Gd)-enhancing lesions on magnetic resonance imaging (MRI) [28]. Gd-enhancing lesions were also shown to be correlated with anti-EBNA1 IgG and EBNA1/VCA IgG ratio [29]. In addition, anti-EBNA1 IgG titers, but not antiVCA IgG levels, were correlated with T2 lesion volume changes during follow-up [29]. Anti-EBNA1 IgG titers were a predictor of change in the Expanded Disability Status Scale (EDSS) score [29]. Anti-VCA IgG levels have been positively correlated with T2 and T1 lesion volume [30,31] and negatively correlated with atrophy parameters (gray matter fraction [GMF] and brain parenchymal fraction [BPF]) [31]. Increased anti-VCA IgG levels were associated with greater decrease in BPF after 3 years [30,31]. In this study, anti-EBNA1 IgG was only negatively associated with GMF, and there was no association of anti-EBV EA IgG antibodies with any of the MRI parameters measured [31]. Although it has been demonstrated that EBV viral load is not associated with overall MS risk [32,33], EBV viral load has been associated with increased disease activity [32]. It has been shown that in patients with clinically isolated syndrome (CIS), the immune response IgG against EBNA1 (but not other EBV antigens) correlates with the baseline number of T2 lesions, the number of lesions meeting the Barkhof criteria at baseline, the number of T2 lesions during follow-up, presence of new T2 lesions, and EDSS score during follow-up. In addition, increased anti-EBNA1 IgG responses predict conversion to MS based on the McDonald criteria [34]. However, no correlation was found between any EBV IgG responses and the number of Gd-enhancing lesions, although this finding was based on a small proportion of their studied patients [34]. Contrary to these studies, another study failed to show a clinical value for serum anti-EBNA-1 IgG levels in association with disease course and clinical disease activity [35]. Elevated EBV–VCA antibodies were also positively associated with other predisposing factors for MS in healthy individuals, including female gender, HLA-DR2, and current smoking status and the total number of pack-years smoked [36]. When both DR15 and high EBV titers are present, the conferred risk of MS is considerably

Epstein–Barr Virus and Multiple Sclerosis: Wrong Place, Wrong Time?

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increased [37–39]. In one study, the relative risk of MS was increased ninefold for female MS patients with DR15 and elevated titers of anti-EBNA1 antibodies and implied an interaction between these two risk factors when compared to that of DR15-negative woman with low anti-EBNA1 titers [37]. A more recent study concluded that smoking is likely to enhance the association between high anti-EBNA titers and an increased risk of MS [40]. In conclusion, EBV seropositivity is likely to be a predisposing factor for the development of MS; however, no direct causative relation to MS has been established. EBV seropositivity is also associated with other autoimmune diseases [14], which might indicate that EBV plays a role in autoimmunity in general.

Is there a specific EBV Strain causing MS? The possibility that different strains of EBV with different biologic activity may be associated with the occurrence of MS has been put forward, but few studies have investigated whether EBV strains differ in MS patients and healthy individuals. In one of the initial studies, Munch et al. [41] used sequencing of a coding EBNA6 region and showed a similar number of repeats in all eight MS patients from a cluster cohort and the majority of random MS patients with more variety in the control cohort. They concluded that MS patients had been infected with the same subtype. Lindsey et al. [42], however, found different sequences of LMP1 both in 11 MS patients and controls. More recently, Brennan et al. found several single nucleotide polymorphisms within the EBNA1 gene and one within the BRRF2 gene, which occurred at marginally different frequencies in EBV strains isolated from MS patients compared to controls. Variations in LMP1 and the sequences of the N and C terminus of EBNA1 were also detected by Simon et al. in 66 MS patients compared to controls [43,44]. However, a longitudinal study by Sitki-Green et al. [45] found different EBV strains in the blood and oral cavity of the same individual, indicating that different strains may also occur in the same host. Also, EBNA1 sequences spanning CD4 T-cell epitopes varied between strains. Individuals may therefore harbor a viral quasi-species, as has been described for human immunodeficiency virus, where areas under selection pressure acquire mutations that allow escape from immune surveillance. Contrasting results on the strain specificity of EBV in MS warrant future studies.

Control of EBV Infection by Host Immune Responses EBV has evolved elegant strategies that enable escape from immune surveillance, allowing the virus to set up latent viral infection and thus reactivate and infect new hosts. EBV starts its infectious cycle in B cells via the binding of the main viral envelope glycoprotein gp350/220 to CD21 (complement receptor 2) [46]. EBV infection

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of epithelial cells depends on the assistance of other viral glycoproteins (e.g., gp42), which appears to be necessary for the binding and fusion of EBV with MHC class II expressing cells [47]. LMP1 and LMP2A, which are latent EBV proteins expressed on infected B cells, mimic the activated CD40 receptor and antigen-activated B-cell receptor (BCR). They play a role in activation, proliferation, and maturation of those B cells into persistent EBV memory B cells [48]. During latent infection of B cells, EBV virion maintains an episomal form. To keep immunologically silent, EBV expresses only a limited repertoire of approximately 11 genes and downregulates surface expression of MHC class II on B cells. The virus is able to transiently switch to the lytic phase, during which approximately 80 genes are expressed encoding proteins for building virions for onward transmission. EBV infection is closely controlled by EBV-specific T cells. The percentage of EBV B cells in the peripheral blood of healthy individuals is between 1 and 10 latently infected B cells within 106 peripheral blood mononuclear cells [49]. It is not yet known if these frequencies are similar in MS patients, which would give us an indication as to whether EBVspecific T-cell responses are altered.

Control of EBV Infection by Virus-Specific CD8 T Cells Circulating cytotoxic CD8 T cells are programmed to eradicate infected B cells [50,51]. Upon infection of antigen-presenting cells, viral proteins undergo proteosomal degradation and the resulting peptides are transported into the endosomal reticulum, where they are loaded onto MHC class I molecules and presented on the cell surface to circulating CD8 cytotoxic T cells. One protein of pronounced immunologic interest is EBNA1, which is a highly immunogenic protein expressed during several latency programs of the virus. It contains a long glycine–alanine repeat sequence, which hinders proteosomal degradation and enables escape from CD8 T-cell recognition [52]. EBNA1 expression in B cells is tightly controlled and kept at levels that allow maintenance of the viral episome, but not high enough to elicit vigorous immune responses. Several studies have investigated EBV-specific CD8 T-cell responses in MS patients, with sometimes paradoxical results. An initial study analyzed 33 MS patients and 33 healthy controls; an increased frequency of CD8 T cells reactive to two out of five HLA-A2- and one HLA-B7-restricted EBV epitope was found in MS patients [53]. A follow-up study with seven HLA-B7-restricted EBV epitopes demonstrated no such difference between 73 MS patients and 32 healthy controls [54]. Increased CD8IFN-γ frequency elicited by 18 HLA class I-restricted EBV peptides was detected in 35 people with CIS, but similar frequencies were found in 73 MS patients and 21 healthy controls [55]. A more recent study described an increased frequency of EBNA1-specific IFN-γ-producing T cells in 28 patients with CIS compared to 30 healthy controls, although the frequency of T cells specific for other EBV-derived immunodominant CD8 T-cell epitopes did not differ between patients and controls [34].

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Other studies used EBV-infected B-cell LCL, which express both latent and lytic phase proteins. One study reported decreased CD8 T-cell responses to EBV as measured by ELISPOT with a lower mean frequency of IFN-γ–expressing peripheral blood mononuclear cells in response to autologous LCL in 34 MS patients compared to 34 healthy controls [56].

Control of EBV Infection by Virus-Specific CD4 T Cells CD4 T-helper cells orchestrate successful immune responses by activating CD8 T-cell function and inducing the maturation of B cells into immunoglobulin-producing plasma cells. CD4 cells recognize exogenous antigen presented by MHC class II molecules on antigen-presenting cells. A study of 21 MS patients and 20 healthy controls showed that MS patients had increased CD4 T-cell responses, mainly of memory phenotype, to a panel of EBNA1-spanning peptides covering the entire C-terminal region [57]. More recently, a cohort of CIS patients was shown to have almost twice the frequency of CD4 T-cell responses (as elicited by an overlapping library of C-terminus EBNA1 peptides) than healthy EBV carriers [34]. Interestingly, 3–4% of EBNA1-specific CD4 T cells in both MS patients and controls also reacted with myelin peptides. EBV encodes proteins that interfere with MHC class II presentation. BGLF5, a lytic program protein that functions as EBV DNase, downregulates surface expression of MHC class I and MHC class II molecules. BZLF1, an EBV immediate-early gene, acts through inhibiting IFN-γ signaling and also causes reduced expression of MHC class II molecules [58]. Surprisingly, intracellular EBV proteins expressed in B cells can bypass the exogenous pathway into the MHC class II pathway [59]. There is no consensus yet as to whether EBV-specific T-cell responses are decreased or increased in MS patients. Opposing results appear to stem mainly from differences in the experimental set-up.

Is EBV a characteristic feature of the MS Brain? Neuropathologic studies have been useful for understanding the driving forces of neuroinflammation, and will also be essential to confirm or refute the idea of viruses as a cause of MS. Early pathologic studies using electron microscopy found the presence of papovavirus and tubular paramyxovirus-like inclusions in active MS lesions [60], which were later described as parainfluenza virus [61], but both types of inclusions were soon found in other conditions and later interpreted as organelle protein artifacts [62,63]. Newer techniques such as in situ hybridization and polymerase chain reaction (PCR) allowed further investigation of viruses and failed to demonstrate the consistent presence of measles, paramyxovirus, or coronavirus [64–66]. Various members of the herpes family, such as varicella zoster virus, herpes simplex virus, and cytomegalovirus, had also been found in MS and control brains [67,68].

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The demonstration of different viral infections in MS brains led to the hypothesis that there may not be a single specific underlying viral trigger to MS, but that several common viral infections may play a role in the pathologic cascade [69,70]. Early studies failed to show consistent presence of EBV within MS brains: Hilton et al. [71] did not find EBV in a cohort of 10 MS patients (including 4 acute, 11 chronic active, 2 chronic inactive, and 4 shadow plaques) using in situ hybridization for EBER, while Sanders et al. [68] found EBV in 27% of MS patients and 38% of controls, and in 5% of active plaques and 10% of inactive plaques using PCR. Suboptimal tissue preservation prevented Opsahl and Kennedy [72] from drawing any conclusions about the presence of EBV in MS brains. Recent pathologic studies have fueled yet again the debate about the involvement of EBV in MS. Serafini et al. [73] reported the presence of EBV-infected B cells and plasma cells by in situ hybridization and immunohistochemistry in the brains of 21 of 22 MS cases, and these were not present in other inflammatory neurologic diseases (primary cerebral vasculitis, viral encephalitis, mycotic meningitis, and encephalopathy of unknown origin). Eight of the MS cases were known to have ectopic B-cell follicles and rich B-cell/plasma cell infiltration, while 12 cases were less infiltrated. Forty percent to 90% of B cells and 50–80% of plasma cells were positive for EBER, with the highest percentage for B cells in ectopic follicles. Of note, the cases with more prominent EBER-positive cell accumulation were cases rich in B cells/plasma cell infiltrates and ectopic B-cell follicles, while less infiltrated cases had fewer and often isolated EBER-positive cells. Viral reactivation, demonstrated by EBNA2 and BFRF1 expression, was restricted to ectopic follicles and active lesions. Other studies made different observations [74–76]. Willis et al. [74] examined 63 paraffin-embedded, formalin-fixed tissue specimens from 12 MS patients and from these selected 23 samples with CD20 B-cell infiltrates. They used a wide range of techniques, including quantitative real-time PCR to detect genomic EBV and EBER1 RNA, in situ hybridization for EBER, and immunohistochemistry for EBNA2 and LMP1 [74]. In addition, 12 specimens were examined for aggregates or B-cell infiltration within the meninges; this was found to be either absent or low, but 3 of these cases did have B-cell aggregates within the brain parenchyma [74]. Real-time PCR detected low-level EBV infection in two of the cases but EBV was undetectable by in situ hybridization [74]. Peferoen et al. undertook a study screening 632 specimens from 94 MS patients, including 11 patients who died before the age of 50; in addition, they studied 12 blocks used in the Serafini study [73,75]. Sixteen of the patients had accumulations of B-cell infiltrates, although follicle-like structures were not present in any specimen. Real-time PCR for EBV DNA and encoded RNA were negative in all tissues examined and EBER was detected only in one tissue sample by in situ hybridization, which also showed lytic cycle markers [75]. Another study screened active MS lesions with overexpression of INF-α for the presence of EBV infection. In situ hybridization with an EBER1/2 probe showed EBV-positive cells in white matter areas of 12 MS lesions and one case of acute disseminated encephalomyelitis (ADEM) [77]. The authors proposed that EBV infection may play a more indirect role via the accumulation of EBV-infected B cells in areas of initial

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inflammation and stress, which may then release viral components (e.g., EBERs), which can bind to Toll-like receptor 3 and thus activate innate immune responses, contributing to sustained or increased inflammation. The most recent study using nested and non-nested real-time PCR to detect EBV-specific and cell-specific transcripts in 5 formalin-fixed, paraffin-embedded and 15 fresh-frozen MS plaques and in single cerebrospinal fluid B lymphocytes and plasma cells did not reveal any evidence of active EBV infection [76]. From these studies it would appear that EBV-positive cells can be detected in active MS lesions, but far less frequently than has been reported at times. However, the presence of these EBV-positive cells may not be specific to the MS brain.

Conclusion The role of EBV in MS pathogenesis remains unknown at present. Further studies are needed to elucidate the role of EBV in this multifactorial disease and its potential contribution to neuroinflammation. Guilt should not be inferred solely from association, but EBV should be kept under close observation.

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[51] Hislop AD, Taylor GS, Sauce D, Rickinson AB. Cellular responses to viral infection in humans: lessons from Epstein–Barr virus. Annu Rev Immunol 2007;25:587–617. [52] Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen PM, Klein G, et al. Inhibition of antigen processing by the internal repeat region of the Epstein–Barr virus nuclear antigen-1. Nature 1995;375(6533):685–8. [53] Höllsberg P, Hansen HJ, Haahr S. Altered CD8 T cell responses to selected Epstein– Barr virus immunodominant epitopes in patients with multiple sclerosis. Clin Exp Immunol 2003;132(1):137–43. [54] Gronen F, Ruprecht K, Weissbrich B, Klinker E, Kroner A, Hofstetter HH, et  al. Frequency analysis of HLA-B7-restricted Epstein–Barr virus-specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy controls. J Neuroimmunol 2006;180(1–2):185–92. [55] Jilek S, Schluep M, Meylan P, Vingerhoets F, Guignard L, Monney A, et  al. Strong EBV-specific CD8 T-cell response in patients with early multiple sclerosis. Brain 2008;131(Pt 7):1712–21. [56] Pender MP, Csurhes PA, Lenarczyk A, Pfluger CM, Burrows SR. Decreased T cell reactivity to Epstein–Barr virus infected lymphoblastoid cell lines in multiple sclerosis. J Neurol Neurosurg Psychiatry 2009;80(5):498–505. [57] Lünemann JD, Edwards N, Muraro PA, Hayashi S, Cohen JI, Münz C, et al. Increased frequency and broadened specificity of latent EBV nuclear antigen-1-specific T cells in multiple sclerosis. Brain 2006;129(Pt 6):1493–506. [58] Ressing ME, Horst D, Griffin BD, Tellam J, Zuo J, Khanna R, et al. Epstein–Barr virus evasion of CD8() and CD4() T cell immunity via concerted actions of multiple gene products. Semin Cancer Biol 2008;18(6):397–408. [59] Taylor GS, Long HM, Haigh TA, Larsen M, Brooks J, Rickinson AB. A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4 T cells. J Immunol 2006;177(6):3746–56. [60] Prineas J. Paramyxovirus-like particles associated with acute demyelination in chronic relapsing multiple sclerosis. Science 1972;178:760–3. [61] Lewandowski LJ, Lief FS, Verini MA, Pienkowski MM, ter Meulen V, Koprowski H. Analysis of a viral agent isolated from multiple sclerosis brain tissue: characterization as a parainfluenzavirus type 1. J Virol 1974;13:1037–45. [62] Hayano M, Sung JH, Mastri AR. “Paramyxovirus-like” intranuclear inclusions occurring in the nervous system in diverse unrelated conditions. J Neuropathol Exp Neurol 1976;35:287–94. [63] Kirk J, Hutchinson WM. The fine structure of the CNS in multiple sclerosis. I. Interpretation of cytoplasmic papovavirus-like and paramyxovirus-like inclusions. Neuropathol Appl Neurobiol 1978;4:343–56. [64] Cosby SL, McQuaid S, Taylor MJ, Bailey M, Rima BK, Martin SJ, et al. Examination of eight cases of multiple sclerosis and 56 neurological and non-neurological controls for genomic sequences of measles virus, canine distemper virus, simian virus 5 and rubella virus. J Gen Virol 1989;70(Pt 8):2027–36. [65] Godec MS, Asher DM, Murray RS, Shin ML, Greenham LW, Gibbs CJ, Jr., et  al. Absence of measles, mumps, and rubella viral genomic sequences from multiple sclerosis brain tissue by polymerase chain reaction. Ann Neurol 1992;32:401–4. [66] Murray RS, Brown B, Brian D, Cabirac GF. Detection of coronavirus RNA and antigen in multiple sclerosis brain. Ann Neurol 1992;31:525–33. [67] Sanders VJ, Waddell AE, Felisan SL, Li X, Conrad AJ, Tourtellotte WW. Herpes simplex virus in postmortem multiple sclerosis brain tissue. Arch Neurol 1996;53:125–33.

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[68] Sanders VJ, Felisan S, Waddell A, Tourtellotte WW. Detection of herpesviridae in postmortem multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol 1996;2:249–58. [69] Reiber H, Ungefehr S, Jacobi C. The intrathecal, polyspecific and oligoclonal immune response in multiple sclerosis. Mult Scler 1998;4:111–7. [70] Allen I, Brankin B. Pathogenesis of multiple sclerosis—the immune diathesis and the role of viruses. J Neuropathol Exp Neurol 1993;52:95–105. [71] Hilton DA, Love S, Fletcher A, Pringle JH. Absence of Epstein–Barr virus RNA in multiple sclerosis as assessed by in situ hybridisation. J Neurol Neurosurg Psychiatry 1994;57:975–6. [72] Opsahl ML, Kennedy PG. An attempt to investigate the presence of Epstein–Barr virus in multiple sclerosis and normal control brain tissue. J Neurol 2007;254:425–30. [73] Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, et  al. Dysregulated Epstein–Barr virus infection in the multiple sclerosis brain. J Exp Med 2007;204:2899–912. [74] Willis SN, Stadelmann C, Rodig SJ, Caron T, Gattenloehner S, Mallozzi SS, et  al. Epstein–Barr virus infection is not a characteristic feature of multiple sclerosis brain. Brain 2009;132(Pt 12):3318–28. [75] Peferoen LA, Lamers F, Lodder LN, Gerritsen WH, Huitinga I, Melief J, et al. Epstein– Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis. Brain 2010;133(Pt 5):e137. [76] Sargsyan SA, Shearer AJ, Ritchie AM, Burgoon MP, Anderson S, Hemmer B, et  al. Absence of Epstein–Barr virus in the brain and CSF of patients with multiple sclerosis. Neurology 2010;74(14):1127–35. [77] Tzartos J, Khan G, Vossenkaemper A, Meager T, Sefia E, Clemens M, et al. The antiviral cytokine interferon-alpha is expressed in acute MS lesions and associated with the presence of Epstein–Barr virus infection. Mult Scler 2009;15(S2):S276.

3 Neutralizing Antibodies and Multiple Sclerosis Amy C. Rauchway Department of Neurology and Psychiatry, Saint Louis University School of Medicine, St. Louis, MO, USA

Immunogenicity of Beta-Interferons Self-antigens are normally tolerated, a process known as immune tolerance [1]. Although therapeutically administered proteins made by recombinant technology closely simulate endogenous proteins, they are potentially immunogenic because of several factors discussed below. Protein-based therapies that have been approved by the US Food and Drug Administration (FDA) for the treatment of multiple sclerosis (MS) are: (1) intramuscular interferon beta-1a (IFN-β1a IM, Avonex), subcutaneous interferon beta-1a (IFN-β1a SC, Rebif), and subcutaneous interferon beta-1b (IFN-β1b SC, Betaseron and Extavia); (2) subcutaneous glatiramer acetate (GA, Copaxone), a synthetic polypeptide; and (3) intravenous natalizumab (Tysabri), a monoclonal antibody [2]. Beta-interferons, which are genetically engineered, protein-based molecules, are classified as biopharmaceuticals [1,3]. Both binding antibodies (BAbs) and neutralizing antibodies (NAbs) have been described as part of the immunologic reaction to beta-interferon treatment [4–7]. BAbs, which include all antibodies that bind to a beta-interferon, occur in up to 80% of treated patients [6]. In general, NAbs are considered a subset of BAbs [7–9] that interfere with receptor binding of beta-interferon at the host’s target cell membranes, preventing the drug’s biologic effects [10]. Moreover, patients with high levels of BAbs may be at elevated risk of developing NAbs [11]. NAbs and BAbs are classified by the assay used in their detection [6,10,12]. Other characteristics, such as site of binding, duration of action, and affinity, may also differ [13]. Factors that play a role in the immunogenicity of a biopharmaceutical are primarily threefold: (1) those related to each agent, such as its chemical formulation, manufacturing method, route of administration, frequency, dose, and duration of treatment; (2) patient characteristics; and (3) unknown factors [1] (Figure 3.1). Differences in the protein sequence, production cell line, and glycosylation pattern between IFN-β1a and -β1b likely affect their immunogenicity [1,14–16]. IFN-β1a is produced in a Chinese hamster ovary cell line [17]. Like human IFN-β, both SC and IM recombinant forms of IFN-β1a are glycosylated and have 166 amino acids. In contrast, IFN-β1b is made in a bacterial strain and is not glycosylated. Furthermore, the N-terminal methionine of IFN-β1b was deleted, resulting in a protein of Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00003-4 © 2011 Elsevier Inc. All rights reserved.

A L C Human N A T

FK

K T K

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Structural properties Sequence variation

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FEBRUARY 1

2

3

4

7

8

9

10 11 12

5

6

13 14 15 16 17 18 25 26 27 28

Other factors

Figure 3.1  Factors that affect the immunogenicity of biopharmaceuticals. Source: Adapted with permission from Nat Rev Drug Discov [1].

Dose

Patient characteristics

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?

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19 20 21 22 23 24

Route of application

Neutralizing Antibodies and Multiple Sclerosis

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165 amino acids. Lastly, the cysteine amino acid at position 17 of IFN-β1b was replaced by a serine [17]. Some patient populations may be more likely than others to develop antiinterferon antibodies. A recent study of a large cohort of MS patients determined that HLA-DRB1*0401 and HLA-DRB1*0408 alleles were highly associated with an increased prevalence of antibodies to beta-interferon [18]. Finally, there remains a group of unknown factors. A dramatic drop occurred in the frequency of NAbpositive patients reported in the safety-extension study of IFN-β1a IM compared to the frequency of NAb-positive patients reported in its pivotal phase III trial. The reduction occurred after the drug was reformulated, but the complete basis for the drop has not been determined [8,9,19].

Interferon Antibody Assays BAb and NAb tests are fundamentally different. BAb tests provide direct evidence of antibody adherence to a target molecule (e.g., beta-interferon), while NAb tests use functional information about the IFN’s bioactivity or ability to induce a biologic response in vitro. Antibody testing is typically performed as a two-stage process [6,7,15], based on the concept that NAbs are a subset of BAbs. Furthermore, testing for BAbs is less expensive and simpler than testing for NAbs [6]. In this approach, a screening test for BAbs is done first. Currently, there are no set standards for BAb testing [9], so the interpretation of positivity is based upon criteria provided by the reference lab performing the test. When BAb titers are detected in the positive range, the specimen is evaluated for NAbs. Binding assays are commonly done by enzyme-linked immunosorbent assay (ELISA) (Figure 3.2). Other methods are Western blotting and radioimmunoprecipitation. In 2005, a European Federation of Neurological Societies (EFNS) Task Force published guidelines on anti-interferon antibody testing in MS. The task force concluded, in part, that (1) measurement of BAbs should be done in specialized laboratories; (2) BAb assays can be reliably done as a screening method before NAb testing; and (3) different assays for BAbs should be evaluated and compared to determine which correlates best with the presence of NAbs [9]. Accordingly, a recent study tested 325 serum samples from patients treated with IFN to compare the sensitivity and specificity among four commonly used binding assays for BAbs for their subsequent detection of NAbs. All specimens were tested with the same four BAb assays followed by the in vitro myxovirus resistance protein A (MxA) assay for NAbs. Among direct ELISA (dELISA), capture ELISA (cELISA), enzyme immunoassay (EIA), and Western blot (WB) assays for BAb detection, the investigators concluded that the cELISA method is the most useful for predicting NAb positivity. They emphasized that BAb testing does not replace NAb testing. Not surprisingly, the correlation between NAb and BAb titers was strongest at the extremes of NAb-negative specimens and high NAb titers [6]. Another recent study tested 50 serum samples for BAbs using a commercially available EIA test kit (Buhlmann Laboratories AG, Allschwil, Switzerland) and

42

Neuroinflammation Substrate HRP

HRP

IFN-β

IFN-β

Steps 1–3 (A)

IFN-β

Step 4 (B)

Step 5 (C)

Figure 3.2  Capture or sandwich ELISA (cELISA). (A) Steps 1–3: (1) attachment of nonhuman capture antibodies against beta-interferon to test plate; (2) application of interferon to capture antibody; (3) binding of BAbs, if present, from the test serum. (B) Step 4: application of horseradish peroxidase (HRP)-conjugated anti-human antibodies. (C) Step 5: HRP enzymatic conversion of a substrate into a chromatically detectable product.

50 other samples for NAbs using a cytopathic effect (CPE) assay to compare the reproducibility of results at three different laboratories. Results demonstrated high reproducibility of BAb testing but variability in NAb titers and status across all three centers [7]. Until recently, detection of NAbs has been performed in vitro in one of two ways. Unfortunately, neither method is standardized in terms of the procedure or the calculating and reporting of results. One method determines IFN’s antiviral capacity by using a CPE. The other method measures MxA production, an IFN-induced protein [9,10]. In the presence of NAbs, the ability of IFNs to prevent viral-induced cell death is diminished in the CPE assay (Figure 3.3), and stimulation of MxA production is decreased or absent in the MxA assay (Figure 3.4). A third assay using gene reporter technology for the detection of NAbs to IFN-β1a and IFN-β1b is now available [20]. Even as early as 1985, a World Health Organization (WHO) committee publication called for an expert panel to evaluate bioassays [21]. The committee made specific recommendations about the content of bioassay reports. A subsequent WHO report in 2004 incorporated recommendations provided by the Standards Committee of the International Society for Interferon and Cytokine Research and reiterated the need to standardize the calculation and reporting of IFN NAb test results [22]. The 2004 report recommended using the Kawade formula to determine the NAb titer [22]. This well-accepted formula expresses NAb potency in 10-fold reduction units

IFN-β IFN-β IFN-β

Invading virus

Invading virus

Invading virus

IFN-β

IFN-β

IFN-β

Cell death

Cell death

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(A)

(B)

(C)

Figure 3.3  The CPE assay. (A) Viral-induced cell death of the A549 test cells in vitro. (B) The antiviral effect of beta-interferon, thereby allowing survival of the A549 test cells in vitro. (C) Antibody neutralization of the antiviral effects of beta-interferon, resulting in cell death of the A549 test cells in vitro.

IFN-β

IFN-β IFN-β IFN-β

IFN-β

IFN-β

IFN-β IFN-β

MxA protein (A)

No MxA protein (B)

Figure 3.4  The myxovirus resistance protein A assay. (A) Ability of beta-interferon to induce MxA production. (B) Interference of NAbs upon the ability of beta-interferon to induce MxA production.

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(TRUs) [23]. A titer of 1 TRU/mL is equivalent to a 10-fold reduction of IFN activity, and a titer of 10 TRU/mL indicates a 100-fold reduction of IFN activity. The titer is calculated by the formula t  f (n–1)/9, where t represents the neutralization titer (TRU/mL), f is the reciprocal of the antibody dilution reached at the endpoint for that particular assay, and n is the IFN concentration in laboratory units (LU/mL). Laboratory units are preestablished on the day of the assay for the value of the relevant interferon such that the median endpoint of bioactivity on a sigmoidal dose– response curve is defined as 1 LU and is expressed as a concentration of LU/mL. Restated, 1 LU/mL is the IFN level that demonstrates a 50% protective effect against the confronted virus in a particular CPE assay [23]. Another commonly used expression of interferon neutralization is neutralization unit per milliliter (NU/mL), which may be equivalent to TRU/mL [15]. For a complete explanation of calculating neutralization titers, the reader is referred to articles by key authors on the topic [23,24]. In the case of NAb testing, the EFNS Task Force was in accordance with the WHO expert committee recommendations for using a validated assay with A549 cells (human lung carcinoma), a fixed amount of IFN, serial dilutions of the test sera [9], and calculation of the titer by the Kawade method. Because the European Medicines Evaluation Agency (EMA) validates a MxA assay, the EFNS Task Force recommended it in preference to the CPE assay. A new report, Recommendations for clinical use of data on neutralizing antibodies to interferon-beta therapy in MS, was published by an international expert panel sponsored by the Neutralizing Antibodies on Interferon Beta in MS (NABINMS) Consortium [25]. To improve the consistency of results among laboratories performing NAb testing, the NABINMS expert panel recommends a validated assay, for example the common EMA, using IFN-β1a for MxA induction regardless of the patient’s disease-modifying beta-interferon, using A549 cells, and reporting titers in TRUs [25]. A summary of the NABINMS key recommendations for NAb testing is provided in the Section Clinical Implications of IFN NAbs. When, as noted above, a fixed amount of IFN is used, the procedure is known as the constant IFN method. A major limitation of the constant IFN method is that high concentrations of serum, for example dilutions less than 1:20, may be either toxic or stimulatory to test cells in culture. Accordingly, another option, known as the constant antibody method [23], uses a fixed dilution of serum to which are added serial dilutions of IFN. Because it is more than 10 times as sensitive as the constant IFN method and is able to detect NAb titers of 1–2 TRU/mL, it has been suggested as a better way to detect and measure NAbs [23].

Prevalence of NAbs to Beta-Interferons The prevalence of NAbs to the IFNs used in MS have been evaluated in large clinical trials (i.e., more than 100 subjects per treatment arm), extension studies, and post hoc analyses of these trials, as well as smaller NAb-specific studies. Data from key clinical trials of relapsing–remitting MS (RRMS), secondary progressive

Neutralizing Antibodies and Multiple Sclerosis

45

Table 3.1  Prevalence of NAbs to Beta-Interferons in Key Clinical Trials Interferon Preparation IFN Dose IFN Dose Name of Study (mcg) Frequency

Definition of Study NAb Positivity Duration (NU/mL) (months)

% NAbPositive Patients

IFN-β1b SC  IFNB Study Group† [26]

250

QOD

2 Consecutive titers 20

36

38

 SPMS European Study Group [27]

250

QOD

2 Consecutive titers 20

36

28

 SPMS North American Study Group [28]

250

QOD

2 Consecutive titers 20

36

23

  BENEFIT [29]

250

QOD

At least 1 titer 60 20

32

  PRISMS-4 [30]

  22   44

TIW TIW

At least 1 titer 48 20

24 14

  PRISMS LTFU [31]

  22   44

TIW TIW

At least 1 titer 72–96 20

21 13

  SPECTRIMS [32]

  22   44

TIW TIW

At least 1 titer 36 20

21 15

  30

QWK

>Placebo

24

22

 European IFN-β1a   30 Dose-Comparison [34]

QWK

At least 1 titer 36 20

 2

  CHAMPS [35]

QWK

At least 1 titer 30 20

 2

IFN-β1a SC

IFN-β1a IM   MSCRG [33]

  30

SPMS, secondary progressive multiple sclerosis; BENEFIT, the Betaferon/Betaseron in Newly Emerging MS for Initial Treatment Study; PRISMS-4, Prevention of Relapses and Disability by Interferon beta-1a Subcutaneously in MS; PRISMS LTFU, PRISMS Long-Term Follow-Up; SPECTRIMS, Secondary Progressive Efficacy Clinical Trial of Recombinant Interferon-Beta-1a in MS; MSCRG, Multiple Sclerosis Collaborative Research Group; CHAMPS, The Controlled High-Risk Avonex MS Trial; QOD, every other day; TIW, thrice a week; QWK, every week. † Reanalysis of data from initial clinical trial.

MS (SPMS), and clinically isolated syndromes (CIS) are shown in Table 3.1. Comparisons of results among trials are limited by differences in methods of NAb assessment, definitions of NAb positivity, testing intervals, and duration of the trials [25]. In some studies, subjects were tested for NAbs after BAbs screening, while in others only NAb testing was performed [19].

46

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The percentage of patients who developed NAbs in clinical trials ranges from 2% to 38% (less than 5% for IFN-β1a IM and 13–38% for the SC IFNs), with IFN-β1b demonstrating the highest percentage of NAb-positive subjects (see Table 3.1). Aforementioned changes made in the production of IFN-β1a IM after its pivotal trial in 1996 led to a consistent decrease in the prevalence of NAb positivity [19]. Surprisingly, the pivotal trial of IFN-β1a SC known as PRISMS demonstrated an inverse dose relationship for NAbs. NAbs occurred in 24% of subjects on low doses (22 mcg SC thrice a week) but only 13% of subjects on high doses (44 mcg SC thrice a week) [36]. A similar inverse relationship was maintained in the trial’s 2-year extension called PRISMS-4, and in the clinical trial for IFN-β1a SC in SPMS known as SPECTRIMS [30,32]. In contrast, data from the EVIDENCE trial, which compared clinical outcomes of subjects with RRMS randomized to either IFN-β1a SC 44 mcg thrice a week or IFN-β1a IM 30 mcg weekly, showed that 25% of patients on high-dose IFN-β1a SC developed NAbs [37]. IFN-β1a SC (Rebif) was recently reformulated in part to reduce its immunogenicity. It is currently available in Europe, but not the United States, as Rebif New Formulation (RNF). A phase IIIb open-label study evaluated the immunogenicity of RNF by comparing the percentage of subjects who developed NAbs at 96 weeks to data from the EVIDENCE (IFN-β1a SC 44 mcg thrice a week versus IFN-β1a IM 30 mcg weekly) and REGARD (IFN-β1a SC 44 mcg thrice a week versus GA 20 mg daily) trials in RRMS [38]. Results showed that RNF had a lower proportion of NAb-positive patients versus the original formulation: 17.4% compared to 21.4% and 27.3% in EVIDENCE and REGARD respectively, although formal hypothesis testing was not performed [38]. Among the beta-interferon preparations, IFN-β1a IM appears to be the least immunogenic [8,9,25]. IFN-β1b SC treatment is associated with higher NAb titers than IFN-β1a SC, although IFN-β1b SC treatment has a higher rate of seroreversion [25]. Reversion of NAb positivity during treatment with beta-interferon will be discussed in the next section.

Interferon Antibody Positivity and Persistence NAb positivity is defined by titer level, persistence over time, or both. Many of the large MS clinical trials used a NAb titer of at least 20 neutralizing units (NU/mL) as the threshold value for positivity. Other studies support the use of higher cutoffs [8]. The IFN-β1b MS Study Group [26] and SPMS trials [27,28] required two consecutive titers of at least 20 NU/mL for positivity, while other trials required a single titer. A recent study defined low, medium, and high NAb titer ranges as 20–100 NU/mL, 102–500 NU/mL, and greater than 500 NU/mL respectively [6]. The lowest NAb titer that produces a biologic effect in patients is unknown [7]. The NABINMS panel report recommended separate cutoffs for IFN-β1a and IFN-β1b as follows: for negative, low/intermediate, and high titers of IFN-β1a (expressed as TRUs), ranges are less than 20, 20–100, and greater than 100 respectively. The IFNβ1b equivalent ranges are somewhat higher: less than 20, 20–400, and greater than 400.

Neutralizing Antibodies and Multiple Sclerosis

47

Studies investigating the duration and risk factors for NAb persistence to betainterferon show that higher titers generally persist longer than lower titers [25]. A presumed mechanism for the development of NAb persistence is that low-affinity antibody binding is followed by high-affinity binding after more months on therapy [9]. Most patients who develop NAbs do so between 9 and 18 months of treatment, although NAbs may develop as early as 1.5 months after initiation of treatment [12,15]. Prior to 1 year of beta-interferon therapy, titer level is unreliable for predicting persistence in comparison to titer levels at 2 years or more. For example, a titer greater than 100 TRU at 2 years is an excellent predictor of persistence [25]. Reversion to BAb- or NAb-negative status may occur either after discontinuation of an IFN or even during continued treatment [7,9]. A higher proportion of patients treated with IFN-β1b serorevert compared to those treated with IFN-β1a [9]. Categories of NAb status persistence used in clinical trials allow for comparison of NAb status with clinical outcomes and are (1) once positive, always positive, which considers a positive titer found at any time in a study as positive for the entire study; (2) interval-positive, which refers to NAb positivity at the end of a specific time interval; and (3) anytime-positive, which refers to a positive titer that is carried forward from that time on for the duration of the study. Interval-positive analysis allows for comparisons when a patient’s NAb status reverts from positive to negative [39].

Clinical Implications of Interferon NAbs Whether NAbs conclusively diminish or abrogate the therapeutic effects of betainterferons has been a subject of much debate [8]. Nevertheless, it appears that persistently high NAb titers are associated with a negative impact upon the therapeutic effects of beta-interferons [9,19,25,30,38]. Data from clinical trials longer than 2 years indicate a negative impact of interferon NAbs on relapse rate and magnetic resonance imaging (MRI) measures [14,19,25]. For example, a reduced therapeutic effect among subjects with NAb positivity (once positive, always positive) was reported in the 1996 reanalysis of the IFN-β1b pivotal trial that analyzed data up to 36 months [26]. Relapse rate and MRI activity were also compared between NAbpositive and negative subjects in a post hoc analysis of PRISMS-4 using intervalpositive and anytime-positive analysis. On interval-positive analysis, a negative effect upon annual relapse rate between NAb-positive and -negative groups was found. Results from the same study showed reduced efficacy on MRI measures for NAb-positive subjects (anytime-positive analysis) [39]. Additional clinical findings from other studies showed that (1) surprisingly, patients who became NAb-positive had reduced relapse rates during the first 6–12 months of beta-interferon therapy [11] and (2) the negative impact of NAb positivity upon relapse rate occurred regardless of the type of beta-interferon used [40]. The essential question for an individual patient is whether IFN treatment has induced an immunologic response that diminished or abrogated the drug’s efficacy. As a complementary test to the measurement of functional bioactivity in vitro,

48

Neuroinflammation

a separate MxA mRNA induction test was developed to monitor a patient’s response in vivo. Studies found the MxA mRNA test correlates with NAb titers and MRI outcome measures. Although the in vivo test is costly and not widely available, it may be a promising adjunct to NAb testing for patients with slightly elevated titers [25]. Methods to reduce NAb levels and restore IFN bioactivity in patients who were NAb-positive and without in vivo bioactivity were recently investigated using monthly, high-dose, pulsed oral methylprednisolone (MP) [41]. The study authors concluded that MP had no significant effect upon either measure. They concluded that changing immunomodulatory therapy remains the best option for patients with NAb-positive status and absent in vivo MxA bioactivity assessments. Three evidence-based reports have been published to guide clinicians about the monitoring and management of NAbs to IFNs (Table 3.2). Although the American Academy of Neurology (AAN) report published in 2007 acknowledged that NAbs in high titers are probably associated with reduced therapeutic efficacy, the AAN report concluded that there was insufficient information to make recommendations in terms of NAb testing (i.e., when and which test to use). In contrast, the EFNS Task Force guidelines (2005) offered specific recommendations on the use of NAb testing for clinical decision making. The consensus opinion given in the NABINMS report (2010) is that NAbs affect therapeutic efficacy at the group level and can be considered in the clinical decision to stop or continue beta-interferon therapy at the individual level. The NABINMS report links recommendations for both NAb assessment and disease-modulating treatment (for patients treated with beta-interferon for 1–2 years) to three categories of clinical disease activity: (1) clinically well, (2) intermediate disease activity, and (3) doing poorly. The panel experts summarize their recommendations: First, in general, even in patients doing well clinically, NAb and/or [in vivo] MxA bioactivity assessments should have therapeutic consequences. Particularly in cases of sustained high-titre NAb positivity and/or lack of MxA bioactivity, a switch to

Table 3.2  Consensus Statements for Clinical Use of NAb Testing EFNS Guidelines

AAN Evidence Report

NABINMS Recommendations†

Test NAbs in specialized laboratories Use validated in vitro NAb assay (CPE or MxA) Calculate NAb titer by Kawade formula Specified intervals for NAb testing Discontinue IFN therapy if sustained high NAb titers

Insufficient information on “when to test, which test to use, how many tests are necessary, and which cutoff titer to apply”

Use validated in vitro NAb assay Base clinical decision making on disease activity, NAb titers, or both Consider use of MxA in vivo assay in patients with low NAb titers Switch therapy if patient is doing poorly

†

Recommendations specifically apply to patients treated with IFN for at least 1–2 years.

Neutralizing Antibodies and Multiple Sclerosis

49

non-interferon-beta therapy should be considered. Second, in patients with intermediate disease activity, continuation of interferon-beta therapy could be considered in NAb-negative patients, whereas high NAb titres and/or lack of MxA bioactivity should suggest a switch to non-interferon-beta therapy. Third, in patients doing poorly a switch of therapy should be initiated, independent of NAb or MxA bioactivity outcomes.

Once the decision is made to change immunomodulating therapy, the NABINMS expert panel advises against switching from one beta-interferon preparation to another because of reported NAb cross-reactivity among beta-interferon preparations [25].

Antibodies to GA Glatiramer acetate (GA, Copaxone) is a heterogeneous mixture of synthetic polypeptides made from four amino acids: L-glutamic acid, L-alanine, L-lysine, and L-tyrosine [42]. Anti-GA antibodies develop in most, if not all, patients treated with GA [19,43,44]. Anti-GA antibodies are assessed by radioimmunoassay or ELISA [43]. Three clinical trials found that anti-GA antibodies (1) peaked 3 months after starting treatment, (2) declined at 6 months to a steady level above baseline, and (3) had no impact upon clinical efficacy [43]. A retrospective study of 42 patients treated with GA for at least 1 year found that anti-GA antibodies had an inhibitory effect on GA-induced T-cell proliferation as well as reversion of GA-stimulated IL-10 and IL-4 anti-inflammatory cytokines [44]. The therapeutic significance and biologic significance of anti-GA antibodies await further investigation [14,19].

Antibodies to Natalizumab Natalizumab is a recombinant, humanized monoclonal antibody produced in murine myeloma cells [45]. Natalizumab’s efficacy in MS is based on results of two phase III clinical trials. It was administered as monotherapy in the AFFIRM trial and as add-on therapy to IFN-β1a in the SENTINEL trial [46]. The grafted murine segment at natalizumab’s binding region is a potential target of anti-natalizumab antibodies [46,47]. Both the incidence and effect of antibodies to natalizumab were investigated in AFFIRM and SENTINEL [46]. Testing for anti-natalizumab antibodies was done at baseline and every 3 months using ELISA and a flow cytometric assay. Results of the two assays showed high concordance with one another [46]. A serum antibody concentration of 0.5 mcg/mL on ELISA was used as a cutoff to define antibody negative (0.5 mcg/mL at all post-baseline visits), transiently positive (at least 0.5 mcg/mL at a single post-baseline visit), or persistently positive (at least 0.5 mcg/mL at two or more post-baseline visits at least 42 days apart or a single assessment with no additional samples). In AFFIRM, 9% of subjects became antibody-positive and in SENTINEL 12% of subjects became antibody-positive. Six percent in each trial were persistently positive

50

Neuroinflammation

[46]. Of those, most developed antibodies within 3 months of treatment [14]. Persistent anti-natalizumab antibodies were associated with a decreased serum natalizumab concentration, increased disease activity on MRI, and an increased incidence of infusion reactions including hypersensitivity. The relapse rate in subjects with persistent antibodies was not different from the placebo group. A reduced benefit was seen on disability progression in AFFIRM only, the significance of which is uncertain [46]. In terms of clinical management, patients with recurrent infusion reactions or breakthrough disease activity should be tested for anti-natalizumab antibodies. Antibody formation is also associated with prolonged interruption of therapy after exposure of less than three doses of natalizumab [48]. The serum test specimen should be drawn just before a natalizumab infusion or no earlier than 2 weeks after infusion [48]. Because antibodies detected during the first 6 months of treatment may disappear, repeat testing is recommended after an additional 3 months. According to the natalizumab prescribing information, the provider should consider the risks and benefits of natalizumab therapy in patients with persistently positive antibodies.

Conclusion The current protein-based immunomodulating therapies for MS have proven clinical efficacy. Even so, some patients on these therapies experience breakthrough disease activity, bringing into question whether the drug is still effective. It is well established that anti-drug antibodies or NAbs induced by beta-interferons diminish or abrogate the bioactivity of interferons in vitro. Conclusive evidence that NAbs also diminish the clinical efficacy of beta-interferons has been more challenging to demonstrate, as discussed in this chapter. Differing approaches to the clinical use of NAb testing are given in the AAN evidence report and EFNS Task Force guidelines. Recently, an international panel with expertise in many aspects of MS developed a set of practical recommendations on the topic of NAbs to beta-interferons. Whether anti-drug antibodies to GA decrease its clinical efficacy is unknown. Persistent anti-natalizumab antibodies are correlated with loss of drug bioactivity, decreased clinical efficacy, and adverse infusion events.

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[36] PRISMS Study Group. Randomized double-blind placebo-controlled study of interferon b-1a in relapsing/remitting multiple sclerosis. Lancet 1998;352:1498–504. [37] Panitch H, Goodin DS, Francis G, Chang P, Coyle PK, O’Connor P, et al. Randomized, comparative study of interferon beta-1a treatment regimens in MS: the EVIDENCE trial. Neurology 2002;59:1496–506. [38] Giovannoni G, Barbarash O, Casset-Semanaz F, King J, Metz L, Pardo G, et al. Safety and immunogenicity of a new formulation of interferon b-1a (Rebif® New Formulation) in a Phase IIIb study in patients with relapsing multiple sclerosis: 96-week results. Mult Scler 2009;15:219–28. [39] Francis GS, Rice G, Alsop JC. for the PRISMS (Prevention of Relapses and Disability by Interferon Beta-1a Subcutaneously in Multiple Sclerosis) Study Group. Interferon beta-1a in MS results following development of neutralizing antibodies in PRISMS. Neurology 2005;65:48–55. [40] Koch-Henriksen N, Sorensen PS, Bendtzen K, Flachs EM. The clinical effect of neutralizing antibodies against interferon-beta is independent of the type of interferon-beta used for patients with relapsing-remitting multiple sclerosis. Mult Scler 2009;15:601–5. [41] Hesse D, Frederiksen JL, Koch-Henriksen N, Schreiber K, Stenager E, Heltberg A, et  al. Methylprednisolone does not restore biological response in multiple sclerosis patients with neutralizing antibodies against interferon-b. Eur J Neurol 2009;16:43–7. [42] Johnson KP. An approach to the treatment of multiple sclerosis. New York, NY: DiaMedica Publishing; 2010. [43] Brenner T, Arnon R, Sela M, Abramsky O, Meiner Z, Riven-Kreitman R, et al. Humoral and cellular immune responses to Copolymer 1 in multiple sclerosis patients treated with Copaxone®. J Neuroimmunol 2001;115:152–60. [44] Salama H, Hong J, Zang Y, El-Mongui A, Zhang J. Blocking effects of serum reactive antibodies induced by glatiramer acetate treatment in multiple sclerosis. Brain 2003;126:2638–47. [45] Biogen Idec Inc., Elan Pharmaceuticals Inc. TYSABRI Prescribing Information. US Food and Drug Administration; May 2010. [46] Calabresi PA, Giovannoni G, Confavreux C, Galetta SL, Havrdova E, Hutchinson M, et  al. The incidence and significance of anti-natalizumab antibodies: results from AFFIRM and SENTINEL. Neurology 2007;69:1391–403. [47] Freedman MS, Pachner AR. Neutralizing antibodies to biological therapies. A “touch of gray” vs a “black and white” story. Neurology 2007;69:1386–8. [48] Coyle PK, Foley JF, Fox EJ, Jeffery DR, Munschauer III FE, Tornatore C. Best practice recommendations for the selection and management of patients with multiple sclerosis receiving natalizumab therapy. Mult Scler 2009;15:S26–36.

4 Animal Models of Multiple Sclerosis Fumitaka Sato*, Seiichi Omura*, Nicholas E. Martinez, Ikuo Tsunoda Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130, USA

Etiology, Clinical Course, and Pathology Etiologies of Multiple Sclerosis and Its Animal Models Multiple sclerosis (MS) is an inflammatory demyelinating disease in the central nervous system (CNS) [1–3]. Although the precise etiology of MS remains unclear, MS has been proposed to be a disease caused by interactions among autoimmunity, virus infections, and/or genetic factors (Figure 4.1). The autoimmune etiology of MS has been supported by clinical findings in which myelin-specific T cells and antibodies have been found in some MS patients [4]. Infiltration of immune cells is observed in the demyelinating lesions [5]. Immunomodulatory drugs, such as interferon (IFN)-β or anti-very late antigen (VLA)-4 antibody, are effective in some patients [6]. Experimentally, sensitization with CNS antigen can induce an autoimmune response, resulting in the inflammatory demyelinating disease, experimental autoimmune (allergic) encephalomyelitis (EAE) [7,8]. Microbial infections, particularly virus infection, have also been associated with MS pathogenesis [9,10]. Clinically, viruses and antiviral immune responses have been detected in some MS patients [11–13]. Experimentally, demyelinating diseases can be induced by viruses, such as canine distemper virus and murine hepatitis

Autoimmunity

Virus infection

MS?

Others

(genetic factors, sex…)

*Drs. Sato and Omura contributed equally. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00004-6 © 2011 Elsevier Inc. All rights reserved.

Figure 4.1  The etiology of MS has several hypotheses.  The most common hypotheses involve viral infections and/ or autoimmune responses. While many other factors have been associated with MS, multiple factors may combine to initiate the disease. Theiler’s murine encephalomyelitis virus (TMEV) and EAE models allow us to address both autoimmune and viral demyelinating disease in combination with other factors that may contribute to MS.

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virus [14,15]. Among viral models for MS, Theiler’s murine encephalomyelitis virus (TMEV) infection has been widely used since 1975 [16]. In the TMEV model, various immune cells play an important role in the pathogenesis of demyelination, as we will discuss in this review. Sex and genetic factors also play a role in MS [17,18]. There are more than 2 million MS patients in the world, with a ratio of women to men of 2.6:1 [19,20]. The incidence of MS is higher in North America and Europe than in Asia; for instance, in Scotland and offshore island, it is 145 to 193/100,000 and in Japan it is 7.7/100,000 [21–23]. A study of MS in monozygotic versus dizygotic twins demonstrated a concordance of 25.9% in monozygotic twins as opposed to 2.3% in dizygotic twins [24]. The human leukocyte antigen (HLA), particularly HLA DRB1*1501, has been reported to influence the severity of MS [25]. Genes, including interleukin (IL)-2 receptor α chain, IL-7 receptor, and tumor necrosis factor (TNF) receptors, may influence the risk of developing MS [25–27]. Other genes, such as the vitamin D receptor gene [28] and the estrogen receptor gene [29], have also been proposed to be factors in susceptibility to MS. Sex and genetic background are also important for disease induction in both EAE and TMEV infection [7,30]. Major histocompatibility complex (MHC) class I antigen is important for resistance in TMEV infection [31,32]. While SJL/J mice have been most widely used as animal models for several immune-mediated diseases, including EAE and TMEV infection, SJL/J mice have genetic abnormalities, such as a defect of dysferlin on chromosome 2q13 [33] and nearly 50% deletion of T-cell receptor (TCR) Vβ [34], and express the I-A but not the I-E class II molecule of the MHC [35].

EAE and TMEV Infection In 1933, EAE was induced in monkeys with multiple intramuscular injections of a rabbit brain emulsion [36]. In the 1940s, when CNS antigen was emulsified in complete Freund’s adjuvant (CFA) and injected into rodents, animals reproducibly developed an acute encephalomyelitis within a few weeks, now known as “active EAE” [37]. Paterson [38] reported passive transfer of EAE by lymphocytes; “passive or adaptive transfer EAE” was obtained by injection of activated encephalitogenic T cells in 1960. Since then, EAE has been induced by injection of various CNS antigens or CNS antigen-specific T cells. TMEV was isolated from the CNS of mice with spontaneous flaccid paralysis of the hind limbs by Max Theiler [39]. Daniels et al. [40] first reported demyelination in TMEV infection. TMEV belongs to the family Picornaviridae that consists of nonenveloped positive sense single-stranded RNA viruses [41]. Based on neurovirulence, TMEV is divided into two subgroups, GDVII and Theiler’s original (TO). The GDVII subgroup is highly neurovirulent in mice, resulting in death within 1–2 weeks [42]. The TO subgroup contains Daniels (DA) and BeAn8386 (BeAn) strains and causes an acute polioencephalomyelitis 1 week after infection (acute phase), followed by an inflammatory demyelinating disease 1 month after infection (chronic phase) [7]. TMEV predominantly infects neurons during the acute phase [43] and infects macrophages and glial cells, including oligodendrocytes (myelin-forming

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cells), during the chronic phase [42,44–46]. Unlike EAE, TMEV induces an inflammatory demyelinating disease in mice only [47].

Clinical Courses of MS, EAE, and TMEV Infection The clinical courses of MS are classified into four types [48,49]: relapsing–remitting (RR), primary progressive (PP), secondary progressive (SP), and progressive–relapsing (PR) (Figure 4.2). RR-MS is defined by disease attacks (“relapses”) with recovery (“remission”) and is the most common (85–90%) [50]. SP-MS is defined by an initial RR disease course followed by disease progression. Approximately 95% of RR-MS patients later develop SP-MS [51]. PR-MS is a progressive disease from the onset, with acute relapses; periods between relapses have continuous progression. PP-MS (10– 15% of patients) progresses continuously from the onset without recovery. While disease activities, including relapse and remission, in RR-MS have been associated with the balance between T helper (Th)-cell subsets such as Th1 and Th2 cells, it is unclear whether immunologic profiles are the same between RR-MS and other forms of MS.

Human

Disability

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Remission

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Mouse Human MOG92–106 in A.SW PP-MS Polioencephalomyelitis

TMEV infection Time

(C)

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Figure 4.2  Clinical courses of MS and its animal models.  (A) In the monophasic model, a single attack is followed by recovery with no relapse. In EAE, MBP-induced EAE and MOG35–55induced EAE develop a monophasic episode with complete or incomplete recovery, respectively. In humans, it may correspond to acute disseminated encephalomyelitis (ADEM) or clinically isolated syndrome (CIS). (B) In RR-MS, disease attack (relapse) and recovery (remission) occur alternately. In mouse EAE, PLP and MOG92–106 induce RR-EAE in SJL/J mice. (C) In SP-MS, the initial RR disease course is followed by disease progression. SP-EAE can be induced by MOG92–106 in SJL/J mice with ultraviolet (UV) radiation or injection of apoptotic cells, or in A.SW mice with Bordetella pertussis (BP) injection. (D) In PP-MS, the disease progresses continuously from the onset without remission. PP-EAE model can be induced by MOG92–106 sensitization in A.SW mice. TMEV infection causes acute polioencephalomyelitis (with no demyelination) during the acute phase followed by chronic progressive demyelinating disease.

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Clinical courses of the animal models for MS are also variable. The monophasic EAE model was first established by injection of myelin basic protein (MBP) or CNS and myelin homogenates [36,52]. However, this monophasic model may be more representative of the human diseases clinically isolated syndrome (CIS) or acute disseminated encephalomyelitis (ADEM) than MS. CIS is a monophasic clinical event involving the CNS that may or may not lead to MS [53]. ADEM is an acute monophasic inflammatory demyelinating disease that occurs after infection or vaccination. Later, by using myelin proteolipid protein (PLP), an EAE model for RR-MS was established [54,55]. More recently, myelin oligodendrocyte glycoprotein (MOG) has been used for EAE induction. In MOG-induced EAE, unlike in MBP- and PLPinduced EAE, not only T cells but also antibodies play a role in demyelination. In addition, MOG35–55 can induce EAE in C57BL/6 mice whose transgene or knockout (KO) strains are readily available. Most of the EAE models develop monophasic or RR disease; only a few PP- and SP-EAE models have been established [56,57]. TMEV-infected mice develop progressive disease.

Pathologies of MS, EAE, and TMEV Infection Pathologically, MS can be divided into several subtypes based on T-cell and macrophage infiltration, antibody and complement deposition, and oligodendrocyte apoptosis [58]. The lesions are segregated into four patterns of myelin destruction. Pattern I is T-cell and macrophage-associated demyelination where T-cell infiltration and activated macrophage/microglia are detected. Pattern II is antibody-mediated demyelination where complement-mediated lysis of antibody-targeted myelin is suggested in addition to the mechanisms of pattern I. Pattern III is distal oligodendrogliopathy-associated demyelination where degeneration of distal oligodendrocyte processes, oligodendrocyte apoptosis, and demyelination are found. Pattern IV is primary oligodendrocyte degeneration, and these lesions are the least common in the MS population. Because of the heterogeneity in pathologic features, each pathology pattern has been speculated to be triggered by several different mechanisms. For example, autoimmune T cells and antibody against myelin have been proposed to be the cause of inflammatory demyelinating lesions (pattern II), while ischemic, toxic, metabolic, and/or viral infections have been speculated to be the cause of oligodendrocyte apoptosis (pattern III). So far, attempts to find the cause in individual MS patients have been unsuccessful; we do not know whether the different etiologies contribute to the different clinical and pathologic phenotypes. Neuropathology of EAE induced with MBP and PLP resembles pattern I, while some MOG-induced EAE is similar to pattern II [49]. Oligodendrocyte apoptosis can be seen in TMEV infection and in some EAE models [7,59]. While different etiologies can cause different neuropathology, one single cause can induce different pathologies and clinical courses depending on the patient’s genetic background. MOG92–106 peptide sensitization in different mouse strains results in different clinical and pathologic outcomes [49]. SJL/J mice with MOGinduced EAE develop an inflammatory demyelinating RR disease with oligodendrocyte apoptosis. In contrast, A.SW mice with MOG-induced EAE develop PP disease accompanied by plaque-like demyelination and antibody deposition in the absence

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of oligodendrocyte apoptosis. In the presence of supplemental Bordetella pertussis (BP), A.SW mice with MOG-induced EAE develop SP disease [49] (Figure 4.2).

Demyelination Versus Axonal Degeneration (Neurodegeneration): Inside-Out Model of MS MS is characterized by demyelination and axonal degeneration in the CNS. The primary target in MS has been believed to be either myelin (myelinopathy) itself or oligodendrocytes (oligodendrogliopathy), and axonal degeneration is regarded as a secondary injury to demyelination. In this hypothesis, anti-myelin immune responses or direct virus infections of oligodendrocytes play an effector role in inflammatory demyelination. If inflammation that causes myelin damage is severe, it can secondarily damage axons. Here, the lesion develops from myelin (outside) to axon (inside), hence the name “Outside-In” model. EAE models induced by myelin antigens support the Outside-In model. However, histologic and neuroimaging studies demonstrated that axonal degeneration in the absence of demyelinating lesions occur in some MS patients [60], which cannot be explained by the classic Outside-In model. In viral models for MS, including TMEV and canine distemper virus infections, axonal degeneration has been shown to precede demyelination [61,62]. In addition, EAE has been shown to be induced by axonal antigens, including neurofilament light protein and contactin-2/transiently expressed axonal glycoprotein 1 (TAG-1) [63,64]. Here, the lesion develops from axon (inside) to myelin (outside), “Inside-Out” model [65–67]. In the Inside-Out model, axonal degeneration can lead to the induction of oligodendrocyte apoptosis, which results in secondary demyelination. Later, degenerated myelin and axonal antigens can be phagocytosed by microglia and macrophages, which can present myelin antigens and induce anti-myelin immune responses. These anti-myelin immune responses, in turn, attack myelin sheaths from the outside. Thus, Outside-In and Inside-Out models can make a vicious cycle, leading to disease progression, unless regulatory mechanisms stop the cascade reaction.

Immunology of MS, EAE, and TMEV Infection CD4 T Helper Cell Subsets CD4 Th cells have been believed to play a central role in the pathogenesis of MS. Naïve CD4 T cells can differentiate into four subsets: Th1, Th2, Th17, and regulatory T (Treg) cells, under distinct cytokine milieus (Figure 4.3). Th1-cell differentiation is initiated by IL-12, which induces T-box expressed in T cells (T-bet) [68]. Th1 cells produce large amounts of IFN-γ and IL-2, mediating cellular immunity such as delayed-type hypersensitivity (DTH) responses. IL-4 enhances the expression of GATA-binding protein 3 (GATA3), driving Th2-cell differentiation [69]. Th2 cells secrete IL-4, IL-5, and IL-13, promoting humoral immunity. The balance between transforming growth factor (TGF)-β and IL-6 acts to induce either retinoic acid receptor-related orphan receptor γt (RORγt), which is a marker for

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Naïve CD4+ T cell

TGF-β

TGF-β + IL-6

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Th17

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RORγ t

T-bet

GATA3

TGF-β, IL-10

IL-17, IL-21, L-22

IFN-γ, IL-2

IL-4, IL-5, IL-13

Figure 4.3  Th-cell subset differentiation.  T-bet activated by IL-12 contributes to Th1-cell differentiation. IL-4 upregulates GATA3, polarizing naïve CD4 T cells into Th2 cells. Th17-cell differentiation is initiated by TGF-β and IL-6, inducing RORγt expression. TGF-β drives Foxp3 Treg cells.

Th17 cells, or transcription factor forkhead box P3 protein (Foxp3), which is a marker for Treg cells [70,71]. Th17 cells produce IL-17A, IL-17F, IL-21, and IL-22. Th17 cells are associated with antimicrobial immunity and autoimmunity. Treg cells secrete TGF-β and IL-10, and regulate the other Th-cell subsets [72].

Roles of Th1 Cells in EAE and TMEV Infection MS has been proposed to be a Th1-mediated disease, since Th1 immune responses have been associated with disease activity in MS patients [73,74]. For example, CD4 T-cell lines developed from cerebrospinal fluid of MS patients produced large amounts of IFN-γ and IL-2 [75]. Th1 cells are also associated with the pathogenesis of EAE. In EAE, large amounts of IFN-γ and IL-2 were secreted from CD4 T cells by in vitro stimulation and IFN-γ-producing T cells were observed in the CNS [76]. Myelin antigen-specific Th1 cells induced EAE upon passive transfer into naïve mice [76–78]. IL-12 neutralization with anti-IL-12 monoclonal antibody (mAb) injection protected susceptible SJL/J mice against mouse spinal cord homogenateinduced EAE [79]. Mice deficient in T-bet sensitized with MOG showed decreased severity of EAE compared with MOG-sensitized wild-type mice [80]. Also, CD4 T cells from T-bet-deficient mice sensitized with MOG produced low amounts of IFN-γ. These results support the hypothesis that Th1 cells are critical for EAE pathogenesis. However, IFN-γ deficient (IFN-γ2/2) mice have been reported to develop more severe EAE. In susceptible B10.PL mice, IFN-γ2/2 mice developed more severe MBP-induced EAE than wild-type mice [81]. Moreover, IFN-γ2/2 mice developed demyelinating disease in BALB/c mice, which are known to be resistant to MBPinduced EAE otherwise [82]. In the CNS of IFN-γ2/2 BALB/c mice, the enhanced infiltration of activated CD4 T cells and neutrophils was observed. Similarly, in the

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EAE-resistant 129/Sv mice, IFN-γ-receptor-deficient (IFN-γR2/2) mice developed severe EAE [83,84]. In addition, MBP sensitization induced lethal progressive EAE with extensive demyelination and neutrophil infiltration in the CNS in otherwise resistant C57BL/6 mice lacking IFN-γR [82]. Thus, IFN-γ may play a protective role in some EAE models. Th1 cells can play both protective and pathogenic roles in TMEV infection. During the acute phase of TMEV infection, Th1 cells seem to control the viral infection, leading to host defense against TMEV [85,86]. IFN-γ neutralization with antiIFN-γ mAb significantly accelerated the onset of disease after TMEV infection [87]. On the other hand, during the early chronic phase of TMEV infection, CD4 T-cellmediated DTH responses to TMEV have been suggested to induce demyelination in a “bystander” fashion [88]. IL-12 neutralization with anti-IL-12 mAb during the early chronic stage of TMEV infection decreased demyelinating disease [89]. During the late chronic phase of TMEV infection, anti-myelin Th1 cells induced by epitope spreading have been proposed to exacerbate the demyelinating disease [90].

Roles of Th2 Cells in EAE and TMEV Infection IL-4 production from Th2 cells can enhance the expression of GATA3, leading to the inhibition of Th1-cell differentiation and suppression of Th1-cell function [91]. In EAE, the adoptive transfer of PLP-specific Th2-cell clones prevented EAE in mice sensitized with PLP in which resistance to EAE is correlated with IL-4 production [92]. Neutralization of IL-4 rendered EAE-resistant BALB/c mice susceptible [79]. The enhancement of Th2 cells delayed the onset and decreased the severity of PLP-induced MOG [93]. Thus, Th2 cells have been proposed to play a protective role in EAE. However, Th2 cells can induce EAE under certain conditions. Lafaille et al. [94] demonstrated that the adoptive transfer of MBP-specific Th2 cells caused progressive EAE in immunocompromised recombination-activating gene-1 (RAG-1) KO or αβ T-celldeficient mice but not wild-type mice. Th2 immune responses have also been proposed to exacerbate demyelinating disease with the enhancement of pathogenic autoantibodies. Tsunoda et al. [56,95] also demonstrated that anti-MOG antibody in favor of Th2 immune responses induced progressive and fatal MOG-induced EAE in A.SW mice. In this model, extensive demyelination with immunoglobulin (Ig) deposition was observed in the CNS. In addition, SJL/J mice sensitized with MOG developed SP-EAE through injection of apoptotic cells. In this system, the enhancement of anti-MOG antibody production appears to be mediated by Th2-biased immune responses [95]. Similarly, in a SP-EAE mouse model induced by ultraviolet (UV) irradiation, high anti-MOG antibody responses were observed with a decrease in Th1 immune responses [96]. Thus, Th2 cells have been shown to have protective and detrimental roles in EAE. In TMEV infection, Th2 cells have been proposed to suppress inflammation and demyelination. Hill et al. [30] demonstrated that IL-4 treatment during the early chronic phase of TMEV infection ameliorated TMEV-induced inflammation and demyelinating disease with a reduction in anti-TMEV antibody responses. Although anti-TMEV antibody can neutralize the virus, it can cross-react with myelin lipid and act as demyelinating antibody [97]. Although Th2 cytokine mRNA has been

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demonstrated in the spinal cord during the chronic phase of TMEV infection [98], it is not clear whether Th2 cytokines play a beneficial role (suppression of encephalitogenic Th1 cells) or a detrimental role (by enhancing production of potential demyelinating antibody [97]) in demyelinating disease.

Roles of Th17 Cells in EAE and TMEV Infection The IL-17 cytokine family is composed of six members, IL-17A through F [99]. IL-17A-producing Th17 cells have been identified as a novel Th-cell subset [100,101]. Th17 cells can be differentiated from naïve CD4 T cells in the presence of IL-6 and TGF-β in mice, and produce IL-17F, IL-21, and IL-22 in addition to IL-17A (see Figure 4.3). IL-17A and F are pro-inflammatory cytokines. IL-17 has been detected in the peripheral blood, brain lesions, and cerebrospinal fluids from MS patients [102,103]. Thus, Th17 cells have attracted considerable attention in MS. Hofstetter et al. [104] first suggested the pathogenic role of Th17 cells in EAE induced with MOG. IL-17 production was detected in splenic T cells post-sensitization, but not from those of naïve mice by in vitro stimulation with MOG. IL-17 neutralization with IL-17-receptor-Fc-hybrid protein or anti-IL-17 mAb injection ameliorated EAE partially. Similarly, Komiyama et al. [105] reported that IL-17deficient (IL-17-/-) mice sensitized with MOG showed delayed onset and decreased severity of EAE compared with MOG-sensitized wild-type mice. Although IL-17 is produced from various cells, the major population of IL-17 producers was Th17 cells during the development of EAE. In addition, upon passive transfer into naïve wild-type mice, CD4 T cells from IL-17-/- mice sensitized with MOG significantly attenuated EAE induction compared with those from MOG-sensitized wild-type mice. Bettelli et al. [106] demonstrated that TGF-β transgenic mice sensitized with MOG had a higher frequency of Th17 cells and developed more severe EAE than wild-type mice. The precise role of Th17 cells in the development of EAE remains controversial. EAE has been considered as a CNS antigen-specific Th1-cell-mediated autoimmune disease, since EAE can be induced by the adoptive transfer of CNS antigenspecific Th1 cells into naïve mice [76–78]. O’Connor et al. [107] demonstrated that the adoptive transfer of myelin antigen-specific Th17 cells into naïve mice induced less severe EAE than that of myelin antigen-specific Th1 cells. In this system, donor Th17 cells were identified only in the CNS of recipient mice when both Th1 and Th17 cells were co-transferred but were not observed in the CNS of recipient mice injected with Th17 cells alone. On the other hand, donor Th1 cells infiltrated the recipient CNS in the absence or presence of Th17 cells. The authors proposed that Th17 cells had a minor role in the induction of EAE compared with Th1 cells. In contrast, Jäger et al. [108] proposed that Th17 cells played a major role in EAE induction. Although the adoptive transfer of MOG-specific Th17 or Th1 cells in vitro led to EAE induction in recipient mice, the Th17 cells induced more severe EAE compared with the Th1 cells. In TMEV infection, Hou et al. [109] demonstrated the pathogenic role of Th17 cells in demyelinating disease. Susceptible SJL/J and resistant C57BL/6 mice were

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infected with the BeAn strain of TMEV. The frequency and number of Th17 cells were higher in mononuclear cells (MNCs) of the CNS from TMEV-infected SJL/J mice than from TMEV-infected C57BL/6 mice. The CNS MNCs from infected SJL/J mice produced large amounts of IL-17 compared with those from TMEV-infected C57BL/6 mice. Although C57BL/6 mice are resistant to demyelinating disease, lipopolysaccharide (LPS) injection can make C57BL/6 mice susceptible to demyelinating disease. LPS injection in TMEV-infected C57BL/6 mice resulted in increased Th17-cell differentiation, IL-17 production from MNCs in the CNS, and inflammatory demyelination. In SJL/J and LPS-treated C57BL/6 mice infected with TMEV, IL-17 neutralization with anti-IL-17 mAb injection reduced the incidence and severity of demyelinating disease. In addition, the injection of anti-IL-17 neutralizing antibody inhibited viral persistence in the CNS and enhanced the function of cytotoxic T cells (CTLs) against TMEV in SJL/J and LPS-treated C57BL/6 mice postinfection. These were in accordance with the findings in EAE where the frequency of IFN-γ-producing CD4 and CD8 T cells was increased in IL-17-deficient mice [105]. Thus, IL-17 production from Th17 cells was associated with the development of demyelinating disease, the enhancement of viral persistence, and the suppression of CTL function in TMEV infection.

Roles of Regulatory T Cells in EAE CD4CD25 Treg cells make up 5–10% of CD4 T cells in mice (2–5% of CD4 T cells in humans) and inhibit the proliferation and cytokine production of effector T cells. Treg cells express Foxp3 and are generated in both the thymus and periphery in vivo [110,111]. Treg cells can be differentiated from naïve CD4 T cells in the presence of TGF-β in vitro (see Figure 4.3) [106]. In humans, the dysfunction of Foxp3 causes a severe autoimmune disease, which is called immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX); Treg cells have been associated with the suppression of autoimmunity [112,113]. Kohm et al. [114] demonstrated that the adoptive transfer of Treg cells isolated from naïve mice ameliorated MOG-induced EAE. The co-injection of MOG-specific T cells with Treg cells attenuated the development of EAE, while the T cells alone induced severe EAE upon passive transfer. Treg cells also suppressed MOG-specific immune responses when encephalitogenic cells from MOG-sensitized mice were cultured with Treg cells in vitro. Similarly, Selvaraj et al. [115] demonstrated that Treg cells generated by TGF-β in vitro inhibited MOG-induced EAE. The adoptive transfer of TGF-β-induced Treg cells suppressed IFN-γ and IL-17 production from encephalitogenic cells stimulated with MOG in vitro. The exact inhibitory mechanisms of Treg cells remain unclear in EAE. Unlike other Th-cell subsets, Treg cells appear to display the suppressive function independent of CNS-antigen specificity. IL-10 production from Treg cells is thought to be critical for the immunosuppressive function in some EAE models, since Treg cells from IL-10-deficient mice failed to prevent the development of EAE [115–117]. The role of Treg cells in EAE has also been investigated by the depletion of Treg cells with anti-CD25 mAb. Montero et al. [118] demonstrated that Treg-cell

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depletion with anti-CD25 mAb prior to MOG sensitization led to the exacerbation of EAE. In this system, the anti-CD25 mAb treatment enhanced IFN-γ production from MOG-specific Th1 cells and serum MOG-specific IgG antibody responses. In addition, Reddy et al. [119] demonstrated that the depletion of Treg cells with anti-CD25 mAb made resistant B10.S mice susceptible to EAE. The anti-CD25 mAb treatment led to an increased incidence and inflammation of the CNS in B10.S mice with PLPinduced EAE, in which mice treated with anti-CD25 mAb showed higher IFN-γ and lower IL-10 production from CD4 T cells compared with control mice. However, caution should be used when interpreting the data in anti-CD25 mAb studies, since CD25 (IL-2 receptor α chain) is expressed not only on Treg cells but also on immature T cells in the thymus [120] and activated T cells. Indeed, daclizumab, a drug that has been shown to be effective in MS, is an anti-CD25 antibody; its suppressive activity has been attributed to inhibition of activated T cells [121,122]. Although there are no reports on the role of Treg cells in TMEV infection, theoretically, Treg cells can play either a beneficial or a detrimental role in TMEV infection. Since Treg cells can suppress the function of effector T cells, the suppression of anti-virus immune responses by Treg cells can result in viral persistence in the CNS, leading to chronic demyelinating disease. Indeed, depletion of Treg cells has been shown to induce more effective viral clearance and enhance anti-virus immune responses in Friend retrovirus-infected mice [123]. On the other hand, the enhancement of Treg-cell activity may ameliorate demyelinating disease. During the late chronic phase of TMEV, anti-virus and anti-myelin immune responses have been proposed to attack myelin and oligodendrocytes, leading to progressive demyelinating disease. Here, if Treg cells control antiviral as well as anti-myelin immune responses, this should inhibit immune-mediated demyelination (immunopathology). Treg cells appear to protect against severe disease after West Nile virus infection in human and mice where antiviral immune responses have been shown to play a pathogenic role [124].

Roles of Th9 Cells in EAE Recently, IL-9-producing Th (Th9) cells have been proposed as a novel Th-cell subset [125,126]. IL-9 was originally identified as a T-cell growth factor and was reported to be associated with Th2-type immune responses, such as parasite infection and asthma [127–129]. Th9 cells can be differentiated from naïve CD4 T cells in the presence of TGF-β and IL-4, and produce large amounts of IL-9 and IL-10. Although IL-10 is an anti-inflammatory cytokine, Th9 cells do not inhibit tissue inflammation or immune responses [126]. Elyaman et al. [130] demonstrated that IL-9 in the presence of TGF-β facilitated Th17-cell differentiation, while IL-9 enhanced the activity of Treg functions in vitro. Thus, Th9 cells may play a pathogenic as well as a protective role in EAE. IL-9-receptor-deficient mice showed more severe EAE than wild-type mice. On the other hand, Jäger et al. [108] demonstrated that passive transfer of MOG-specific Th9 cells induced EAE, in which demyelination with massive parenchymal cell infiltration was observed in the spinal cord.

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Roles of Natural Killer T Cells in EAE and TMEV Infection Natural killer T (NKT) cells have been shown to play a role in both viral infections and autoimmune diseases. This subset of immune cells expresses natural killer cell markers and an invariant TCR (Vα14 in mice and Vα24 in humans) that binds to CD1d molecules presenting a glycolipid antigen, including α-galactosylceramide (α-GC) [131]. Stimulation of the NKT cells results in a rapid secretion of cytokines, such as IL-4 and IFN-γ, that can regulate immune responses. The high levels of CD1 expression present in CNS lesions and overall high lipid content in the CNS make NKT cells likely candidates for regulation of CNS diseases [132–134]. In MS, several studies have suggested a beneficial role for NKT cells in pathogenesis [135–137]. In EAE, there is evidence that NKT cells play a protective role. Singh et al. [138] were able to protect against EAE in mice that were given α-GC to activate NKT cells. In this study, α-GC was unable to protect against EAE in CD1d, IL-4, or IL-10 KO mice, indicating that α-GC presentation on CD1d molecules and secretion of IL-4 and IL-10 were necessary for the suppression of EAE [138]. The Th1 response was suppressed by activating NKT cells with α-GC, which shifted the immune system to a Th2 response. Furthermore, transgenic mice with increased numbers of NKT cells exhibited reduced clinical signs of EAE even in the absence of extrathymic CD1d [139]. EAE has also been suppressed in an NKT-cell-dependent manner through the alteration of the gut flora [140]. While NKT cells can suppress the monophasic or RR form of EAE by invoking Th2 responses, they may contribute to the fatal progressive form of the disease. In MOG-induced EAE, A.SW mice (with inducible NKT cells) have been shown to develop the fatal progressive disease, while SJL/J mice (with reduced NKT cell numbers) develop moderate RR disease [56]. NKT cells also play regulatory roles in TMEV infection. CD1d-deficient (CD1d-/-) mice do not facilitate development of NKT cells in the thymus and are essentially NKT-deficient mice. CD1d-/- mice infected with the neurovirulent GDVII virus had an increased number of viral antigen-positive cells, more rapid weight loss, and higher mortality rates than wild-type mice [141]. Although wild-type BALB/c mice infected with DA virus had no demyelination, 60% of CD1d-/- mice developed demyelination [141]. These results suggest that the CD1d molecule and thus likely NKT cells are important in regulating the immune response and clearing viruses in the CNS. In another study, anti-Vα14 antibody was used to deplete NKT cells at various time points in mice infected with the DA virus. If the antibody was administered early, it was able to delay the onset of demyelination, while late or weekly administration resulted in more severe demyelination [142]. Similar to the CD1d-/- mice, the Vα14-depleted mice had increased demyelination and viral antigen-positive cells in the CNS [142]. Together, these studies suggest that NKT cells play contrasting roles in TMEV infection over the disease course.

Other Pathomechanisms in EAE and TMEV Infection MHC class I-restricted virus-specific CD8 CTLs are important in virus clearance during the acute phase of TMEV infection. CD8 T cells have also been shown to

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play a suppressive/regulatory role against demyelination in both EAE and TMEV infection [7]. More recently, however, CD8 T cells have been demonstrated to play a pathogenic role in EAE and TMEV models. Although oligodendrocytes do not express MHC class I molecules on their cell surface in steady state, they can be activated by inflammatory cytokines, such as IFN-γ, to express MHC class I mole­cules on their surface. Here, oligodendrocytes present myelin or viral antigen on MHC class I molecules and can be directly attacked by CTLs during the inflammation, which can lead to demyelination. Myelin-specific and nonspecific CD8 T cells have been shown to induce EAE [143,144]. TMEV-specific CD8 T cells have also been shown to play a pathogenic role upon passive transfer [145,146]. Like other immune effectors, antibody can play dual roles in MS. Antiviral antibody has been shown to neutralize TMEV in vitro. Adoptive transfer of antiTMEV antibody can eradicate virus from the CNS of infected nude mice [147]. On the other hand, since anti-TMEV antibody can cross-react with myelin lipid, antiTMEV antibody can function as demyelinating antibody [148]. Both anti-MOG antibody and anti-TMEV antibody have been shown to enhance demyelinating disease in vivo, although the administration of the antibody alone is not sufficient to cause demyelination. A direct TMEV infection in neurons and oligodendrocytes can lead to axonal degeneration and demyelination [149]. During the acute phase of TMEV infection, TMEV antigens are identified predominantly in neurons of the gray matter, but not in the white matter. Later, axonal degeneration, but not demyelination, is observed in the spinal cord white matter without the infiltration of inflammatory cells. Thus, early neuronal infection seems to result in wallerian (axonal) degeneration in TMEV infection [150]. During the chronic stage of TMEV infection, TMEV antigen was observed in oligodendrocytes; this suggests that direct oligodendrocyte infection may result in demyelination. Indeed, Roos et al. [151] demonstrated that TMEV infection induced demyelinating disease in nude mice. In addition, direct TMEV infection has been shown to cause demyelination in vitro [152]. Thus, direct lytic infection of oligodendrocytes can lead to demyelination.

Conclusions The “Two-Stage” Disease Theory MS and its animal models appear to be influenced by the interactions among various immune cells. In the RR disease course, Th1 and Th17 cells play a major role in relapse, whereas Th2, Treg, and NKT cells are associated with remission. In this context, enhanced Th1 and Th17 immune responses, including epitope spreading, as well as decreased Th2, Treg, and NKT cell function can lead to the SP disease course. On the other hand, other effector mechanisms, such as axonal degeneration or production of autoantibodies enhanced by Th2 immune responses, axonal degeneration, or neurodegeneration, have been considered to play a central role in the development of SP-MS (Table 4.1).

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Table 4.1  Possible Roles of Immune Effectors in MS, EAE, and TMEV Infection Effectors

Cell Types

Role

Clinical Course

Cytokine and Marker

CD4 T cells Th1

DTH, Th2↓, Disease onset Th17↓, IgG2a (c)↑, Relapse/progression anti-viral CTL↑ (epitope spreading)

IFN-γ, IL-2, T-bet

Th2

Th1↓, IgG1↑, IgE↑, Remission anti-viral Ig↑ Disease progression

IL-4, IL-5, IL-13, GATA3

Th17

Th1↓

IL-17A, IL-17F, IL-21, IL-22, RORγt

Treg

Th1↓, Th2↓, Th17↓ Remission Virus persistence

TGF-β, IL-10, Foxp3

NKT cells

CD1d-restricted NKT cells and others

Regulatory Virus killing

Disease suppression Virus suppression

IFN-γ, IL-4, NK1.1, CD3, Vα14 TCR

Antibody

B cells

Demyelinating Ig Extracellular virus neutralization

Disease progression

Ig, CD19, B220

Epitope spreading Virus persistence

CTL, cytotoxic T lymphocyte; DTH, delayed-type hypersensitivity; Foxp3, forkhead box P3; GATA3, GATA-binding protein 3; IFN, interferon; Ig, immunoglobulin; IL, interleukin; NKT, natural killer T; RORγt, retinoic acid receptorrelated orphan receptor γt; T-bet, T-box expressed in T cells; TGF, transforming growth factor; Th, T helper; Treg, regulatory T.

As discussed earlier, we have accumulated much information on which cell types/ mechanisms play effector or protective roles in MS and its animal models. However, we still do not know which factors contribute to the switching from the RR disease course to the SP disease course. MS has been suggested to be a heterogeneous “twostage” disease that could switch from an inflammatory to a degenerative phase (Figure 4.4) [153,154]. The two-stage disease theory is based on descriptive findings that inflammation and axonal degeneration appear to correlate with clinical signs during the RR and SP courses, respectively. One should keep in mind, however, that inflammation and axonal degeneration can influence each other, while the two processes can occur independently. Thus, the two-stage disease can be induced solely by inflammation alone or a combination of two mechanisms. For example, severe inflammation in the early disease stage can lead to axonal degeneration in the late stage. Here, we would like to propose a possible immunopathologic mechanism for the two-stage disease theory. MS patients develop the RR disease course when there is a balance between inflammatory (Th1 and Th17 cells) versus regulatory (Th2, Treg, and NKT cells) cells, where the former contributes to relapse and the latter contributes to remission. If MS is a “one-stage” disease, the disease progresses when the balance between inflammatory versus regulatory skews toward inflammatory.

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“One-stage” disease theory SP

RR

Disability

Th1 Th17 Th1 Th17

Th1 Th17 Th2 Treg NKT

Th2 Treg NKT

(A)

Time

Th2 Treg NKT

“Two-stage” disease theory RR

SP Anti-axonal antibody? Neurodegeneration?

Disability

Biological switch

(B)

Th1 Th17

Th1 Th17

Inflammatory Regulatory

Th2 Treg NKT

Th2 Treg NKT

Time

Figure 4.4  In both “one-stage” (A) and “two-stage” (B) disease theories of MS, the RR disease courses are induced by the balance between inflammatory (Th1 and Th17 cells) versus regulatory (Th2, Treg, and NKT cells) cells, where the former contributes to relapse and the latter contributes to remission. During the SP phase of the “one-stage” disease theory, immune responses skew more toward inflammatory. Here, the pathomechanisms of SP-MS are basically the same during the disease course; excessive anti-myelin inflammatory responses result in secondary axonal degeneration (neurodegeneration). On the other hand, in the “two-stage” disease theory, the central effector mechanisms change when the RR disease course shifts to the SP course. Here, a new effector mechanism, such as the increased autoantibody production promoted by Th2 cells or the axonal degeneration mediated by virus infection, exacerbates demyelinating disease, leading to disease progression. Here, the switch that triggers production of anti-axonal antibodies can be axonal damage by virus infection or a change in the cytokine profile by IL-4-producing NKT cells.

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Here, the pathomechanism of MS does not change between the RR course and the SP course: excessive anti-myelin inflammatory responses result in secondary damage to axons (neurodegeneration). On the other hand, in the two-stage disease theory, there is a change in effector mechanisms when the RR disease course switches to the SP course. For example, generation of anti-axonal antibody during the course can lead to disease progression with the help of Th2 cytokines, rather than pro-inflammatory cytokines. Here, the switch that triggers production of anti-axonal antibody can be axonal damage by virus infection or a change in the cytokine profile by IL-4producing NKT cells. In this latter stage, the role of each cytokine and immune cell can be opposite to that in the early stage. For example, IFN-γ plays an effector role in the early stage, while it plays a protective role by inhibiting Th2 cytokines and antibody production in the late stage.

Microarray Analysis and Future Prospective Analyses of gene expression profiles using microarray provide comprehensive information about gene expression at a given time point [155]. To study the molecular mechanisms of pathogenesis in MS, microarray analyses would provide systemic information other than the immune responses reviewed here, such as axonal degeneration and neuronal apoptotic pathways. In MS, microarray analyses have been performed using mainly peripheral blood lymphocytes. Several reports showed that various genes related to the immune responses, apoptosis, and cell cycle progression were upregulated or downregulated. For example, Singh et al. [156] reported that IFN-β treatment upregulated genes related to immune responses, such as Mx1, Ifi35, and Ifit1, in peripheral blood MNCs from RR-MS patients. Satoh et al. [157] reported that pro-apoptotic genes, such as Nr4a2, Tcf8, and Cyp1a2, were upregulated and anti-apoptotic genes, such as Hspa1a and Bag1, were downregulated in T cells isolated from the blood of MS patients. However, access to the CNS parenchyma is limited by its nature [158]. In EAE models, there are several reports using microarray analyses [159], with various experimental conditions (different stages of disease, animal strains and species, and antigens) and tissue samples (CNS versus lymphoid). In most microarray analyses in EAE, genes related to the immune responses, such as cytokines (Aif1, Ifng, and Il10), chemokines (Ccr2, Cxcl1, and Rantes), and complement components (C1qa, C1qb, and C3), have been shown to be upregulated in the CNS [160–163], while treatment with therapeutic agents, such as lovastatin and N-acetylcystein amide (AD4) [161,164], can normalize the gene expression levels. Pertussis toxin, which is an adjuvant used in some EAE models, has been shown to upregulate genes related to angiogenesis, such as Ccl2 and Cxcl1, following in vitro exposure of brain microvascular endothelial cells [165]. In TMEV infection, there is only limited information about gene expression profiles based on microarray analyses. Rubio et al. [166,167] reported that genes related to immune responses, such as Cxcl1, IFN-α family genes, and Ifnb, were upregulated in TMEV-infected astrocyte cultures. Navarrete-Talloni et al. [168] reported that TMEV infection altered the expression of various genes related to immune response, such as Cd55, Cd68, and Cd209a, in the acute phase in the deep cervical

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lymph nodes and gene transcription decreased during the chronic phase. Ulrich et al. [169] demonstrated that in the CNS of SJL/J mice infected with TMEV, genes related to the immune response, such as Cd28, Cd40, Cd86, Il4r, and Tlr4, were upregulated, and genes related to lipid and cholesterol biosynthesis, such as Dhcr7, Nsdhl, and Fdps, were downregulated during the chronic phase. A bioinformatics approach that can associate the data among comprehensive microarray analyses and immunologic, clinical, and histologic findings will lead to clarification of the roles of immune cells as well as elucidation of novel pathomechanisms.

Acknowledgments Drs. Sato and Omura contributed equally. This work was supported by the National Institutes of Health (R21NS059724, P20-RR018724).

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5 Neuroimaging of Multiple Sclerosis: An Update

Alexandra Lopez-Soriano1, Robert Zivadinov1,2 1 

Department of Neurology, Buffalo Neuroimaging Analysis Center, State University of New York, Buffalo, NY, USA 2  Department of Neurology, The Jacobs Neurological Institute, State University of New York, Buffalo, NY, USA

Conventional Magnetic Resonance Imaging Conventional magnetic resonance imaging (MRI) techniques have become an important tool for supporting a diagnosis of multiple sclerosis (MS), for ruling out MS mimickers, and for monitoring disease evolution in clinical trials. MRI is highly sensitive for detecting demyelinating plaques in the brain and spinal cord, but similar abnormalities can be found in other diseases and even in healthy subjects. Therefore, to increase the specificity of MRI findings, several diagnostic criteria have been proposed based on the demonstration of lesions disseminated in space and in time in the appropriate clinical context and after exclusion of alternative causes [1–4].

Inflammatory MRI Metrics MS plaques are usually at least 3 mm in size, ovoid, perivenular, following the vessel axis, and can occur in white matter (WM) and gray matter (GM) in the brain, spinal cord, or optic nerves. There are some locations considered characteristic for MS, including juxtacortical, periventricular, corpus callosum, infratentorial, and spinal cord. Spinal cord lesions are more common in the cervical than in the thoracic cord, and characteristically their length is two or fewer vertebral segments, and they are asymmetrical on axial sections [5]. Cortical lesions are very difficult to identify on conventional MRI sequences due to their small size and poor lesion-to-tissue contrast [6,7]. MS lesions are hyperintense on fluid-attenuated inversion recovery (FLAIR) and dual echo proton density (PD) and T2-weighted (PD/T2) MR sequences. Depending on the stage of evolution of a lesion and its degree of edema, demyelination/ remyelination, and axonal loss, lesions show more or less hypointensity on T1-weighted images. Newly active lesions represent transient breakdown of the blood–brain barrier (BBB) and can show nodular or ring-like contrast enhancement (Figure 5.1). Contrast enhancement can last from 1 to 16 weeks, but most of the Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00005-8 © 2011 Elsevier Inc. All rights reserved.

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Figure 5.1  Enhancing lesions typical of MS on axial T1-weighted postcontrast scans after gadolinium injection (0.1 mmol/kg). Two types of enhancing lesions are present in this 33-year-old man with RRMS: (A) homogenous lesions (hyperintense on T1-weighted images) and (B) open-ring lesions (with hypointense center and external hyperintense rim).

enhancement disappears over 4–6 weeks [8]. Persistent gadolinium enhancement for more than 3 months is unusual for MS lesions. Preexisting lesions on T2-weighted images can show contrast enhancement on T1-weighted images as a sign of inflammatory reactivation with BBB disruption [9]. Gadolinium enhancement is more common in younger patients than in older MS patients of all disease subtypes [10]. Some studies have also described a possible compartmentalization of inflammation in the progressive stage of MS behind a “repaired” BBB, which may lead to a chronic progressive active tissue injury without gadolinium enhancement [11]. Fifty percent of the T1 hypointense lesions resolve within 4 weeks, and a similar proportion of the hypointensities still visible after a month will disappear over the following 6 months [10,12]. T1 hypointense lesions persistent after 6–12 months are called chronic black holes and represent persistent tissue damage (demyelination and axonal loss). T1 hypointense lesions are more common with longer disease duration (Figure 5.2). On the other hand, most of these lesions remain hypointense with certain hyperintensity on T2-weighted images [10]. Apart from focal lesions, conventional T2-weighted images can show diffuse and more subtle signal hyperintensities in WM referred to as diffuse abnormal white matter (DAWM). DAWM presents extensive axonal loss, decreased myelin density, and chronic fibrillary gliosis [13].

Use of MRI for Diagnosis of MS MRI is able to demonstrate spatial and temporal dissemination of demyelinating plaques in the brain and spinal cord and is able to detect clinically silent lesions.

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Figure 5.2  Axial T1-weighted images in a 49-year-old woman with secondary progressive MS. On T1-weighted images, multiple lesions are hypointense or appear as “black holes.”

This is why MRI is integrated in the diagnostic scheme of MS and used to monitor treatment efficacy in clinical trials. International panel MRI criteria for supporting a diagnosis of MS [3] were revised in 2005 [4] to increase their sensitivity in the first mono-symptomatic attack, also known as clinically isolated syndrome (CIS), while maintaining high specificity. The McDonald criteria revised in 2005 consider dissemination in space on either baseline or follow-up MRI when three or more of the following four parameters are fulfilled: (a) nine or more T2 lesions or one or more gadolinium-enhancing lesions; (b) three or more periventricular lesions; (c) one or more juxtacortical lesions; (d) one or more posterior fossa lesions or spinal cord lesions. Any number of cord lesions can be included in the total lesion count. These criteria also consider dissemination in space when demonstrating two or more subclinical lesions consistent with MS on MRI, plus positive cerebrospinal fluid (CSF) findings (detection of oligoclonal bands or raised immunoglobulin G index). On the other hand, dissemination in time is fulfilled when one or both of the following conditions are demonstrated on MRI: (a) one or more gadolinium-enhancing lesions at least 3 months after CIS onset if not related to CIS and (b) a new T2 lesion with reference to a baseline scan obtained at least 30 days after CIS onset. Recently, these criteria have also been reviewed to simplify them and increase their sensitivity for an early diagnosis in CIS [1]. The new criteria propose one criterion for dissemination in space: presence of one or more asymptomatic T2 lesions in two or more of the four locations considered characteristic for MS (juxtacortical, periventricular, infratentorial, and spinal cord), excluding all lesions in the brain stem or spinal cord in cases of brain stem or spinal cord syndromes (concept of symptomatic region). The new proposal for dissemination in time includes the following: (a) simultaneous presence of asymptomatic gadolinium-enhancing and

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Figure 5.3  Axial FLAIR image shows widespread WM lesions in the periventricular region (A). WM lesions may also be seen at the cortical level (B).

nonenhancing lesions at any time and (b) a new T2 and/or gadolinium-enhancing lesion on follow-up MRI irrespective of the timing of the baseline scan. MS is still a clinical diagnosis, however, and these criteria should be applied in the proper clinical context because they have not been tested in cohorts with clinically atypical syndromes or other multifocal WM diseases. Likewise, the performance of these criteria in populations under 14 years of age or older than 50 years has not been tested [1]. In 2006, an international group of neurologists and radiologists provided guidelines for a standardized MRI protocol for the diagnosis and follow-up of MS patients [14]. In this consensus, conventional MRI sequences were recommended because they are generally available and widely used in the management of MS patients. For brain MRI, the following sequences were recommended: (a) sagittal fast FLAIR, which is sensitive to early MS lesions, such as those in the corpus callosum; (b) axial fast spin-echo (SE) or turbo SE PD and T2-weighted sequence (FSE PD/T2), which is more sensitive to infratentorial lesions than FLAIR; (c) axial fast FLAIR (Figure 5.3), which is especially sensitive to WM lesions in the periventricular and juxtacortical–cortical regions; and (d) axial gadolinium-enhanced T1 with a standard dose of 0.1 mmol/kg injected over 30 s and the scan starting a minimum of 5 min after start of the injection. Axial pregadolinium T1 was considered optional, but it is widely accepted as being performed routinely. Recommended slice thickness for the above sequences was 3 mm or less, with no inter-slice gap. Also, the 3D-T1 sequence was considered optional, as it is used in some centers for atrophy measures (partition thickness 1.5 mm or less). For spinal cord examination, the guidelines recommended the following sequences when acquired without a preceding enhanced brain MRI: (a) precontrast sagittal T1-weighted image; (b) precontrast sagittal FSE PD/T2, the PD series being better at depicting some lesions less apparent on heavily T2-weighted

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series; (c) precontrast axial FSE PD/T2 through suspicious lesions; (d) postcontrastenhanced sagittal T1-weighted image, with a standard dose of 0.1 mmol/kg injected over 30 s and the scan starting a minimum 5 min after start of the injection; and (e) postcontrast-enhanced axial T1-weighted image through suspicious lesions. When spinal MRI is acquired immediately following an enhanced brain MRI, the recommended sequences are the following: (a) postcontrast sagittal T1-weighted image; (b) postcontrast sagittal FSE PD/T2; (c) postcontrast axial T1-weighted image through suspicious lesions; and (d) postcontrast axial FSE PD/T2 through suspicious lesions. Recommended section thickness for sagittal sequences of the spinal cord is 3 mm without gap. The examinations should be performed on a closed 1.0 T or higher field MRI scanner to optimize image quality and tissue contrast. Considering the process of diagnosing suspected MS, brain MRI findings tend to be more common and characteristic for MS than those in the spinal cord. However, if the brain MRI results are equivocal, spinal cord MRI can sometimes be helpful because age-related T2 hyperintense lesions do not occur in the spinal cord, unlike in the brain. Some MRI findings are atypical for early MS [2], and exclusion of other pathologies should be performed—for example, vascular entities (vasculitis, embolic disease, amyloid angiopathy, CADASIL), vitamin B12 deficiency, infectious, neoplastic, or other non-MS idiopathic inflammatory-demyelinating processes. Some examples of those atypical findings are symmetric distribution of lesions, ill-defined lesion margins, absence of periventricular or corpus callosum lesions, punctiform parenchymal enhancement, diffuse WM involvement, predominance of lesions at the cortical–subcortical junction, large infiltrating brain stem lesions, mass effect, multiple lesions in basal ganglia, cortical or lacunar infarcts, hemorrhages or microhemorrhages, meningeal enhancement, selective involvement of the anterior temporal and inferior frontal lobe, persistent gadolinium enhancement and continued enlargement of lesions, simultaneous enhancement of all lesions, or diffuse abnormalities in the posterior columns of the cord.

Clinical/Radiologic Paradox Conventional MRI techniques are the most important paraclinical tool for the diagnosis and management of MS, as well as for monitoring the efficacy of experimental treatments. However, there is generally a poor correlation between clinical findings and the radiologic extent of involvement shown on conventional MRI techniques [6,15]. This clinical/MRI paradox is in part related to the fact that not all lesions are equivalent in terms of their functional impact. Some lesions in strategic areas in the brain may lead to very significant disability, whereas widespread alterations in nonstrategic areas may be clinically silent. Other reasons for this paradox have been already noted in relation to conventional MRI techniques: (a) limited specificity for the different pathologic substrates of MS [11] and (b) inability to detect and quantify the extent of damage in normal-appearing WM and normal-appearing GM. Another limitation of these techniques is the difficulty of assessing the reparative mechanisms and compensation by cerebral adaptation or plasticity. To overcome these limitations, in the past decade advanced sequences and new quantitative and nonconventional MRI

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techniques have been developed and applied to provide multidimensional information [16,17]. Some of these approaches include measures of hypointense T1 lesions and atrophy, measures of the changes within individual lesions on T2-weighted sequences by using voxel-wise dynamic lesion change mapping and subtraction techniques [18], magnetization transfer imaging (MTI), diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI), proton MR spectroscopy (MRS), functional MR imaging (fMRI), susceptibility-weighted imaging (SWI), and use of new contrast agents. Also, the use of higher field MRI scanners, including high-field 3.0 T and ultra-high-field 7.0 T, increases the sensitivity for detection of pathology.

Advanced Sequences, New Contrast Agents, High-Field MRI, Quantitative and Nonconventional MRI Techniques Three-Dimensional Fluid-Attenuated Inversion Recovery, Double Inversion Recovery Imaging, and T1-Weighted Imaging (SPGR, PSIR, MPRAGE) To increase the detection of cortical lesions, some improvements in inversion recovery sequences have been studied. The choice of slice thickness influences the ability to identify cortical–juxtacortical lesions. Diminishing the slice thickness to 1.5 mm in the 2D-FLAIR sequence significantly improved the sensitivity and precision of detecting cortical and juxtacortical lesions on a 1.5 T scanner [19]. In relation to this, the use of 3D sequences implies a special design for acquiring thin sections with a higher signal-to-noise ratio and small, nearly isotropic voxel dimensions, which also allows postprocessing without losing image quality, in contrast to 2D sequences [20]. A comparison study of conventional 2D-FLAIR and single-slab 3D-FLAIR sequences showed a significant increase in detection of MS lesions in general (supratentorial and infratentorial; study not centered specifically on cortical lesions) (Figure 5.4) [21]. Three-dimensional double inversion recovery imaging (3D-DIR) is probably the most effective technique today for detecting type I and II cortical lesions [22]. Apart from suppressing the signal from the CSF, this sequence uses an additional inversion pulse to suppress the signal from WM, showing superior delineation of GM and better contrast between lesions and the surrounding normal-appearing GM, which shows a slightly attenuated signal itself. Thus, 3D-DIR shows an increased sensitivity to intracortical lesions and better distinction between mixed WM and GM lesions from purely intracortical or juxtacortical lesions than 3D-FLAIR and T2-weighted SE sequences [23]. 3D-DIR showed an average increase of 152% in the detection of intracortical lesions per patient compared with 3D-FLAIR and more than 500% when compared with T2-weighted SE [23]. Other 3D sequences using high-resolution T1-weighted images, such as T1-weighted 3D-spoiled gradient-recalled-echo (SPGR) [24], T1-weighted phasesensitive inversion recovery (PSIR) [25], or 3D magnetization-prepared rapid acquisition gradient echo (MPRAGE) [26], also allow the detection of cortical lesions and their classification as purely intracortical, mixed GM and WM, or juxtacortical. These sequences have high spatial resolution and contrast between GM and WM and

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Figure 5.4  Comparison of 2D-FLAIR (A) versus 3D-FLAIR (B). 3D-FLAIR shows significantly better delineation of the lesions than 2D-FLAIR.

provide a clearer delineation of the GM–WM boundary than DIR sequences. Lesions appear hypointense on these T1 sequences. Apart from improving the ability to categorize cortical lesions, the combined analysis of both DIR and these high-resolution T1-weighted sequences increases the reliability of lesion detection when confirming the presence of a lesion in both sequences and allows rejection of artifacts [25]. However, when comparing the amount of cortical lesions reported on these sequences and those in histopathologic studies, 80% or more of type III cortical lesions remain undetected even with 3D-DIR or 3D-MPRAGE techniques [22,23,27,28]. When comparing 3D-DIR with T1-weighted 3D MPRAGE, the former showed better detection of intracortical and mixed WM and GM lesions [20]. Some cortical lesions detected on the T1-weighted sequence were hardly or not at all visible on 3D-DIR, and vice versa. Some reasons for this may be possible increased sensitivity of the T1-weighted sequence for detecting more chronic lesions over 3D-DIR and/or the regional variation of the signal throughout the cortex on 3D-DIR sequences [20,22]. 3D imaging techniques allow quality reconstructions to select optimal viewing planes and to compare and register with other sequences, which is valuable for longitudinal studies. Moreover, 3D sequences such as 3D-DIR, 3D-FLAIR, and 3D-MPRAGE have been improved with the implementation of single-slab, isotropic versions instead of multi-slab, which implies a reduction of acquisition time as well as a reduction of slice-profile and flow artifacts from blood or CSF, especially in the posterior cranial fossa [20,29]. These improvements facilitate the use of those sequences in clinical practice. Single-slab 3D sequences DIR and FLAIR showed increased detection of both GM and WM lesions, as well as infratentorial lesions, compared to 2D and 3D T2-weighted sequences. 3D-DIR showed the highest detection of intracortical and mixed WM and

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GM lesions but demonstrated fewer small lesions in the deep WM than 3D-FLAIR. The latter could be explained by a partial suppression effect and relatively low signalto-noise ratio due to the two inversion pulses of the 3D-DIR sequence itself [20]. In conclusion, technical developments of inversion recovery sequences allow an increased detection of cortical, WM, and infratentorial MS lesions with reasonable acquisition times and the possibility of reformatting the images with high quality. For these reasons, single-slab 3D-DIR and 3D-FLAIR could substitute for T2-weighted SE sequences in routine clinical practice and research [23]. The increase of lesion volume (LV) detection is important when assessing and monitoring disease activity in the clinical setting and research trials. The improvement of detection of infratentorial lesions also has prognostic implications because these lesions predict a high risk for earlier and clinically relevant long-term disability in patients with initial findings suggestive of MS [30]. The possibility of detecting cortical lesions, although limited in comparison to histologic findings, allows for quantifying cortical LV and assessing its clinical relevance (physical and cognitive impairment and epilepsy) [31], as well as its role in studying the pathophysiology of MS [32]. Cortical lesions detected by DIR are more common at later MS stages, when the CSF shows an inflammatory profile (presence of IgG oligoclonal bands), and in males, but they could be found from disease onset. They have also been associated with more pronounced brain tissue damage (increased T2-LV and decreased brain parenchyma fraction) and a higher clinical disability score [27]. In a 2-year longitudinal study in primary progressive MS, cortical LV at baseline correlated with increasing cortical atrophy and disability accumulation and was an independent predictor of these changes [33]. When comparing 2D-DIR with 2D-FLAIR and T2-weighted turbo SE sequences at 1.5 T and a high field of 3.0 T, DIR at 3.0 T seems to be the sequence with the highest overall sensitivity in the detection of focal MS lesions, with especially higher sensitivity in cortical and infratentorial lesions. The DIR sequence has a better contrast ratio between lesions and normal-appearing brain tissue than the other sequences; it also takes better advantage of the higher signal-to-noise ratio provided by 3.0 T [34–36]. However, taking into account the cortical LV detected histopathologically, the sensitivity of MRI using DIR remains relatively low even at 3.0 T. Despite this, the DIR sequence at 3.0 T could become a valuable clinical tool because the cortical lesions detected with MRI correlate with clinical outcome measures such as cognitive impairment and physical disability [27,33,37]; on the other hand, infratentorial lesions are a prognostic parameter [30]. 3D acquisition techniques at 3.0 T are being developed and assessed in MS patients and show promising results. They are high-quality images with well-tolerated acquisition times and suitability for postprocessing techniques, thanks to the volumetric characteristics of the data [38].

New Contrast Agents Conventional gadolinium contrast agents are used to detect new or reactivated inflammatory lesions with disruption of the BBB, which leads to accumulation of the contrast agent in the interstitial space. However, there is heterogeneity in the

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inflammatory processes in MS and there is inflammatory activity not detected with gadolinium contrast agents despite the presence of BBB disruption [11]. New MR contrast agents are able to track some cell subsets, such as macrophages, and allow us to study the presence of those cells in MS lesions, which implies a different approach for assessing inflammation, a cell-specific mechanism [39,40]. These contrast agents include super-paramagnetic iron oxides (SPIOs) and ultra-small superparamagnetic iron oxides (USPIOs) composed of nanoparticles made of stabilized iron oxides with very high T1 and T2 relaxivities [41]. These particles circulate in the blood pool and are phagocytosed by monocytes and macrophages. Preclinical studies of the experimental autoimmune encephalomyelitis (EAE) rat model of MS revealed in vivo the presence of macrophages in brain lesions [42–45]. Several phase II imaging studies in patients with relapsing–remitting MS (RRMS) described the characteristics of MS lesions using USPIO compared with gadolinium contrast. Enhancing lesions were seen as high signal intensities on T1-weighted images [40,46] and showed three different patterns: focal, ring-like, and isointense that were previously T1 hypointense [40]. On T2-weighted images, preclinical studies and some clinical studies showed a signal decrease by the lesions [46]. However, other clinical studies showed a lack of signal decrease on post-USPIO T2/T2* weighted images, suggesting that this effect depends on the type of USPIO particles, their concentration, and their ability to be incorporated into the cells [40]. When comparing USPIO enhancement with gadolinium enhancement, the former was more common than the later, enhanced for a longer period of time, and, in some cases, preceded gadolinium enhancement [40]. Both preclinical and clinical studies showed a spatial and temporal mismatch between these two types of enhancement, which could be related to differences in the underlying pathology and/or the stage of the lesions. These two distinct patterns of enhancement provide complementary information about the inflammatory process, with increased BBB permeability seen with gadolinium contrast and, on the other hand, the entrance of USPIO-labeled monocytes into the central nervous system (CNS), with or without a certain component of passive extravasation of nonmacrophage-incorporated USPIO across an open BBB, which cannot be excluded [40,47]. Apart from being pathophysiologically and clinically relevant, USPIOs have the potential to be a valuable tool for assessing the efficacy of new therapeutics, as shown in some preclinical studies [45,48]. Other new MR contrast agents are being developed and assessed in preclinical studies [49,50]—for example, the use of super-paramagnetic antibodies specific for cell surface antigens, which allow labeling and imaging of any cell with unique surface markers such as different inflammatory cells in the CNS: CD4 T, CD8 T, and Mac1 cells [51]. Other MR contrast agents in development that could be useful for studying MS are called responsive/activated or smart contrast agents, which undergo an important change in relaxivity upon activation. Therefore, for instance, it is possible to detect myeloperoxidase activity in inflamed tissues in vivo by using a myeloperoxidase-sensitive contrast agent [52]. Finally, another example of an experimental contrast agent is gadofluorine M, an amphiphilic macrocyclic gadolinium complex that allows visualization of subtle BBB alterations not visible with conventional gadolinium enhancement [50,53].

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High-Field-Strength MRI Magnetic field strengths higher than 1.5 T are considered high-field MRI scanners, usually 3.0 T scanners. Field strengths beyond 4.0 T are considered ultra-high-field scanners, usually 7.0 T. The main advantages of high field strengths are the increased signal-to-noise ratio and chemical shift. The higher signal-to-noise ratio can be used either for reducing scan time without losing diagnostic quality or for increasing spatial resolution. Higher field strengths also imply changes in T1 relaxation time leading to an increased postcontrast signal of the gadolinium-enhancing lesions [36]. Therefore, several studies comparing 1.5 and 3.0 T field strength scanners from different vendors confirmed that conventional brain sequences at 3.0 T offer higher lesion detection rates compared to 1.5 T (Figure 5.5) [54–58]. From a clinical point of view, this improvement is important for prognostic purposes, although it does not imply an earlier diagnosis of lesion dissemination in time and, therefore, definite MS [59,60]. On the other hand, no significantly higher sensitivity in the detection of spinal cord lesions has been proven when comparing 1.5 T with 3.0 T [61]. Nonconventional MR techniques such as MRS also take advantage of the increased chemical shift, which provides a higher spectral resolution. A higher chemical shift together with increased signal-to-noise ratio implies an improvement of the quantification of metabolite concentrations [62,63]. High-field MRI also implies an increased magnetic susceptibility effect, which, despite the artifacts, implies improvement of techniques such as SWI, susceptibilityweighted perfusion, blood oxygen level-dependent (BOLD) fMRI, and DTI [36].

Figure 5.5  Evaluation of T2 hyperintense lesions in patients with RRMS on FLAIR images. 1.5 T scanner (A) shows lower T2-LV than 3 T (B) (arrow).

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Some of the disadvantages of high magnetic fields are the heterogeneity of the field, higher radiofrequency power deposition, and the need to adapt imaging protocols from lower field strengths. When considering ultra-high field strengths, these drawbacks remain as challenges to be solved, especially for in vivo applications [36]. One of the interesting applications of ultra-high-field MRI in MS have been highresolution postmortem MR studies to perform histopathologic correlations of GM and WM lesions with MRI findings. A spinal cord study performed at 4.7 T showed increased sensitivity in the detection of lesions when compared to lower field strength [64]. On the other hand, a postmortem study to compare detection of intracortical lesions between 1.5 T and 4.7 T showed sensitivity below 10% for both fields [65]. Still another postmortem study performed at 8 T showed increased detection compared to 1.5 T [66]. A study performed in a small group of MS patients at 7 T demonstrated the possibility of characterizing different cortical lesion types according to histopathology [67]. An increased susceptibility effect at ultra-high field strengths can be useful for studying special features of MS pathology such as iron deposition in basal ganglia or MS lesions without injection of contrast media [36,68,69].

Quantitative MR Methods and Nonconventional MRI Techniques Brain Atrophy Measurements Brain atrophy is the best-accepted imaging biomarker of neurodegeneration and progression of disability in MS [70]. It is more severe in progressive forms and advanced stages of the disease, but it can also be present early in the disease process [70–72]. Massive cortical demyelination and diffuse axonal loss in the normal-appearing WM, which contribute to loss of brain volume, are present in the later stages of MS [73]. By using MRI, brain atrophy can be detected in vivo and measured in a reproducible manner by automated or semi-automated methods [15]. When considering brain volumes as measurements of brain atrophy (tissue loss), it is important to be aware of some limitations and confounding factors in relation to transient changes in water content. For example, relative dehydration or recent treatment with anti-inflammatory agents such as corticosteroids, natalizumab, interferon-beta, or immunoablation followed by stem-cell transplantation leads to a decrease in cerebral volume called pseudo-atrophy [74]. On the other hand, remyelination, reactive gliosis, or edema in relation to inflammation and new lesions may mask reductions in brain volume or an increase in brain volume [39,74]. Dynamic changes in brain volume in MS can be considered a composite of volume-gaining and volume-losing processes that are influenced by the extent of inflammatory, neurodegenerative, and remyelinating processes and the potency of anti-inflammatory therapy as well as its neuroprotective effects. In MS patients, atrophy is not limited to the WM; in fact, the GM seems to be affected more than the WM, especially the basal ganglia and thalamus [75–77]. GM atrophy develops at a much faster rate than WM atrophy [78–80]. CNS atrophy is a moderate but significant predictor of neurologic impairment that is independent of

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the effect of conventional MRI lesions [81,82]. Including GM atrophy in the assessment of patients with MS may further improve the usefulness of MRI measurements [83–85]. The relationship between spinal cord atrophy and disability is strong [86]. Total and regional brain volumes are further decreased in patients with a confirmed diagnosis of MS, particularly in those in advanced stages of the disease [87]. Further research is needed to understand the interrelationship between demyelination, neuronal and axonal loss, neurodegeneration, and cerebral volume changes [39].

Magnetization Transfer Imaging MTI measures the interactions between protons in free fluids and protons bound to macromolecules such as myelin and axonal membranes by use of the magnetization transfer ratio (MTR) [88]. Although the MTR is not a specific measure of a determinate pathologic substrate, in MS patients a decreased MTR most likely indicates demyelination and axonal loss in lesions and normal-appearing brain tissue, as shown in postmortem pathologic correlations [89,90]. A reduced proton exchange, or decreased MTR, may also be due to edema, gliosis, and inflammation [91], whereas an elevated proton exchange, or increased MTR, is evidence of possible remyelination or resolution of edema. MTI is probably the technique best adapted to measuring the extent of remyelination in patients with MS [90,92,93]. A voxel-by-voxel comparison of MTR changes in serial MRI scans allows monitoring of demyelination and remyelination in vivo [94–97]. Different lesions can thus be characterized as predominantly demyelinating or remyelinating, and, even within the same lesion, heterogeneity in myelination status over time can be observed (Figure 5.6). The degree of MTR alteration can be considered a marker of lesion severity, being more pronounced in patients with progressive MS and in black holes. MTR alteration can precede T2-visible lesion formation and tends to worsen with disease progression [98]. MTR decreases are related to cognitive impairment and can predict accumulated neurologic disability [15].

Magnetic Resonance Spectroscopy Proton magnetic spectroscopy (1H-MRS) has the unique ability to measure metabolites in tissues in vivo noninvasively. This technique allows the quantification of metabolic changes in lesions and normal-appearing brain tissue and complements the structural information provided by conventional MRI [39,99]. The most relevant metabolites in MS patients and CIS are the following: N-acetyl-aspartate (NAA) is considered a marker of neuro-axonal viability; myoinositol is a marker of glial cell activity; choline is a cell membrane marker; creatine is a marker of energy metabolism; lipids are components of cell membranes; lactate is a product of anaerobic glycolysis; and glutamate, glutamine, and GABA (gammaaminobutyric acid) are neurotransmitter amino acids. A decrease in NAA is associated with axonal/neuronal damage or dysfunction. An increase in choline and lipids is associated with myelin breakdown, remyelination, and inflammation. Increased choline concentration is seen when cell density increases. Increased myo-inositol levels suggest glial proliferation and astrogliosis.

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Figure 5.6  From left to right: FLAIR image, MTR map, and raw voxel-wise dynamic MTR map. The raw voxel-wise MTR map shows areas of MTR stability (yellow), decrease of MTR (red), and increase in MTR (green) associated with lesions and normal-appearing brain tissue in a patient with MS over 1 year. Note the T2 lesion (arrow) corresponding to the red area (decrease of MTR) on the voxel-wise MTR map (arrow) over the follow-up, possibly reflecting demyelination. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this book.)

Increased glutamate levels suggest glutamate excitotoxicity. A higher concentration of lactate is related to inflammatory or ischemic conditions. In active lesions there is an increase of creatine, choline, myo-inositol, glutamate, lactate, and lipids. NAA may be low or slightly decreased and can be partially restored after the acute phase. The changes in these metabolite levels are dynamic and variable over time and should be interpreted with caution. After the acute phase, lactate, choline, lipids, and glutamate return to normal levels. In chronic nonenhancing lesions, NAA is reduced and myo-inositol is increased (Figure 5.7). An elevated concentration of myo-inositol is not present in other age-related WM lesions [100]. In definite MS patients, NAA is decreased in normal-appearing WM and myoinositol is increased, suggesting axonal damage or dysfunction and increased glial cell activity. In patients with CIS, NAA and myo-inositol have prognostic relevance because a lower concentration of the former and a higher concentration of the latter have been demonstrated in patients converting to definite MS when compared to healthy controls [36]. High magnetic field strengths imply a higher signal-to-noise ratio and improved spectral separation. This allows more accurate and reproducible quantification of metabolites, as well as the study of regions such as the infratentorial brain and the spinal cord [36]. Due to high technical demands and limitations of MRS, this technique is not routinely used for assessing and monitoring MS patients in clinical practice or trials [99]. Some guidelines for using 1H-MRS in multicenter clinical MS studies have been proposed [101]. Single-voxel technique instead of multivoxel acquisition was recommended because the former is easier to standardize. According to these guidelines,

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Figure 5.7  MRS imaging in normal control (left) and MS patient (right). LC model was used to estimate the value of the spectra. The NAA/Cr ratio is higher in the normal-appearing white matter of the normal control and lower in the T1 hypointense lesion of the MS patient.

short echo-time acquisition should be used to measure metabolites such as myoinositol, lipids, or amino acids because of their short T2 relaxation times. Long echo times should be used to monitor NAA because these acquisitions are less influenced by gradient-induced distortions and the baseline is better delineated. The deep central WM centered on the corpus callosum, where much of the visible MR pathology occurs in MS, was also recommended.

Diffusion-Weighted and Tensor Imaging Diffusion imaging, which includes DWI and DTI, measures Brownian motion of water molecules in a fluid system and allows inferences to be made about the orientation, size, and geometry of axonal fibers and the major WM tracts in lesions and normal-appearing brain tissue [102–105]. DWI and DTI are acquired by applying DWI magnetic field gradients in many directions. Disruption of WM tracts and axonal membrane permeability implies an increase in water diffusivity measured by increased apparent diffusion coefficient (ADC) and mean diffusivity (MD), as well as a decrease in fractional anisotropy (FA), which measures a directional

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preponderance of diffusion [15]. Abnormalities in diffusivity patterns have been seen in both focal lesions and normal-appearing WM and normal-appearing GM. MS plaques show increased MD and ADC values and decreased FA, which are nonspecific changes in relation to demyelination, gliosis, inflammation, axonal contraction, and axonal loss [15]. Diffusion techniques also show those abnormal parameters, although less prominently, in normal-appearing WM and normal-appearing GM, suggesting the presence of subtle microstructural changes. Acute MS plaques may also show a transient decrease in ADC, probably in relation to the swelling of myelin sheaths, a certain degree of cytotoxic edema, or dense inflammatory cell infiltration. Diffusion abnormalities become more pronounced with increasing disease duration and neurologic impairment. Diffusion MRI is a sensitive marker of subtle microstructural changes, and it has been shown to be sensitive to the evolution of MS damage over short periods of time; however, the precision and accuracy of diffusion parameters to monitor longitudinal MS-related changes need further research [105]. The reason for this is that diffusion techniques remain challenging in terms of image acquisition, reproducibility across different sequences and scanners, and postprocessing methods [36]. Most diffusion techniques are based on single-shot echo planar imaging sequences, which allow fast image acquisition but are also prone to geometric distortions, limited signal-to-noise ratio, and low spatial resolution. Some of these drawbacks could be overcome by high-field MRI with higher signal-to-noise ratio, which can be applied to reduce scan time and distortions as well as to increase spatial resolution. High magnetic field scanners improve the study of the optic nerve, spinal cord, and GM, which are technically more difficult to study at lower field strengths. Diffusion tensor tractography methods also benefit from higher spatial resolution [36]. Tractography methods studying connectivity between different structures or GM regions, together with functional MRI, could lead to a better understanding of pathologic changes, functional reserve, and brain plasticity [39].

Functional MRI Functional MRI is an emerging technique that assesses brain activation patterns by using BOLD contrast, which is based on differences in deoxygenated hemoglobin concentrations in the blood, in brain regions secondary to neuronal activity [106]. This technique allows a noninvasive spatial localization of brain function. In MS patients, abnormal activation has been described, as well as the presence of adaptive and/or compensatory mechanisms from early stages of the disease [36,107]. This functional reorganization during task performance could be shown, for example, as increased activation of ipsilateral and contralateral cortices and recruitment of parallel existing pathways or distant sites. However, as the disease progresses, brain reserve is exceeded and activation decreases in relation to previous stages. fMRI could be used to monitor the efficacy of rehabilitation or pharmacologic therapies that promote neuroplasticity [15,39]. High magnetic fields with increased sensitivity to susceptibility differences and higher spatial resolution can acquire a higher BOLD signal that allows detection of areas of activation not shown at standard field strengths [36,39].

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Susceptibility-Weighted Imaging SWI is a new neuroimaging technique that uses tissue magnetic susceptibility differences to generate a unique contrast, different from that of spin density, T1, T2, and T2* [108]. SWI involves the use of both magnitude and phase images from a high-resolution, 3D fully velocity-compensated gradient recalled echo sequence. Phase masks are created from the MR phase images, and multiplying these with the magnitude images increases the conspicuousness of the smaller veins and other sources of susceptibility effects (such as iron deposits), which are depicted using minimal intensity projection (minIP) (Figure 5.8). SWI correlates with brain iron content, perhaps ferritin specifically. The phase images are useful in differentiating between diamagnetic and paramagnetic susceptibility effects of calcium and blood, respectively. The use of SWI in MS has gained increasing attention in the past few years, especially with the use of highfield and ultra-high-field MRI. Although the use of SWI in MS is in its infancy, the detection of vein abnormalities and areas of iron deposition in the brain are two main topics of interest. Previous studies in MS have identified iron accumulation in deep GM [109] and plaques [110], but they did not establish whether iron deposition was

Figure 5.8  SWI in a patient with secondary progressive MS. The upper row represents images with a single dose of gadolinium (0.1 mmol/kg). The lower row shows the SWI images with a triple dose of gadolinium (0.3 mmol/kg). From left to right: high-pass-filtered phase image, SWI image, and minimum intensity projection (minIP). The SWI images show focal areas of hypointensity representing iron deposition (note the advantages of triple versus single dose in visualization of these hypointensities). The minIP images are used to reconstruct the vein architecture in the brain (again, note advantages of single versus triple dose in visualization of the veins).

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a primary phenomenon in MS pathology or was secondary to chronic inflammation in MS. Iron deposition in MS patients may derive from myelin/oligodendrocyte debris or destroyed macrophages, or it can be the product of hemorrhages from damaged brain vessels [111,112]. In addition, iron overload may also lead to oxidative mitochondrial injury through Fenton reaction and release phospholipid-rich cellular membrane elements in MS [113]. The mechanism of direct damage to the CNS by iron might be related to oxidative stress and the generation of toxic free radicals [113]. Recently, it was proposed that iron deposits in MS are a consequence of altered cerebrospinal venous return and chronic insufficient venous drainage, but this suggestion needs further research to be proved [112,114]. Transferrin carries iron from the blood into tissues, while ferritin stores excess iron atoms that are not immediately engaged in metabolic activities. Free iron generally occurs when iron levels exceed the capacity of transferrin to bind the iron in the CNS. An excess of iron contributes to a number of CNS neurodegenerative processes because free ferrous iron reacts with peroxides to produce free radicals, which are highly reactive and can damage DNA, proteins, lipids, and other cellular components [112]. Both free and bound iron lead to toxic effects in the cells. Because the concentration of transferrin receptors is physiologically higher in the deep GM structures [112,114], iron is usually stored in these areas of the brain as a result of aging and other pathologic processes. Iron is a paramagnetic substance that reduces T2 relaxation time on MRI, resulting in hypointensity on T2-weighted images. Bakshi et al. [115,116] showed almost a decade ago the presence of deep GM T2 hypointensity in patients with MS. These authors recently reported that the deep GM T2 hypointensity is present even at first symptom onset [117]. Various imaging techniques have been used to evaluate the amount of deep GM iron deposition, including T2 hypointensity [115,116], relaxometry [118,119], magnetic field correlation [120], and SWI [68,69,110]. SWI is a unique MRI technique that offers a way to visualize tissues affected by iron deposition in the form of ferritin, deoxyhemoglobin, or hemosiderin. Therefore, measurement of iron deposition in the deep GM structures of MS patients on SWI may be an important biomarker of the disease process from the earliest phases of the disease and can predict iron content more accurately than other available imaging techniques [68,110,121,122]. Recent studies identified the pulvinar nucleus of the thalamus as well as entire thalamus, globus pallidus, caudate, and hippocampus as the deep GM structures with increased iron content in MS patients [68,69]. Recently published studies detected SWI lesions not seen on conventional MRI [68,110]. In one study, phase images showed contrast in 74% of the 403 lesions, increasing the total lesion count by more than 30% and showing distinct peripheral rings and a close association with the vasculature [68]. In another study, Haacke et al. [110] found a total of 75 lesions seen only with conventional imaging, 143 only with SWI, and 204 by both. From the iron quantification measurements, a moderate linear correlation between signal intensity and iron content (phase) was established. In a pilot study, we used a manual region-of-interest approach that identified SWI magnitude and phase lesions [69]. Phase-visible lesions were further subdivided into nodular, ring-shaped, scattered, and cortical subcategories. Magnitude lesions were also assessed. We attempted to evaluate the lesions associated with veins [68]

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but determined that the assessment was not reliable in most of the scans. All lesion masks were co-registered into the subject-specific upsampled FLAIR space, and an overlap with respect to T2, T1, and magnitude intersections of phase lesions was automatically obtained. The mean number of phase and magnitude lesions was more than 50% lower compared to T2 and T1 lesions. The phase and magnitude lesions largely did not overlap with T2 and T1 lesions. The most representative phase lesion type was nodular. The highest iron concentration was detected in the phase lesions and in the overlapping intersections of phase T2, T1, and magnitude lesions. All in all, these preliminary findings suggest that phase lesions and their overlaps with T2, T1, and magnitude lesions represent an important new lesion category in MS patients. Ultra-high-field 7.0 T MRI has provided sophisticated imaging capabilities by virtue of increased signal intensity and enhanced susceptibility effects, fundamental quantities underlying image resolution and contrast, respectively. On high resolution, susceptibility-sensitive T2*-weighted images at ultra-high-field 7.0 T MRI, most lesions are associated with centrally coursing veins. We recently developed an objective method for quantifying venous vasculature in brain parenchyma on SWI and applied this technique in 62 MS patients and in 22 age- and sex-matched healthy controls imaged on a 3 T GE scanner using precontrast SWI [69]. A subset of MS patients (50) and healthy controls (7) underwent a SWI postgadolinium contrast sequence (0.1 mmol/kg Gd-DTPA with a 10-min delay). An in-house–developed segmentation algorithm, based on a 3D multiscale line filter, was applied for vein segmentation. Absolute volumetric measurement for total vein vasculature was performed in milliliters and the relative venous intracranial fraction (VIF) was obtained to correct for head size and amount of brain atrophy. The size of individual veins was measured in millimeters and four groups were created according to their mean diameter: less than 0.3 mm, 0.3–0.6 mm, 0.6–0.9 mm, and more than 0.9 mm. Voxel brain average distance-from-vein maps were also calculated, with a higher distance indicating fewer veins. A significantly lower absolute venous volume was detected in MS patients compared to healthy controls, both in precontrast (67.5 versus 82.7 mL, 18.3%, P<0.001) and postcontrast (70.4 versus 87.1 mL, 19.1%, P<0.011) images. The VIF was significantly lower in MS patients (P<0.001). The highest mean diameter difference was found for the smallest veins (<0.3 mm), both on precontrast (P<0.001) and post-contrast (P<0.018) images. The distance from veins was also significantly higher in MS patients (P<0.001). We showed altered visibility of venous vasculature on SWI pre- and postcontrast images in MS patients. To summarize, SWI at ultra-high magnetic field allows the visualization of microvascular structures and the detection of iron deposition in brain structures and lesions and improves the detection rate of MS lesions in vivo. Therefore, this could be an important tool for further research.

Conclusions Although conventional MRI is an important tool in the diagnosis and management of MS, the ability of conventional MRI techniques to accurately assess inflammatory

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and neurodegenerative changes is limited. Advanced MRI sequences, new contrast agents, nonconventional and quantitative MRI techniques, and high- and ultra-highfield scanners provide more sensitive tools for studying the pathophysiology of MS and for monitoring disease evolution and activity.

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6 Role of IL-12/IL-23 in the

Pathogenesis of Multiple Sclerosis Yuhong Yang1,2, Amy E. Lovett-Racke1,2, Michael K. Racke1,2,3 1 

Department of Neurology, Ohio State University Medical Center, Columbus, OH, USA 2  Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Medical Center, Columbus, OH, USA 3  Department of Neuroscience, Ohio State University Medical Center, Columbus, OH, USA

Introduction Multiple sclerosis (MS) is a T-cell-mediated autoimmune disease of the central nervous system (CNS) with a complex genetic background. Although the precise etiology of MS is still unknown, it is generally accepted that MS begins with the formation of acute inflammatory lesions that were mediated by autoreactive T cells and B cells because of the breakdown of the blood–brain barrier (BBB). The demyelinating plaques were dominated by activated T cells and macrophages associated with oligodendrocyte destruction [1]. Early studies suggested that the IFN-γ-producing Th-1 CD4 T cells, which were driven by IL-12, played an essential role in mediating disease, whereas recent data have indicated that a new CD4 T-cell lineage, Th-17 cells, driven by IL-23, were critical to the development of disease. Experimental auto­ immune encephalomyelitis (EAE) is an autoimmune disease characterized by relapsing paralysis and CNS inflammation and demyelination [2]. EAE has been used as a model for MS for several decades, since it has clinical and immunopathologic similarities to MS. EAE can be induced either by immunization with myelin proteins emulsified in complete Freund’s adjuvant (CFA) or by adoptive transfer of myelin-specific CD4 Th-1 cells, but not Th-2 cells, into naïve wild-type recipient mice [3–10].

IL-12 p40 Family IL-12 and IL-23 are two heterodimeric cytokines belonging to the p40 family, which plays an important role in regulating T-cell responses [11–13]. They both are hetero­ dimers of two subunits. IL-12 is a heterodimer of p35 and p40, whereas IL-23 is a heterodimer of p19 and p40. They share a common subunit, p40. Both IL-12 and Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00006-X © 2011 Elsevier Inc. All rights reserved.

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IL-23

p35

p40

p19

p40

IL-12Rβ2

IL-12Rβ1

IL-23R

IL-12Rβ1

Figure 6.1  Structural diagram of IL-12 p40 family.  Both IL-12 and IL-23 belong to IL-12 p40 family. They share a common subunit, p40. IL-12 is a heterodimer of p35 and p40, whereas IL-23 is a heterodimer of p19 and p40. IL-12 receptor consists of IL-12 receptor β1 chain and IL-12 receptor β2 chain, while IL-23 receptor consists of IL-12 receptor β1 chain and a second chain called IL-23 receptor.

IL-23 were mainly produced by antigen-presenting cells (APCs), including macrophages, dendritic cells, and B cells, and exerted their biologic functions on CD4 T cells. IL-12 receptor and IL-23 receptor are heterodimeric receptors. They share a common receptor subunit, the IL-12 receptor β1 chain. IL-12 receptor consists of the IL-12 receptor β1 chain and IL-12 receptor β2 chain, while the IL-23 receptor consists of the IL-12 receptor β1 chain and a second chain called the IL-23 receptor (Figure 6.1). IL-12 drives naïve CD4 T cells to differentiate into the IFN-γproducing Th-1 lineage, while IL-23 promotes the IL-17-producing Th-17 lineage. Both IL-12-driven Th-1 cells and IL-23-driven Th-17 cells are believed to contribute significantly to the pathogenesis of MS and EAE [14,15].

T-Helper-Cell Lineages and Immune Deviation CD4 helper T lymphocytes differentiate into different types of T-effector cells in the periphery in response to different pathogenic microorganisms as a result of recognition of these organisms by the innate immune system [16]. It has been suggested for more than two decades that there are two different types of CD4 T-helper (Th) cells, Th-1 and Th-2 cells [17]. The Th-1 cell subset mainly produces IFN-γ, IL-2, and GM-CSF, while the Th-2 cell subset produces IL-4, IL-5, and IL-13 [18–21]. Th-1 cells were generated to control infections by intracellular pathogens, including viruses and bacteria, and cytokines produced by Th-1 cells mediated delayedtype hypersensitivity (DTH) inflammatory responses and have been implicated in a variety of autoimmune diseases, including MS [22–24]. Conversely, Th-2 cells control infections by extracellular microbes, and cytokines produced by Th-2 cells mediate helper T-cell functions for antibody production and mediate the immunopathology of allergic responses. Correspondingly, autoimmune diseases can be divided into those mediated by Th-l cells, with primarily inflammatory manifestations, and those mediated by Th-2 cells, whose manifestations are secondary to autoantibody containing immune complexes [25]. Immune deviation was a term used to characterize an immune response where Th-2 cells predominate, and one approach

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Figure 6.2  CD4 T-helper-cell lineages.  Three different CD4 T-helper-cell lineages, Th-1, Th-2, and Th-17 cells, have been identified so far. Naïve CD4 T cells are differentiated into Th-1 lineage, when the environment is rich in IFN-γ and/or IL-12. The Th-1 subset mainly produces IFN-γ, IL-2, and GM-CSF. The Th-1 transcription factors include T-bet, STAT1, and STAT4. The Th-17 subset mainly produces IL-17, IL-22, and IL-21. IL-6, TGF-β, and IL-1β are cytokines involved in Th-17 differentiation, and ROR γt, RORα, Batf, and STAT3 are transcription factors within Th-17 pathway. IL-23 promotes and maintains Th-17 differentiation. Th-2 differentiation is induced by IL-4, and Th-2 cells mainly produce IL-4, IL-5, and IL-13. GATA3 is the transcription factor for Th-2 cells.

to the immunotherapy of inflammatory autoimmune disease, including MS, was the antigen-specific deviation of an immune response dominated by a Th-1 response to a Th-2 response [26]. More recently, Th-17 cells have been identified as a new CD4 T-cell lineage. In vivo, Th-17 cells were driven by IL-23, although in vitro they were induced by TGF-β and IL-6. Th-17 cells also have been shown to be critical in the development of autoimmune diseases (Figure 6.2).

IL-12 and Th-1 Differentiation Pathways Cytokines play a critical role in the differentiation of Th cells, and Th-1 differentiation involves the actions of IFN-γ and IL-12 [27,28]. A series of cellular events mediated by different transcription factors control the development of Th cells (Figure 6.3). T-bet is the master transcription factor for Th-1 cell differentiation, in concert with other transcription factors, including STAT1 and STAT4, which decide

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Figure 6.3  Molecular pathway of Th-1 differentiation.  IFN-γ binds to the constitutively expressed IFN-γ receptor on the surface of naïve CD4 T cells, resulting in the phosphorylation and translocation of STAT1 (pSTAT1). pSTAT1 contributes to T-bet transcription, which promotes STAT1 transcription. pSTAT1 and possibly T-bet activate the transcription of IL-12Rβ2. IL-12 binds to the IL-12R, resulting in STAT4 phosphorylation and translocation. pSTAT1, pSTAT4, and T-bet all bind to the IFN-γ promoter and initiate IFN-γ production and contribute to the full differentiation of a Th-1 cell.

the phenotypic fate of Th-1 cells. IL-12 was first identified and purified in 1989 as a soluble 70-kDa anionic glycoprotein, with two subunits at 40 and 35 kDa (which were referred as p40 and p35, respectively). IL-12 was shown to stimulate natural killer (NK) cells to produce INF-γ [29]. It is mainly produced by APCs and exerts immunoregulatory effects on T and NK cells. Th-1 cells develop when the cytokine milieu is rich in IFN-γ and IL-12 [28,30–35]. Macrophages and dendritic cells produce IL-12 to polarize naïve T cells to Th-1 cells, in response to some bacterial and parasitic infections [32,33,36–38]. IL-12 promotes Th-1 differentiation by activating the STAT4 signaling pathways [39], and IL-12- or STAT4-deficient mice do not produce Th-1 cells [40–42]. IFN-γ signals through the STAT1 pathway, and STAT1deficient mice display a complete lack of responsiveness to IFN-γ and are highly sensitive to infection by microbial pathogens and viruses [35]. T-bet, which stands for T-box expressed in T cells, a novel member of the T-box family of transcription factors was isolated using an IL-2 promoter-reporter and a

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cDNA library prepared from activated Th-1 cells in a yeast one-hybrid screen [43]. T-bet was defined by the presence of a highly conserved T-box DNA-binding domain [44–46]. T-bet was shown to be expressed in IFN-γ-producing Th-1 and NK cells. Ectopic expression of T-bet transactivates the IFN-γ gene and induces endogenous IFN-γ production. Retroviral gene transduction of T-bet into polarized Th-2 T cells redirects them into Th-1 cells, as evidenced by the simultaneous induction of IFN-γ and repression of IL-4 and IL-5 [47]. Although T-bet is expressed in all three type of IFN-γ-producing cells (CD4, CD8, and NK cells), it is only required for control of IFN-γ production in CD4 and NK cells, but not in CD8 cells, which makes it a very attractive target for MS therapy [7,8,48]. T-bet induces Th-1 differentiation by targeting chromatin remodeling to individual IFN-γ alleles and by inducing IL-12 receptor β2 expression, without apparent assistance from IL-12/STAT4. IL-12/STAT4 appears to serve as a growth signal and trans-activator to prolong IFN-γ production, suggesting that lineage-determining cytokines may also mediate the selective survival of a lineage [49,50]. T-bet is induced by IFN-γ and STAT1 signaling during T-cell activation. T-bet expression is strongly dependent on IFN-γ signaling and STAT1 activation but is independent of STAT4. However, IL-12- and IL-18-induced IFN-γ production remained STAT4dependent, despite ectopic T-bet expression. Ectopic T-bet expression selectively induced expression of IL-12Rβ2 [51]. When the environment is rich in IFN-γ, IFN-γ binds to the constitutively expressed IFN-γ receptor on the surface of naïve CD4 T cells, which leads to the activation of STAT1. Downstream of STAT1, the expression of T-bet is induced [51,52], and T-bet acts to induce remodeling of the repressed IFN-γ locus and induce expression of IL-12Rβ2, the inducible chain of IL-12 receptor. T-bet also promotes the transcription of STAT1, and thus a positive feedback loop exists between these two transcription factors that initially drive a CD4 T cell toward a Th-1 phenotype. IL-12 in the microenvironment binds to the IL-12R, resulting in STAT4 phosphorylation [53,54]. Another cytokine that influences Th-1 development is IL-18, whose receptor is related to the IL-1 receptor family [55,56]. IFN-γ production was markedly reduced in IL-18-deficient mice, despite normal IL-12 induction [57].

IL-12 and T-Cell Encephalitogenicity The observation that myelin-specific CD4 Th-1 cells were sufficient to induce EAE focused MS research on these IFN-γ-producing Th-1 cells and IL-12, the cytokine that potently induces IFN-γ production and promotes Th-1 differentiation. IL-12 showed strong effects in enhancing T-cell encephalitogenicity, accelerating disease onset and increasing disease severity [58]. In adoptively transferred EAE, the disease course was more severe and prolonged when recombinant IL-12 was added to lymphocytes from immunized mice during in vitro restimulation. Similarly, mice treated with recombinant IL-12 (rIL-12) in vivo developed a more severe and prolonged course of disease after adoptive transfer. In contrast, mice treated with neutralizing

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IL-12 antibody after adoptive transfer were partially protected from disease, with only 40% of mice developing mild disease, and these mice were completely free of paralysis [59]. Even in the normally EAE-resistant mouse strain, B10.S, IL-12 was able to restore the IFN-γ production in myelin basic protein (MBP)-reactive T cells, with the subsequent induction of their ability to transfer EAE to naïve recipients [60]. Similar to what has been observed in the adoptive transfer EAE model, in the active immunization EAE model, IL-12 administration mimics staphylococcal enterotoxins (SEs) in inducing spontaneous relapses and in enhancing the severity and frequency of spontaneous relapses, whereas IL-12 neutralizing antibody blocks SE-induced and subsequent relapses of EAE and prevents spontaneous relapses [61].

Myelin-Specific Th-1 Cells in MS Although myelin-specific T cells were found in both MS patients and healthy individuals, which raised questions as to the relevance of these cells in MS patients, it does appear that myelin-specific T cells from MS patients are more likely to have a Th-1 phenotype [62–65]. Subsequently, several studies demonstrated that although healthy individuals had myelin-specific T cells, these cells were naïve, whereas MS patients had activated and memory myelin-specific T cells, indicating that these cells had been previously activated in vivo [66–68]. In addition, a clinical trial with an altered peptide ligand from MBP, which was intended to downregulate myelin-specific T cells, actually exacerbated disease in several MS patients. The increase in MS disease activity was associated with increased frequency of MBP-specific T cells that produced IFN-γ, suggesting that MS is mediated by myelin-specific Th-1 cells [69].

IFN-γ in MS/EAE Some studies focused on IFN-γ as the pathogenic molecule in EAE and MS, since IFN-γ is the hallmark of Th-1 immune responses. IFN-γ played an important role in MS pathogenesis, as increased production of IFN-γ preceding clinical attacks had been observed [70,71]. Furthermore, the inflammatory process within the CNS was characterized by increased IFN-γ expression, and disease activity correlated with IL-12 production [72–75]. Treatment of MS patients with IFN-γ led to an exacerbation of the disease [76,77], suggesting a pathogenic role for IFN-γ in MS. Surprisingly, the data from mouse studies contradicted what has been observed in human studies [78,79]. Instead of ameliorating disease, anti-IFN-γ antibodies enhanced EAE in SJL/J mice, with increased morbidity and mortality and earlier onset of disease. Systemic administration of IFN-γ did not improve or worsen clinical outcome, but delayed disease onset. Adoptive transfer of MBP-specific lymph node cells (LNC) pretreated with anti-IFN-γ antibody resulted in more severe disease [78,80,81]. Genetically deficient mice were used to determine if IFN-γ is necessary for the induction of EAE. Ferber et al. crossed IFN-γ-deficient mice (H-2b) to an EAEsusceptible mouse strain, B1O.PL (H-2u). EAE was induced in IFN-γ-deficient mice

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and wild-type littermates. Most of the mice developed EAE with comparable kinetics and severity regardless of their IFN-γ genotype. Histologic analyses of the CNS of IFN-γ-deficient mice revealed massive infiltrates composed of lymphocytes, macrophages, and granulocytes [82]. Similarly, IFN-γ R-deficient mice were found to be highly susceptible to EAE [80–83]. The conclusion drawn from these mouse studies was that IFN-γ was not necessary for EAE and, by extension, MS. However, recent studies revealed that aside from its classic role in Th-1 responses, IFN-γ has a role in regulating many aspects of T-cell biology, by cross-regulation of cellular responses to other cytokines and inflammatory factors [84]. IFN-γ signaling might be a very important part of the T-regulatory system in mice, which controls pathogenic T-effector functions. This finding is consistent with what has been observed in IFN-γ-deficient mice in that the activated CD4CD44 high cells showed significantly increased in vivo proliferation and significantly decreased ex vivo apoptosis [85]. Moreover, several studies that specifically suppressed IFN-γ in the myelin-specific T cells before transfer into recipient mice demonstrated that altering the signaling pathway that results in IFN-γ production in CD4 T cells decreases the encephalitogenic capacity of these cells [7,26,86]. In addition, STAT4 and T-bet, transcription factors in the Th-1 cell differentiation pathway, have been shown to be critical for EAE induction [7,87–89].

Genetically Deficient Mice of the p40 Family To determine the contribution of IL-12 in the pathogenesis of EAE, mice lacking IL-12 were generated. Surprisingly, IL-12 p40 mice were resistant to EAE, but IL-12 p35 mice were fully susceptible to EAE [90,91]. This confusion led to the intense study of IL-23 and its potential role in MS and EAE. Mice lacking IL-12 and/or IL-23 were compared to determine the contributions of IL-12 and IL-23 in EAE. Only IL-23 p19 (lacking IL-23) [92] and IL-12 p40 mice (lacking both IL-23 and IL-12) were resistant to EAE. By contrast, p35 mice that lacked only IL-12 were highly susceptible to EAE [90,91]. IL-12 signals through a heterodimeric receptor (IL-12R β1/IL-12R β2), whose β2-chain is upregulated on activated, autoreactive Th-1 cells. Contrary to the expectation that the absence of IL-12R β2 would protect mice from EAE, it was found that mice deficient in IL-12R β2 developed earlier and more severe disease, with extensive demyelination and CNS inflammation. Compared to wild-type mice, mice deficient in IL-12R β2 exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-α, GM-CSF, IL-17, IL-18/ IL-18Rα, and NO. Significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12R β2-deficient mice was observed compared to wild-type mice, suggesting that increased IL-23 and other inflammatory molecules may be responsible for the increased severity of EAE [93]. However, IL-12R β1, a common subunit shared by both IL-12 receptor and IL-23 receptor, was shown to be required in the induction of EAE. IL-12R β1-deficient mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th-2 response. IL-12R

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β1-deficient APCs drive CD4 T cells to an Ag-induced Th-2 phenotype, whereas wild-type APCs drive these CD4 T cells toward a Th-1 type response. IL-12R β1-deficient CD4 T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-γ and TNF-α. Decreased levels of IL-12 p40, p35, and IL-23 p19 mRNA expression were found in IL-12R β1-deficient APCs [94]. The conclusion drawn from these data was that IL-23, and not IL-12, was the critical cytokine for autoimmune inflammation of the brain, and this focused additional studies on IL-23. This also led to the discovery of a new CD4 T-cell lineage, Th-17 cells. However, we still need to take IL-35, a new cytokine member in the IL-12 family, into consideration when we evaluate the EAE data from genetically deficient mice of the IL-12 family [95]. IL-35 is a heterodimer of Epstein–Barr virus-induced gene 3 (EBI3) and IL-12 p40 [96,97]. EBI3 was first identified on the plasma membrane of EBV-transformed B lymphocytes and on transfected cells [98]. Different from IL-12 and IL-23, which were predominantly expressed by APCs and promoting T-cell differentiation and proliferation, IL-35 was highly expressed by mouse Foxp3 Treg cells but not by resting or activated effector CD4 T cells, and was required for maximal Treg suppressive activity [97]. While the fact that p19- and/or p40-deficient mice were resistant to EAE suggested that Th-17 cells were fully pathogenic, it did not exclude a role of IL-12driven Th-1 cells in EAE, since it is clear that p35-deficient mice lack both IL-12 and IL-35, which means that p35-deficient mice have not only an impaired Th-1 differentiation pathway, but also impaired Treg function. As a result, IL-23-driven Th-17 cells could be fully pathogenic alone, when the suppressive effects of IL-35 on T-cell proliferation were eliminated in those mice. However, encephalitogenic T cells from immunized wild-type mice caused indistinguishable disease when adoptively transferred to wild-type, p19-deficient, or p19/p35 double-deficient recipient mice, demonstrating that EAE can develop in the complete absence of IL-23 once encephalitogenic cells have been generated. Furthermore, MOGspecific T cells from p19-deficient mice could induce EAE in wild-type recipient mice by adoptive transfer, although the disease onset was delayed and disease severity was much lower [99]. Further studies are needed to determine the relative contribution of IL-12 and IL-23 in EAE, and it will be helpful to identify the contribution of IL-35 by comparing disease in p40-deficient mice, which have intact IL-35 but lack both IL-12 and IL-23, with p19/p35-deficient mice, which lack all three cytokines.

The Discovery of IL-23 IL-23 was discovered in 2000. A novel sequence distantly related to the p35 subunit of IL-12 was identified in a computational screen and was termed as p19. It had no biologic activity by itself; however, it was biologically active when combined with the p40 subunit of IL-12. This novel composite factor of p19 and p 40 was termed IL-23. Natural p19p40 was expressed by activated dendritic cells and was shown to

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activate STAT4 in PHA blast T cells [100]. IL-23 binds to IL-12R β1 chain but not IL-12R β2 chain. IL-23R, a novel member of the hemopoietin receptor family, was identified as another subunit of the receptor for IL-23 2 years later. IL-23R pairs with IL-12R β1 chain to confer IL-23 responsiveness on cells expressing both subunits [101]. The fact that the IL-12 R β1 chain is constitutively expressed by CD4 T cells suggests that the IL-23R is an important factor controlling the T-cell response to IL-23. However, most of IL-23R published data are at the message level, since there is not a good flow cytometry antibody for mouse IL-23R. Flow cytometry data from GFP-IL-23R mice showed IL-23R expression on a very small population of CD4 TCR αβ cells [102], which raised the question of how IL-23 exerted its strong effects on driving a highly pathogenic CD4 population, if it was expressed on such a small portion of CD4 T cells. Further studies of this unique IL-23R at the protein level will greatly enhance our understanding of IL-23 function in T cells.

Critical Role of IL-23 in EAE The study of the biologic functions of IL-23 revealed its unique role in autoimmune inflammation and led to the identification of a new CD4 Th-cell lineage, designated Th-17 cells, which are distinct from the previously reported Th-1 and Th-2 populations by producing IL-17. IL-17 was known to be critical for host defense against extracellular bacteria and fungi but was also implicated in the pathogenesis of autoimmune diseases. Though originally it exhibited some activities similar to IL-12 in increasing the production of IFN-γ by CD4 cells [101], IL-23 was distinct from IL-12 in its ability to induce strong proliferation of mouse memory T-cell populations, whereas IL-12 had no effect on memory cells. Murine IL-23 was first shown to stimulate memory cells to secrete IL-17 soon after the discovery of IL-23 in 2003 [103]. Later, IL-23 was found to be essential for the expansion of a pathogenic CD4 T-cell population, Th-17 subset, which is characterized by the production of IL-17A, IL-17F, IL-6, and TNF. These IL-23-driven autoreactive Th-17 cells identified a unique expression pattern of proinflammatory cytokines and other novel factors, distinguishing them from IL-12-driven Th-1 cells. They are highly pathogenic and essential for the establishment of the organspecific inflammation associated with CNS autoimmunity [104]. Studies from p19 transgenic mice showed that the expression of p19 in multiple tissues induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 months of age. Bone marrow transfer experiments further pointed to hemopoietic cells as the source of biologically active p19 [105]. At almost the same time, mice lacking IL-23 were used to determine the contributions of IL-23 in EAE. IL-23 p19-deficient mice (lacking IL-23) [92] and IL-12 p40-deficient mice (lacking both IL-23 and IL-12) were resistant to EAE. By contrast, p35-deficient mice that lacked IL-12 and IL-35 were highly susceptible to EAE [90,91]. Likewise, mice treated with antibodies to IL-23 failed to develop EAE [106]. These data suggested that IL-23 was a critical cytokine for autoimmune inflammation of the brain, although the detailed molecular mechanisms for this effect are still unknown.

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IL-23 Expression in CNS Both p19 and p40 subunits of IL-23 have been detected in CNS-resident cells. The p40 subunit was shown to be produced by macrophages/microglia in MS plaques more than 10 years ago, suggesting the existence of IL-23 and/or IL-12 in plaques [75]. The mRNA of p19 was detected in resident microglia and infiltrating macrophages [92]. The expression of IL-23p19 mRNA was highly induced in adult mouse microglia by stimulation with IFN-γ and lipopolysaccharide (LPS). In EAE, there was an early peak of IL-23p19 mRNA expression found in CD11b CNS APCs [107]. Since the CNS-resident microglia and macrophages are one of the main sources of inflammatory cytokines within the CNS during EAE, Becher et al. generated mice in which p40 was deleted from the CNS parenchyma, but not the systemic immune compartment, to determine the functional role of macrophage/microglia-derived p40. The p40deficient mice had decreased EAE severity upon adoptive transfer, indicating that IL-23 produced by CNS-resident cells controlled T-cell encephalitogenicity during the effector phase of EAE. However, the absence of p40 from the CNS has little impact on the degree of inflammation, since histologic analysis revealed significant inflammation in the spinal cords of p40-deficient mice that was comparable to that of wild-type mice with significantly more severe EAE, suggesting that p40 subunit expression in CNS is not required for the infiltration of inflammatory cells into the CNS, although it altered the degree of encephalitogenicity of those cells. Expression profiles of the CNS lesions showed an increase in Th-2 cytokines when compared with mice that develop EAE in the presence of CNS IL-12 and/or IL-23 [108]. Similar to findings in mice, increased IL-23 levels has been observed in MS patients. A significant increase in mRNA expression and protein production of both subunits of IL-23 was found in lesions compared with nonlesioned tissue. Activated macrophages and microglia were shown to be an important source of IL-23p19 in active and chronic active MS lesions. Elevated IL-23p19 expression was also detected in dendritic cells in MS patients [109,110], preferentially located in the perivascular cuff of active lesions [110]. This upregulation of IL-23 p19 expression in human microglia was suggested to be regulated by p38 MAP kinase and NF-kappaB signaling pathways [111]. Consistent with this abnormality, increased IL-17 production was found by T cells from MS patients [109], and IL-17 mRNA was found to be 18-fold higher in MS plaques than brains of control patients without CNS pathology by microarray analysis [112]. IL-17-producing lymphocytes were also detected in the cerebrospinal fluid of MS patients [113].

Th-17 Differentiation Pathways Many investigators have focused their research on this new CD4 lineage, Th-17 cells, since it was the first revision of the Th-1/Th-2 hypothesis since 1986. A number of studies have been published in the past several years regarding the factors controlling Th-17 differentiation (Figure 6.4). IL-23 was not the primary differentiation

Role of IL-12/IL-23 in the Pathogenesis of Multiple Sclerosis Nonencephalitogenic Th-17 pathway TGFβ

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Figure 6.4  Molecular pathway of Th-17 differentiation.  Nonencephalitogenic Th-17 pathway: TGF-β and IL-6 induce IL-17 production from naïve CD4 T cells. IL-6 binds to the IL-6 receptor and activates the phosphorylation and translocation of STAT3. pSTAT3, ROR γt, RORα, and Batf bind to the IL-17 promoter to initiate IL-17 transcription. TGF-β signals through Smad proteins to suppress T-bet expression and block IFN-γ production. The Th-17 phenotype depends on constant exposure to TGF-β, and Th-17 cells differentiated by TGF-β and IL-6 are not encephalitogenic. Encephalitogenic Th-17 pathway: IL-6 signals through the STAT3 pathway to induce IL-17 production in the presence of antibodies to IFN-γ, IL-4, and IL-12. Th-17 cells differentiated through this pathway are encephalitogenic, and T-bet is required for their encephalitogenicity.

factor for the generation of Th-17 cells from naïve T cells in vitro, although previous data showed that highly pathogenic Th-17 populations generated in vivo were IL-23-driven. TGF-β and IL-6 were shown to induce the differentiation of mouse Th-17 cells in vitro from naïve T cells, in combination with T-cell antigen receptor (TCR) stimulation [114,115], although TGF-β was originally thought to be an antiinflammatory cytokine by inducing in vitro differentiation of Foxp3 T-regulatory cells (Tregs) [116,117]. However, TGF-β appeared to be a key cytokine for both Th-17 and Treg differentiation. It promotes Th-17 differentiation at low concentrations with IL-6 and IL-21, whereas at high concentrations it represses IL-23R expression and favors Foxp3 Treg cells [118]. Other than IL-23, IL-1β appeared to be crucial for in vivo differentiated antigenspecific Th-17 cells, since the induction of Th-17 cells by immunization with antigens and adjuvants was abrogated in IL-1 receptor type I-deficient (IL-1RI(/))

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mice [119]. The involvement of IL-21 in Th-17 differentiation is still controversial. Several studies suggested that similar to IFN-γ, the autocrine cytokine important for Th-1 commitment, IL-21 was the essential autocrine cytokine for Th-17 differentiation [120–122]. Mouse Th-17 cells highly expressed IL-21; conversely, IL-21 promoted Th-17 differentiation by inducing ROR γt expression. During the process of Th-17 differentiation induced by TGF-β and IL-6, IL-6 induced expression of both IL-21 and IL-23R, and the IL-21 production in Th-17 cells was STAT3-dependent [121]. IL-21 potently induces ROR γt expression and IL-17 production to form a positive feedback loop to sustain Th-17 differentiation [121]. IL-6, IL-21, and IL-23 act sequentially in conjunction with TGF-β to induce ROR γt-dependent Th-17 differentiation [122]. IL-21 deficiency impairs the generation of Th-17 cells and results in partial protection against EAE [120]. Furthermore, studies of IL-6-deficient mice suggested that IL-21 may initiate an alternative pathway to induce Th-17 differentiation independent of IL-6. IL-21 cooperates with TGF-β to induce Th-17 cells in naive IL-6-deficient T cells and that IL-21-receptor-deficient T cells are defective in generating a Th-17 response [123]. However, a study in IL-21-deficient mice and IL-21R-deficient mice contradicted these data by showing that the differentiation of IL-17-producing CD4 T cells, their recruitment to inflamed organs, and the development of EAE were not affected in both IL-21R-deficient mice and IL-21-deficient mice [124]. IL-2 was found to inhibit Th-17 differentiation [125]. Genetic deletion or antibody blockade of IL-2 promoted differentiation of the Th-17 cells. Absence of IL-2 or disruption of its signaling by deletion of STAT5 resulted in enhanced Th-17 cell development. IL-10 negatively regulates IL-17 production by both macrophages and T cells. Under Th-17 polarizing conditions, IL-17-producing cells were increased significantly in IL-10-deficient and IL-10R-deficient splenocytes. The addition of recombinant IL-10 significantly decreased the percentage of IL-17-producing CD4 T cells [126]. γδ T cells are another important source of innate IL-17, when activated by IL-1β and IL-23, and act in an amplification loop for IL-17 production by promoting IL-17 production by CD4 T cells and increased susceptibility to EAE. IL-17-producing γδ T cells were found at high frequency in the brains of mice with EAE [127].

Molecular Mechanisms Regulating Th-17 Pathway The molecular mechanisms underlying the differentiation of Th-17 cells are still not completely understood, although several transcription factors have been implicated as involved in this process (see Figure 6.4). STATs play a very important role in regulating T-cell differentiation. STAT3 was thought to be one of the key regulators of Th-17 differentiation [125,128–130]. STAT3 is required for programming Th-17 cells by TGF-β/IL-6, and IL-23-driven IL-17-secreting phenotype. It was required for ROR γt expression in TGF-β/IL-6 differentiated Th-17 cells. Retroviral transduction of a constitutively active STAT3 enhanced IL-17 production from those cells [128]. IL-6, IL-21, and IL-23 all activate STAT3. A hyperactive form of STAT3 promoted Th-17 differentiation, whereas this differentiation process was greatly impaired in STAT3-deficient T cells [129]. Conversely, the deletion of the STAT3

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inhibitor, Socs3, results in elevated numbers of Th-17 cells [131]. Several studies focused on the molecular mechanisms by which STAT3 regulates Th-17 differentiation. Chromatin immunoprecipitation assays showed that STAT3 directly bound to the IL-17A and IL-17F promoters [131]. However, it is not clear whether STAT3 regulates or interacts with other transcription factors essential for Th-17 differentiation, including ROR γt, the aryl hydrocarbon receptor (AHR), and IRF4. Although originally it was shown that IL-17 production is independent of STAT4, STAT6, and T-bet [132], data from conditional knockout mice demonstrated that STAT4 was partially required for the development of IL-23-driven Th-17 cells, but not for TGF-β plus IL-6-primed IL-17-secreting cells, and was absolutely required for IL-17 production in response to IL-23 plus IL-18 [128]. Furthermore, T-bet and STAT1 may not be required for the initial production of IL-17, but optimal IL-17 production induced by IL-23 requires the presence of T-bet [106]. Similar to this finding, it was also shown that the encephalitogenicity of Th-17 cells was dependent on the T-bet expression in those cells [9]. The master transcription factor controlling Th-17 differentiation was thought to be the orphan nuclear receptor ROR γt [133]. ROR γt is a thymus-specific isoform of the retinoic acid receptor-related orphan receptor (RORγ) and has been shown to be essential for the generation of fetal lymphoid tissue inducer cells (LTi) [134], and required for the development of lymph nodes and Peyer’s patches [135]. ROR γt was expressed in populations of intestinal lamina propria T lymphocytes, most of which constitutively produce IL-17, and that these cells were absent in ROR γt-deficient mice. ROR γt was shown to be required for Th-17 differentiation in vitro, and forced expression of ROR γt induces IL-17 expression in naïve CD4 T cells. Reduced severity of EAE and absence of infiltrating Th-17 cells were observed in ROR γt-deficient mice [133]. ROR γt directly binds to the IL-17 promoter, and this binding is sufficient for activation of the minimum promoter in the HEK 293T cells [136]. The optimal transcription of the IL-17 gene required a 2-kb promoter and at least one conserved enhancer sequence, CNS-5. Both cis-regulatory elements were able to bind ROR γt and Runx1. Runx1 acts together with ROR γt during IL-17 transcription [137]. However, the ROR γt defect does not completely abolish Th-17 differentiation, suggesting additional factors might be involved. RORα was shown to direct Th-17 differentiation together with ROR γt. Th-17 cells highly expressed RORα, induced by TGF-β and IL-6 in a STAT3-dependent manner. Overexpression of RORα promoted Th-17 differentiation, and RORα and ROR γt co-expression synergistically drove greater Th-17 differentiation, whereas RORα deficiency resulted in reduced IL-17 expression in vitro and in vivo, and double deficiencies in RORα and RORγ entirely impaired Th-17 generation in vitro and completely inhibited EAE disease expression [138]. One recent study showed that activator protein 1 (AP-1) transcription factor Batf directly regulates the IL-17 promoter, and by doing so controls Th-17 cell differentiation [139]. AP-1 transcription factors are dimers of JUN, FOS, MAF, and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains (but BATF contains only a basic region and leucine zipper) and are considered to be inhibitors of AP-1 activity. Batf-deficient mice, which have normal Th-1/Th-2 differentiation and defective Th-17 differentiation, are resistant to EAE.

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Batf-deficient T cells fail to induce ROR γt, and even overexpression of ROR γt cannot fully restore IL-17 production in Batf-deficient T cells. BATF was shown to bind directly to the conserved intergenic elements in the IL-17A/F locus and to the IL-17 promoters, demonstrating that Batf has a critical role in Th-17 differentiation, supposedly in synergy with ROR γt. The interferon-regulatory factors (IRFs) are transcription factors regulating type I interferon production [140], Toll-like receptor signaling [141], and Th-cell differentiation [142]. IRF4 was recently shown to be critical for the generation of Th-17 cells, although originally it was thought to be essential for Th-2 development [143–146]. IRF-specific siRNA blocked Th-17 differentiation of wild-type cells in vitro. IRF4-deficient Th cells failed to differentiate into Th-17 cells. IRF4-deficient Th cells had less expression of ROR γt and increased expression of Foxp3. IRF4-deficient mice were resistant to EAE because of the altered regulation of both Th-17 differentiation and T-regulatory cell differentiation. AHR is a ligand-dependent transcription factor best known for mediating the toxicity of dioxin. Ligand-activated transcription factor AHR was also identified as a regulator of Th-17 cell differentiation in mice [147,148] . AHR regulates both Treg and Th-17 cell differentiation in a ligand-specific fashion. AHR activation by 6 formylindolo[3,2-b]carbazole promoted Th-17 cell differentiation and increased the severity of EAE, whereas AHR activation by its ligand 2,3,7,8-tetrachlorodibenzop-dioxin induced functional Treg cells that suppressed EAE. AHR is also expressed in human Th-17 cells [148].

The Plasticity of Th-17 Cells The lineage differentiation of naïve CD4 T cells has been considered to be an irreversible event [30,149,150]. However, Th-17 cells differentiated in vitro with TGF-β and IL-6 appeared to be more plastic than traditional Th-1 and Th-2 cells [9,151– 154]. In vitro TGF-β/IL-6-inducing Th-17 cells required constant exposure to TGF-β for sustained expression of IL-17F and IL-17A. Even cells polarized in Th-17 cell conditions for 3 weeks failed to maintain IL-17A and IL-17F expression and were converted to Th-1 or Th-2 cells in the presence of IL-12 or IL-4, respectively. In the absence of TGF-β, both IL-23 and IL-12 acted to suppress IL-17 and enhance IFN-γ production in a STAT4- and T-bet-dependent manner. These results suggested substantial developmental plasticity of Th-17 cells differentiated in vitro, distinct from the stable lineage commitment of Th-1 and Th-2 cells.

In Vitro Differentiation of Encephalitogenic Th-17 Cells Several observations raised the question whether IL-23-driven Th-17 cells generated in vivo and the TGF-β/IL-6-induced Th-17 cells generated in vitro are actually the same, although they both express IL-17. The first difference they have is their difference in encephalitogenicity. Although the combination of TGF-β and IL-6 in vitro induces a

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significant amount of IL-17 production in CD4 T cells, these TGF-β/IL-6-induced Th-17 cells are not pathogenic after adoptive transfer, which is completely different from those highly pathogenic, in vivo differentiated, IL-23-driven Th-17 cells described originally [9,104]. In addition, IL-17 production in TGF-β/IL-6-induced Th-17 cells differentiated in vitro is dependent on constant exposure to TGF-β/IL-6 and is blocked by IFN-γ/IL-4 signaling. These T cells converted to Th-1 or Th-2 cells in response to IL-12 or IL-4 exposure, respectively. However, Th-17 cells generated in vivo maintain their IL-17 memory upon subsequent in vitro culture, even in the absence of IL-23, showing a stable and distinct lineage of Th cells [153]. These findings suggested that there might be more than one molecular pathway through which IL-17 is induced from naïve T cells. Th-17 cells differentiated from different pathways are not all the same in terms of the encephalitogenicity in EAE, because the end product IL-17 is not the only factor responsible for the encephalitogenicity of T cells. IL-17 contributed partially to the encephalitogenicity, but other factors within this molecular pathway also contributed. This idea was supported by the data from both IL-17 knockout mice and transgenic mice. Neither the T-cell-driven overexpression of IL-17A nor its complete loss had a major impact on the development of clinical disease [155]. EAE severity was decreased in IL-17 knockout mice but not totally abolished, suggesting that there were other important factors accounting for the encephalitogenicity of those cells. Among all the possible factors during T-cell differentiation, the transcription factor T-bet is very special in this process. T-bet is the master transcription factor in Th-1 cell differentiation, but it is also shown to be required for the optimal IL-17 production by the IL-23-driven pathogenic Th-17 cells [106]. Moreover, T-bet was found to directly regulate transcription of the IL-23R and influence the fate of Th-17 cells, which depend on optimal IL-23 production for survival [8]. More importantly, in vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited disease [7,8]. All these data suggested that T-bet might be an important factor within the differentiation pathway to decide the encephalitogenicity of CD4 T cells. However, it has been shown that TGF-β is a powerful suppressor of T-bet expression. As a result, T-bet expression was completely suppressed in TGF-β/IL-6-induced Th-17 cells, which lack the ability to adoptively transfer EAE. In a search for ways to generate encephalitogenic Th-17 cells in vitro, Yang et al. found that IL-6 alone could induce IL-17 production, although IFN-γ production was also induced. So instead of adding TGF-β, which has been shown to suppress T-bet, we used IL-6 plus antibodies to IFN-γ, IL-12, and IL-4 to generate antigen-specific Th-17 cells from naïve T-cell-receptor transgenic T cells, which showed strong encephalitogenicity after adoptive transfer [9].

Human Th-17 Cells One of the major differences between the differentiation of human Th-17 cells and mouse Th-17 cells is the effect of TGF-β on Th-17 differentiation. TGF-β promoted IL-17 production from murine CD4 cells, but its effects on human Th-17 differentiation remained controversial.

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IL-23 and IL-1β were shown to induce the development of human Th-17 cells expressing IL-17A, IL-17F, IL-22, IL-26, IFN-γ, CCL20, and ROR γt. In situ, those Th-17 cells express IL-23 receptor and the memory T-cell marker CD45RO [156]. Similar to this study, another study confirmed that the polarization of human Th-17 cells was induced by IL-1β, but enhanced by IL-6 and suppressed by TGF-β and IL-12 [157]. In contrast to those two studies, Yang et al. showed that TGF-β and IL-21 promoted the differentiation of human naïve CD4 T cells into Th-17 cells, whereas IL-1β and IL-6 induced IL-17A secretion from human central memory CD4 T cells [158]. Manel et al. showed that TGF-β, IL-1β and IL-6, IL-21, or IL-23 in serum-free conditions was necessary and sufficient to induce IL-17 expression in naïve human CD4 T cells from cord blood, suggesting that similar cytokine pathways are involved in this process in mice and humans [159]. Other than the differentiating cytokines, Evans et al. showed that cell–cell contact with Toll-like receptor-activated monocytes in the context of T-cell-receptor ligation was required for the expression of IL-17 from precommitted precursors present in human peripheral blood [160]. Another difference between human and mouse Th-17 cells is the existence of IL-17/ IFN-γ double-positive cells in humans. Human CD4 T cells producing both IL-17 and IFN-γ were observed in MS [161] and other autoimmune diseases [162]. However, the significance of IL-17/IFN-γ double-positive cells is still not clear. Human IL-17producing cells have distinct cell surface markers. The chemokine receptor CCR6 was one of the main surface markers for human Th-17 cells [163]. CCR6 and CCR4 have been identified as the surface markers for human memory Th-17 cells, whereas CCR6 and CXCR3 identified human Th-1 cells and human IL-17/IFN-γ double-positive cells [162,164]. CD161, the equivalent of murine NK1.1, a molecule expressed on the surface of NK, NKT, and some CD4 and CD8 T cells, was reported to be consistently expressed on human Th-17 cells [165,166]. Surface IL-17A expression was also observed as another surface marker for human Th-17 cells [167]. Another interesting aspect of human Th-17 cells came from unexpected findings in human Treg cells [168]. Human memory Tregs were observed to secrete IL-17 ex vivo and constitutively express ROR γt. These IL-17-secreting Tregs express high levels of CCR4 and CCR6 and low levels of CXCR3 and CD161 [169]. Similarly, human regulatory T cells (CD4(pos)CD25(high)Foxp3(pos)CD127(neg)CD27(pos)) were shown to differentiate into IL-17-producing cells with high levels of ROR γt and CCR6, when stimulated by monocytes, in the presence of rhIL-2/rhIL-15. This differentiation process was enhanced by exogenous IL-1β, IL-23, and IL-21, whereas IL-6 or TGF-β had no effects [170]. Human peripheral blood and lymphoid tissue contain a significant number of CD4FOXP3 T cells expressing CCR6, which have the capacity to produce IL-17 upon activation. They co-express FOXP3 and ROR γt and strongly inhibit the proliferation of CD4 responder T cells [168]. Similar to what is observed in mouse, RORc2, the human counterpart of ROR γt, was identified as the master transcription factor of human Th-17 programming [171,172]. Human Th-17 cells were able to infiltrate into the CNS and cause inflammation. Kebir et al. showed that Th-17 lymphocytes transmigrate efficiently across BBB endothelial cells, highly express granzyme B, kill human neurons, and promote CNS inflammation through CD4 lymphocyte recruitment [173]. IL-17 and IL-22

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receptors were found to be expressed on BBB endothelial cells in MS lesions, and IL-17 and IL-22 were shown to disrupt BBB tight junctions in vitro and in vivo.

Distinct Type of Inflammation Induced by Th-1 or Th-17 Cells Although adoptive transfer of both Th-1 and Th-17 cells induced EAE that was almost clinically indistinguishable, the pathology showed that Th-1 and Th-17 actually induced different types of inflammation with distinct histopathologic features and immune profiles in CNS, which made it unlikely that Th-17 cells are the only players in driving tissue damage in autoimmune diseases. It was shown that IL-12-driven disease was characterized by macrophage-rich infiltrates, whereas neutrophils and granulocyte colony stimulating factor (GCSF) were prominent in IL-23-driven lesions. The monocyte-attracting chemokines CXCL9, 10, and 11 were preferentially expressed in the CNS of mice injected with IL-12p70modulated T cells, whereas the neutrophil-attracting chemokines CXCL1 and CXCL2 were upregulated in the CNS of mice given IL-23-modulated T cells [174]. Not only were the inflammatory features different, but the lesion location in the CNS also seemed to be different between Th-1-induced inflammation and Th-17induced inflammation. The lesion localization in the CNS correlated with severe clinical outcomes, so this may be a crucial determinant of clinical outcomes in MS. Data from EAE studies showed that Th-1 and Th-17 seemed to have different infiltrating localization in the CNS. T cells that are specific for different myelin epitopes generate populations characterized by different Th-17 to Th-1 ratios, and the Th-17:Th-1 ratio of infiltrating T cells determines where inflammation occurs in the CNS. T cells infiltrated into brain parenchyma and caused inflammation when Th-17 cells outnumbered Th-1 cells. However, at low Th-17:Th-1 ratios, T-cell infiltration proceeded into the spinal cord, but not brain. This finding suggested critical differences in the regulation of inflammation in the brain and spinal cord by Th-1 and Th-17 cells [175].

Anti-p40 Therapy The consensus that MS is a T-cell-mediated autoimmune disease and Th-1 and Th-17 cells play critical roles in the disease pathogenesis has led to the clinical use of immunomodulatory therapies by targeting molecules in the Th-1 and/or Th-17 pathway. IL-12p40 has been chosen as the target for developing new therapy since IL-12p40 is the common subunit shared by both IL-12 and IL-23, and IL-12 p40- or IL-23 p19deficient mice are resistant to EAE induction. Anti-p40 therapy aims at targeting both the IL-12/Th-1 pathway and the IL-23/Th-17 pathway with one antibody. Treatment with neutralizing antibody against IL-12 p40 showed protective effects in rodent and nonhuman primate EAE models. SJL/J mice given neutralizing antiIL-12 monoclonal antibodies are protected from EAE [176]. Treatment of mice with

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an antibody to murine IL-12 after adoptive transfer completely prevented paralysis, with only 40% of the mice developing mild disease, while mice treated with rmIL-12 in vivo after the transfer of antigen-stimulated LNC developed a more severe and prolonged course of disease [59]. IL-12 neutralizing antibody blocks SE-induced and subsequent relapses of EAE and prevents spontaneous relapses [61]. Treatment with monoclonal anti-IL-12 antibody after immunization with MOG35–55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing–remitting EAE both clinically and histologically [177]. The common marmoset (Callithrix jacchus) is a neotropical primate species that shares significant immunologic similarity with humans [178]. The marmoset is highly susceptible to EAE, offering a valid preclinical model for evaluation of the efficacy of new therapies against MS that are inactive in lower species [179]. Marmoset monkeys treated with the IL-12p40 antibody from day 14 after EAE induction with human myelin failed to develop EAE. A protective effect on the neurologic dysfunction as well as on neuropathologic changes normally observed in the brain and spinal cord of EAE-affected animals has been observed with this neutralizing anti-IL-12p40 antibody [180]. To further determine if anti-human IL-12/23 p40 antibody has effects on established ongoing disease, EAE was induced by active immunization with rhMOG. Lesions developing in the cerebral white matter were visualized and characterized with standard magnetic resonance imaging techniques. Treatment with the antibody was initiated after active brain white matter lesions were detected on T2-weighted images, to mimic the treatment of MS patients. Changes in the total T2 lesion volume and T2 relaxation times were significantly suppressed in monkeys treated with anti-IL-12p40 antibody. Moreover, the time interval to serious neurologic deficit was delayed [181]. To explore the clinical efficacy of targeting the IL-23 immune pathway, Chen et al. tested whether blocking IL-23 function by IL-23p 19-specific antibody can inhibit EAE. Their data showed that anti-p19 antibody treatment reduced the serum level of IL-17 as well as CNS expression of IFN-γ, IP-10, IL-17, IL-6, and TNF mRNA. Treatment during active disease inhibited proteolipid protein (PLP) epitope spreading and prevented subsequent disease relapses. Thus, therapeutic targeting of IL-23 effectively inhibited inflammatory pathways driving CNS autoimmune inflammation [106]. With all the promising data from anti-p40 therapy in rodents and nonhuman primates, repeated subcutaneous injections of a fully humanized monoclonal antibody against IL12/23 p40, ustekinumab, was used to treat patients with relapsing–remitting multiple sclerosis (RRMS) to assess the drug’s safety, efficacy, and pharmacokinetics. Ustekinumab treatment did not show a significant reduction in the primary endpoint, the cumulative number of new gadolinium-enhancing T1-weighted lesions on serial cranial MRI through week 23, for any dosage groups versus placebo [182]. The failure of the ustekinumab trial was surprising, given the successes of antiIL-12p40 antibody in Crohn’s disease [183] and moderate to severe chronic plaque psoriasis [184,185]. One explanation might be that this antibody did not penetrate the CNS, where the inflammation was, as readily as it does in the skin and gut, although a dose-dependent increase in ustekinumab serum concentration was seen in patients.

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Another possibility was that the subject population and the methods chosen for this study might obscure the true effect. The facts that the median disease duration was 0.4–10.1 years and the median expanded disability status score (EDSS) score was 0–6.5 indicated that some patients with advanced disease were included in this trial. It is unlikely that anti-IL-12/IL-23 p40 antibody would work well in patients with advanced disease, since both cytokines exerted their functions mainly on the differentiation of pathogenic CD4 T cells [186].

T-bet: A Potential Prognostic Marker and Therapeutic Target in MS As a transcription factor involved in both the IL-12/Th-1 and IL-23/Th-17 pathways, T-bet serves as an attractive therapeutic target for MS. It has been shown that T-betdeficient mice were resistant to the development of EAE by active immunization [88]. In vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited EAE [7]. Moreover, suppression of T-bet has also been shown to ameliorate disease in established EAE. Therapeutic administration of T-bet siRNA significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of MBP-specific Th-1 autoreactive T cells. The decrease of T-bet levels was observed in MS patients under IFN-β treatment. The mRNA expression of IFN-γ, IL-17, T-bet, and ROR γt on peripheral blood mononuclear cells (PBMCs) from 36 MS patients before and after 1 year of IFN-β treatment was determined. The levels of IL-17 and ROR γt remained similar in all MS patients. IFN-β induced significant decreases in IFN-γ in all patients, while decreases in T-bet were detected only in responders. Higher pretreatment T-bet levels allowed prediction of the clinical response in the first year, suggesting that T-bet expression might be a potential prognostic marker of treatment response to IFN-β [187].

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[165] Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S. The phenotype of human Th17 cells and their precursors, the cytokines that mediate their differentiation and the role of Th17 cells in inflammation. Int Immunol 2008;20:1361–8. [166] Cosmi L, De Palma R, Santarlasci V, Maggi L, Capone M, Frosali F, et  al. Human interleukin 17-producing cells originate from a CD161CD4 T cell precursor. J Exp Med 2008;205:1903–16. [167] Brucklacher-Waldert V, Steinbach K, Lioznov M, Kolster M, Holscher C, Tolosa E. Phenotypical characterization of human Th17 cells unambiguously identified by surface IL-17A expression. J Immunol 2009;183:5494–501. [168] Voo KS, Wang YH, Santori FR, Boggiano C, Arima K, Bover L, et al. Identification of IL-17-producing FOXP3 regulatory T cells in humans. Proc Natl Acad Sci U S A 2009;106:4793–8. [169] Ayyoub M, Deknuydt F, Raimbaud I, Dousset C, Leveque L, Bioley G, et  al. Human memory FOXP3 Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t. Proc Natl Acad Sci U S A 2009;106:8635–40. [170] Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I. Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 2008;112:2340–52. [171] Crome SQ, Wang AY, Kang CY, Levings MK. The role of retinoic acid-related orphan receptor variant 2 and IL-17 in the development and function of human CD4 T cells. Eur J Immunol 2009;39:1480–93. [172] Unutmaz D. RORC2: the master of human Th17 cell programming. Eur J Immunol 2009;39:1452–5. [173] Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, et  al. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med 2007;13:1173–5. [174] Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM. IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 2008;205:1535–41. [175] Stromnes IM, Cerretti LM, Liggitt D, Harris RA, Goverman JM. Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells. Nat Med 2008;14:337–42. [176] Constantinescu CS, Hilliard B, Ventura E, Wysocka M, Showe L, Lavi E, et  al. Modulation of susceptibility and resistance to an autoimmune model of multiple sclerosis in prototypically susceptible and resistant strains by neutralization of interleukin-12 and interleukin-4, respectively. Clin Immunol 2001;98:23–30. [177] Ichikawa M, Koh CS, Inoue A, Tsuyusaki J, Yamazaki M, Inaba Y, et  al. Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing–remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide. J Neuroimmunol 2000;102:56–66. [178] Brok HP, Bauer J, Jonker M, Blezer E, Amor S, Bontrop RE, et al. Non-human primate models of multiple sclerosis. Immunol Rev 2001;183:173–85. [179] t Hart BA, Amor S, Jonker M. Evaluating the validity of animal models for research into therapies for immune-based disorders. Drug Discov Today 2004;9:517–24. [180] Brok HP, van Meurs M, Blezer E, Schantz A, Peritt D, Treacy G, et al. Prevention of experimental autoimmune encephalomyelitis in common marmosets using an anti-IL12p40 monoclonal antibody. J Immunol 2002;169:6554–63.

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7 Spinal Cord Injury and its

Relationship to the Development or Worsening of Clinical Multiple Sclerosis Peter O. Behan1,2, Abhijit Chaudhuri3 1 

Division of Clinical Neuroscience, Faculty of Medicine, University of Glasgow, Glasgow, Scotland, UK 2  School of Life Sciences, Glasgow Caledonian University, Glasgow, Scotland, UK 3  Department of Neurology, Queen’s Hospital, Romford, Essex, England, UK

Introduction Under the principles of epidemiology, one needs to show a very high certainty— often 95%—to prove a statistical association. One must be able to say that 95 times out of 100 an experiment is performed, it will have the same result. Yet, it is important to make one vital distinction: although finding such an association almost certainly ensures a cause-and-effect relationship, the failure of an epidemiological study to detect the association with the scientist’s rigid standard of proof does not mean the link does not exist [1]. Multiple sclerosis (MS) is one of the most common causes of chronic neurologic disease in young adults. The cause is unknown and even the pathogenesis is poorly understood [2–7]. Indeed, the previously held tenet that the disorder was immunologically mediated and of autoimmune etiology has been not only questioned but also robustly challenged [3,4]. It cannot be emphasized often enough that the cause of the disease is unknown and, since its exact pathogenesis is likewise poorly understood, it is not surprising that therapeutic endeavors have met with total and abysmal failure [8–10]. This has not prevented dramatic and serious attempts at therapy, often with high mortality and morbidity [11–15]. For nearly a century the scientific community has adhered to the dogma of an autoimmune etiology and has been blinkered to the fact that experimental allergic encephalomyelitis (EAE) is not a model for the disease but for another unrelated condition in humans: acute disseminated encephalomyelitis (ADEM) [3,16]. New data using a variety of sophisticated neuroradiologic techniques show that the disease initially occurs in areas of the brain that are metabolically compromised [17], Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00007-1 © 2011 Elsevier Inc. All rights reserved.

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and these data, coupled with the finding of atrophy and neuronal death away from the plaque, in the normal-appearing white matter [18], leave little doubt that the illness is now best seen and considered as a neurodegenerative disorder [19,20]. Genetics clearly plays a role, but despite the most modern analysis, no specific genetic locus has been found [21–25]. Familial studies [26] and the findings of disease associations [3,27,28] clearly implicate as yet unknown genetic mechanisms in its pathogenesis. Taking these data into consideration and critically looking at the known facts surrounding MS, we are unable to understand why the longstanding and wellrecognized relationship with trauma is denied [29,30]. Evidence that other serious treatments given in error and causing gross worsening is often overlooked [31]. In accepting that the disease may be immunologically mediated, neurologists treat their patients with expensive, dangerous, immunosuppressive, immunomodulating agents [15,32–35], yet they deny that specific focal trauma may aggravate existing disease or precipitate the illness de novo in someone predisposed to develop MS [29,30]. For these reasons we have reviewed the literature on the association of specific focal trauma to the development of MS and have argued that this association is not only true but that it also provides further help with understanding the pathogenesis of the disease and aids in the development of management that may prevent further attacks.

Historical Review That MS may be precipitated de novo or worsened after trauma has been amply and well documented in the neurologic literature since the 19th century [36]. Indeed, very eminent neurologists and neuropathologists have written on this unique clinical aspect. The distinguished Edinburgh physician James Dawson, in his classical work on MS, described how one of his nine patients had suffered head trauma prior to developing the disease [37]. In the 19th century, Mendel [36] was perhaps the first to describe four cases of MS that occurred within a year following trauma. He conjectured that in patients predisposed to the disease, trauma might be very relevant. Von Hoesslin [38] found that trauma was involved in precipitating the onset of the illness in 11.4% of cases and that the trauma had occurred usually within 2 months prior to the onset of the disease. In 1946 McAlpine [39] claimed that 5.5% of his series had sustained prior trauma. Indeed, “both Von Hoesslin and McAlpine concluded that in certain cases of multiple sclerosis there can be little doubt of the close relationship between trauma and the appearance of signs of the disease” [39]. Further large series such as that of Adams, Sutherland, and Fletcher found in a study of 389 cases that trauma preceded the illness in 10% [40]. The Association for Research in Nervous and Mental Diseases in the United States also was of the opinion that “In a small percentage of cases it [multiple sclerosis] appears to be excited by trauma but trauma itself cannot cause it but may, apparently, awake a disease process potentially existent” [41]. In 1950 Keschner [42], in his classic study of 255 cases, stated that trauma

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was an exacerbating and aggravating factor. A number of distinguished neurologists, including Kinnier Wilson [189] and Henry Miller [43], also held this belief. Miller reported his findings in 1964 and stated in a classic study of seven cases that “In all seven cases neurological symptoms were clearly related to the site of the injury” [43]. The cases cited in the previous paragraph are usually the ones claimed for advocating in the past this putative association. However, other cases and more detailed larger studies have been reported that in our opinion are more important since here the patients were studied not only clinically but often pathologically. They make a very strong case for demonstrating not only this unique association but also the putative pathogenic mechanisms involved. In this paper we will refer to the now-classic studies of Gonsette [44,45]. Gonsette’s seminal work has been confirmed by others, including Riechert [46], and recent magnetic resonance imaging (MRI) studies of evolving similar lesions make these findings of fundamental importance in understanding the pathologic mechanisms in MS [47]. Prof. Brinar studied a patient with MS and showed serially the development of plaques around the path of the trocar by MRI studies, thus further confirming Gonsette [44,45,47]. Other works that are monumental landmarks in illustrating this association are those of the late Lord Brain [48] and the scholarly studies of Oppenheimer at Oxford [49]. Perhaps one of the most compelling pieces of evidence to confirm this association has been the clinical studies and trials that arose from the experiments from Oxford on tuberculous meningitis conducted by the late Dr. Honor Smith [50,51]. What must be stressed here is that the findings of Gonsette, which are scientifically compelling, have been reproduced on several occasions. We have already alluded to Prof. Brinar and his studies in a patient with MS in which deformation of plaques after breakdown of the blood–brain barrier (BBB) was observed by MRI scans. A number of other reports have also confirmed that patients with MS, who have had operations on their brain to reduce tremor, have shown clinical deterioration secondary to the genesis of new plaques [52–54]. It should also be pointed out that the detail given by Prof. Gonsette appeared in two seminal papers. The first, published in 1966, gave an overview, but the second, detailed work in 1972 put paid to criticisms that new plaques were not degenerated and denied any argument that the plaques were not related to the breakage of the BBB and adjacent to the line of the trocar. The literature contains many examples of the association of trauma with MS, but it is strange to note that these up to now have never been analyzed critically. Sibley [29], for example, in his prospective study of trauma and MS (a) does not give any attention to BBB breakdown [44–46], (b) omits the critical studies mentioned [50,51], and (c) most importantly skips over the only positive finding he had in his report—that is, that there was a significant statistical association between the worsening of MS and electrical injuries [29,55,56]. We will comment critically on these neglected data since they are of an impressive number and are of extreme importance; they not only fill a niche in the natural history of MS but also give important clues as to its pathogenesis [57]. In a tortuous article Goodin [58] tried to show that there was no statistical association between trauma and the onset of MS and based his theoretical reasoning on

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MS having an immunopathologic foundation. The argument he puts forward, particularly mentioning the trafficking of T cells, has not been proved and is pure conjecture. Furthermore, in the paper the authors do not stress the works of Smith and Gonsette, or indeed the relationship of electrical injuries. In studying very large numbers of patients who developed MS after trauma to their thoracic and cervical spinal cord, Poser postulated that one of the major mechanisms underlying this occurrence was BBB breakdown [57,59]. This theory of Poser has now gained strong and compelling support from a wide variety of evidence. Our work therefore consists of a systematic analysis of the relationship of cerebral vessels to MS plaques, how increased permeability and BBB breakdown is the initiating and essential component of plaque formation, and how trauma can bring this about by disrupting the barrier, and it also examines the evidence both in animals and in humans of the effects of trauma on BBB permeability. Finally, the opposing claims that trauma does not precipitate MS or make established disease worse will also be analyzed critically.

Relationships of Plaques to Blood Vessels It has long been pointed out by a number of investigators that the plaques of MS bear a special relationship to blood vessels [60]. Döring was one of the first to voice the opinion that the plaques, which are found adjacent to the lateral ventricles, also have a specific relationship to blood vessels [61]. These early investigators also drew attention to the fact that the small blood vessels within plaques may or may not have inflammatory cells, but virtually all active plaques will have macrophages. Dawson, one of the first serious students of MS, showed that there was a clear relationship between the development of plaques and blood vessels [37]. “In all regions, including the cerebral cortex, the lesions are almost invariably perivenous in location. In the case of some cortical and spinal cord plaques, the central vein may be located in the subarachnoid space” [62]. The proximity of the plaque to the ventricular system and the fact that it has a central vein or venule has led many investigators to speculate about the possibility of a pathogenic agent, such as an enzyme or immunoagent, that diffuses from the cerebrospinal fluid (CSF) or blood into the brain [63]. In a serial section study, Fog [64] showed that plaques wrapped themselves around small veins over considerable distances, and it would seem that the plaque arises around such vessels [40,64]. All investigators of MS have come to the same conclusion that the plaque is based around small blood vessels, usually venules [62]. This will explain why areas rich in such venules, such as the optic nerve or cervical spinal cord, are prominent sites of MS plaque involvement.

Breakdown of the BBB in Plaque Formation The BBB was originally conceived as a rigid wall between the central nervous system and the periphery in which this barrier regulated and protected the microenvironment

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of the brain. It was thought that this barrier consisted only of endothelial cells lining capillary venules and the foot processes of astrocytes. The astrocytes and their foot processes are essential for the induction and maintenance of the tight junctions in endothelial cells [65–67]. It has now been shown that the BBB is a dynamic, highly complex structure in which the astrocytes during development and life induce the formation of tight junctions in the endothelial cells. The astrocytes maintain the BBB phenotype, which is a highly complex molecular structure [65]. Indeed, the BBB may be compromised and damaged secondary to many different types of lesions, particularly metabolic changes in astrocytes. Furthermore, the BBB is a very sensitive structure, as shown by the fact that peripheral inflammation may increase its permeability, and it is now a recognized and accepted fact that BBB breakdown is the first and integral step in MS plaque formation. It is essential in maintaining cerebral homeostasis, in which it selectively transports different substances and nutrients by a variety of mechanisms, including surface transporters [65–67]. Suffice to say that its molecular structure and function are beyond the scope of this paper, except that the BBB can be damaged via a number of different mechanisms. How it is damaged in MS is unknown, but we have shown that irrespective of the precise type of damage that occurs in ordinary MS, any form of damage that opens the BBB seemingly affects the progress and development of plaques [67]. Clearly the breakdown of the BBB is not due to myelin destruction, since it has been established that there is alteration of this barrier outside myelin-containing structures during attacks of MS [68]. One of the first investigators to draw attention to the fact that these vessels within the plaque had increased permeability was Tore Broman [69,70]. In two seminal publications, he described how having removed the brain from a MS patient at postmortem, he perfused the cerebral vessels with a solution of trypan blue, followed by normal saline and formaldehyde solutions. He detected that the dye passed out and stained the tissue around pathologically altered vessels that were found to lie within the areas of demyelination. Indeed, he even commented on the “shadow” plaques— that is, he drew attention to the incomplete demyelination that seems to occur at the edge of the plaque, suggesting to him that some substance diffused through the more permeable vessel and caused the demyelination. He also showed enormous prescience in noting that there was more extensive dye passage around small ramifications than from the main trunk of the vessel. He further noted “the vessels with disturbed permeability are usually washed clean of blood corpuscles in all the ramifications and at the same time they are stained blue, which proves that there has been a perfect circulation and no obstruction by thrombosis” [69]. In discussing the possible mechanisms he quite clearly drew attention to the fact that damaged brain tissue (i.e., from an infarction or other causes of cerebromalacia in themselves) would not give rise to secondary vascular permeability: “However, only those where the endothelial layers subjected to a lesion—seems to produce a temporary disorder in the vascular permeability” [69]. It is a well-recognized phenomenon that cerebral edema can cause demyelination [71,72]. Damage to cerebral endothelial cells is also found in guinea pigs in the Herxheimer reaction with the Forssman antigen. Guinea pig brains in which cerebral endothelial cells are damaged specifically by the Forssman reaction show

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demyelination [73]. The Forssman antigen is a unique protein that is situated in the endothelial cells that line the brain vessels of the guinea pig, and it is also found on the kidney cells of rabbits. By taking rabbit kidney and injecting it into sheep, antibodies are produced against this protein. Toxic vascular lesions using this antibody thus cause demyelination by damaging cerebral endothelial cells [69,73,74]. Gonsette et al. [44,45] also showed in guinea pigs that by intracarotid injections of detergents, endothelial cell damage would occur. While he and his colleagues used ionized detergents, the effect was identical to that of endothelial cell damage from other causes. What is important is that the antibodies injected damaged the endothelial cells, and this resulted in the development of immediate clinical signs. The cerebral endothelial cells became swollen, perivascular edema occurred, and depending on the titer of the antibody or strength of the detergent, the animals displayed weakness or died. Further histopathologic analysis of these lesions showed them to have a clear perivascular topography, and in appearance they bore a striking relationship to other human so-called demyelinating diseases (i.e., ADEM) [16,75]. These experiments clearly show that BBB lesions play a role in determining not only the localization but also the pathogenesis of demyelination. Demyelination in both EAE and its human counterpart ADEM classically occurs in a perivascular distribution with localized edema involving an area 1–2 mm in diameter around the cerebral venules. While these experiments have been conducted in animals, they show that identical disease clinically and pathologically may occur in humans where the endothelial cells are damaged by endotoxin or a variety of other toxic processes [76]. Poser’s original idea that trauma somehow affected the BBB and as a result demyelination occurred has been amply confirmed, particularly by the large number of MRI studies that demonstrated BBB alteration in MS [77–83]. Pozzilli et al. [84] reached the same conclusion from their positron emission studies. It is now an established fact that BBB alteration occurs as an obligatory event in the initiation of demyelination in MS.

Inflammatory Cell Infiltrates The plaque (i.e., the area of demyelination) is always centered around a small blood vessel, usually a vein but occasionally an arteriole. There may or may not be a mild inflammatory infiltrate of lymphocytes in the established region. This point needs to be stressed since apart from the analogy with EAE, the claim that MS is an autoimmune disease mainly lies with the demonstration of such infiltrating cells [62,85]. Serious students of MS such as Lumsden found that the myelin disintegrated in the absence of any such cells; indeed, several investigators have shown that such cells are not present in up to 30% of all cases [63,69,86–89]. Dawson [37] noted initial myelin pallor when the lesion contained only lipid-laden macrophages. Electron microscopic studies have strongly suggested that the myelin breakdown is also concomitant on the presence and arrival of infiltrating macrophages [62]. Inflammatory cells are not seen in these early MS lesions [62].

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However, some lymphocytes may be a normal occurrence in the perivascular spaces. Similar infiltrates, in fact often greater than that found in MS, may occur in a wide variety of pathologic conditions, including trauma, infarcts, and tumor and following infections [89–91]. “The number of lymphocytes that leave the perivascular compartment and enter the parenchyma is very small, both in absolute numbers and relative to the number of macrophages present” [62]. Similarly, lymphocytes are found in a large number of conditions that have no bearing on immunologic etiology, such as adrenoleukodystrophy and amyotrophic lateral sclerosis, and may be found years later in the brain and spinal cord of patients who have previously had poliomyelitis [89–92]. Furthermore, doubt for a pathogenic role for inflammatory cells is seen in patients with MS who have inflammatory cells in parts of their brain that contain no myelin, such as the retina and in areas away from plaques [68,85]. As we have pointed out, “Reliance on the presence of these mild inflammatory infiltrates as the prime pathological process in the disease therefore seems to be totally unfounded” [3,4].

Astrocytic and Other Cellular Changes The initial hypercellularity in the plaque is due to the proliferation of astrocytes and infiltrating microglia. The gliofibrillogenesis begins simultaneously with demyelination. Often astrocytes here show gross hypertrophy. With breakdown of the myelin into “myelin balls,” there is an associated vigorous astrocytic response, and this may be the earliest pathologic abnormality observed in demyelination [62,63]. We know that microglia appear not to be involved until there is breakdown of myelin, whereas reactive astrocytes appear almost simultaneously to the myelin disintegration [37,63,85]. Several authors have commented on this remarkable response of astrocytes in MS. Indeed, “Simultaneously gliofibrillae appear and there is astrocyte swelling. Such changes in astrocytes bear a relationship to the mechanism of the development of a glioma” [3]. Both Lumsden and Dawson [37,63] thought that demyelination occurred first and then simultaneously there was astrogliofibrillogenesis with involvement of the microglia and hematogenous cells and lastly invasions by scant lymphocytes. It cannot be stressed enough that astrocytes are cells that usually reflect abnormalities of metabolism, whether they be primarily local or systemic. The function of these cells is yet incompletely known, including their methods of division; indeed, their division on tissue culture is atypical [93]. What is known is that in demyelination, there is a dramatic alteration in their number and morphology. The accumulating data leave little doubt that they are intimately involved in the process of demyelination. They also play a role in controlling the BBB [65–67].

Effect of Trauma on the BBB There is a vast literature on the effects of trauma to the head and neck in animals and humans.

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Poser cites Gudmundsson, who in 1955 noted the appearance of Evans blue at the superior angle of the lateral ventricles of the rabbit brain that had been subjected to a mild concussional trauma to the parietal lobe (unpublished observations) [94]. Further and more detailed studies were carried out by Ommaya et al. [95] at the National Institute of Neurological Diseases and Blindness in the early 1960s. These authors looked at monkeys in which their necks were protected or free, who were then subjected to trauma. This allowed them to measure the degree of brain damage with particular reference to the presence or absence of cervical mobility. The animal brains were studied by cerebral angiography in the acute post-concussive phase, and BBB breakdown was also measured by injecting sodium fluorescein; the authors measured both the patterns and the degree of fluorescence. They showed that the BBB was broken down at a distance from the site of trauma. When the monkeys did not wear a cervical collar, the BBB was seen to be broken down in the medulla oblongata and in the cervical cord. These experiments were repeated and confirmed by Rinder and Olsson in 1968 [96]. Other researchers, including investigators in Glasgow, showed that BBB breakdown occurred with disruption of endothelial cells in the white matter of baboons which were subjected to lateral head acceleration movements [97]. Again, similar observations in humans had been made by the Oxford neuropathologist Oppenheimer in 1968 [98]. Following these seminal papers, a number of researchers carried out studies of experimental concussional trauma to the spinal cord of animals [99–101]. Their findings revealed that the BBB was broken down, a finding that had been studied using the electron microscope by Bakay et al. [102] in 1977. In essence, therefore, with the use of quite gross microscopic techniques such as the visual demonstration by fluorescence of fluorescein injected compounds and the visualization of Evans blue and other dyes in the brain, such findings hint at quite a marked disruption of BBB function. “However, as is the case with the clinical aftermath of concussion in the human, it is now well recognised and established fact, that even small degrees of head injury can produce concussion that may not be visually demonstrable by such techniques, but its occurrence in whom is accompanied by definite clinical abnormalities” [102]. An elegant study by Lossinsky et al. [103] showed that in the mouse there was permeation of intravenously administered horseradish peroxidase (HRP) through the endothelial cells of small vessels in the brain following different degrees of concussion. Kobrine et al. [104] showed similar findings when they carried out critical experiments on rhesus monkeys in which the trauma was directed at the cervical spinal cord. Perhaps one of the most compelling studies of the effect of trauma, as one finds in whiplash injury in humans, was that demonstrated by Domer et al. [105] in 1979. They induced hyperextension injuries identical to whiplash injury in humans in the monkey, which produced breakdown of the BBB in the cervical spinal cord and also in other areas of the brain far removed from the site of injury [105]. In other words, the lesions that occur in which the BBB permeability is increased are found not only at the site of injury, as one would expect in whiplash injury, but also away from the spinal cord, in the brain stem and cerebral hemispheres. These findings explain why new MS plaques occur in such areas away from the direct site of focal trauma. It readily explains such symptoms as optic neuritis.

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In 1978 and again in 1982 Povlishock et al. [106,107] confirmed by sophisticated techniques that the BBB is broken down after varying degrees of trauma. These experiments and others cited have proved beyond doubt that “Increased vascular permeability resulting from local tissue injury has been an established concept. This leakage is presumably due to direct tissue injury and secondary release of endogenous compounds which contribute to continuing permeability” [99]. Clearly in the human, experimental studies such as those reported for animals cannot be carried out, but a number of clinical, postmortem situations and radiologic studies have shown that what occurs in animals under experimental conditions is exactly referable to the findings in humans after trauma. Perhaps the best evidence for this is seen in patients who have serial MRI studies carried out following trauma [77–81,98]. The ultimate sophisticated technique (i.e., positron emission tomography [PET]) has also been used to confirm the findings reported in humans and also in animals [83]. The brain of fatal cases where head injury had occurred showed similar findings on detailed microscopic examination [98,108,109]. Another important feature is that the concussion need not be severe to bring about clinical and pathologic events; indeed, minor whiplash injury may be very effective in causing BBB breakdown in humans, with resulting clinical effects. This was well recognized by Ommaya, one of the most eminent investigators in the field of head injury. In 1968 he stated, “Direct impact to the head is not necessary for brain injury; whiplash injuries can also cause brain damage” [95]. Flanders et al. [110] found that “The degree of the associated bone and soft tissue injury has no bearing on the extent of the spinal cord injury or neurologic deficit”. Anatomically these lesions produced by trauma are not only superficial but also occur deep in the cerebral white matter, a finding that has been amply demonstrated by MRI [108,110–112].

Studies of Stress on the BBB “Stress may be defined as the cumulative, biological reaction mounted by an organism in response to acute or chronic noxious stimuli—which may be physical or psychological, i.e. toxic, infectious, chemical or traumatic” [113]. In looking at whiplash injury, one first has to consider the localized effects of daily minor trauma on the cervical spinal cord [49,114]. These latter references contain many citations as to the various mechanisms by which trauma to the cervical cord may damage the BBB. Studies in monkeys have also demonstrated that such specific trauma can cause BBB breakdown not only locally but also generally throughout the brain, particularly in the areas surrounding the ventricles posteriorly [105]. Whiplash injury occurring unexpectedly and associated in the majority of cases with acute trauma to the neck and often to the body and head, with severe emotional upheaval, due to a car crash and the possibility that others might be injured, can clearly be construed as a very severe form of acute, physical, psychological, and specialized stress to the nervous system. Stress has long been shown to have a deleterious effect on MS, and indeed such a view has now become acceptable to those who earlier denied any relationship between such trauma and the development of MS [58].

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The body therefore can clearly be expected to demonstrate the many changes that occur during acute stress, and of particular importance in this regard has been the recent findings that acute stress, even of a mild nature, may not only cause BBB changes but may also affect the hypothalamic-pituitary-adrenal axis (i.e., the main governing brain apparatus for controlling steroids). Such stress can also cause changes in gene expression for the production of essential neurotransmitters and other peptides involved in brain metabolism [115]. Recent research, particularly from Israel, has shown that following acute stress, changes occur in gene expression in nerve cells; such genes control the expression of a variety of different neurotransmitters, including acetylcholine [115]. Stress-induced changes in the expression of neurotransmitters in turn influence the expression of other neuronal genes affecting the production of almost all neurochemicals and their metabolites. Those most widely studied have included the biogenic amines, neuropeptides, and inhibitory amino acid neurotransmitters and their receptors [116,117]. Of the neurotransmitter systems, serotoninergic transmission is thought to be the most sensitive in stressful situations, its metabolism being altered in the central nervous system [117]. 5-hydroxytryptamine is known from a variety of studies to be involved in increasing BBB permeability [117–120]. Furthermore, trauma is one of the events that produce changes in the level of cytokines, and these are considered to be involved in the pathogenesis, initiation, and continuation of demyelination [121,122]. There are innumerable papers showing that trauma may be associated with elevation in the serum and in the CSF of different cytokines [121–127]. The production of IL-6, a specific cytokine, is increased in many brain diseases, particularly MS, and such cytokines are increased after trauma [128–131]. Of enormous interest is the fact that trauma to the cervical spinal cord may be followed not only by demyelination in that area but also widespread demyelination throughout the nervous system, particularly periventricularly in the occipital horns. One wonders, therefore, what might be the scientific explanation for the widespread distribution of demyelination found in humans after cervical whiplash injury when the predominant area of focal trauma is to the cervical spinal cord. There is now compelling scientific evidence, gleaned particularly from animal experiments, that lesions of the cervical spinal cord are associated with more widespread lesions throughout the brain [105]. Domer et al. [105], in their experiments on monkeys, showed that the lesions were widely distributed throughout the brain and were not only found in the areas damaged by the hyperextension/hyperflexion injury. Furthermore, Schmidt and Grady [132] found that specific lesions to the brain produced diffuse generalized BBB breakdown in the rat. Tanno et al. [133] found similar abnormal permeability of the brain vessels to microinjected IgG and HRP after focal injury to the rat: the lesions occurring in the brain were generalized and widespread, involving both hemispheres. It is known that cytokines will increase the permeability of cerebral blood vessels. Further evidence that cytokines may be the mediators after this type of injury was shown by findings of increased tumor necrosis factor (TNF) and IL-6 in the CSF of 14 patients after head injury [134,135]. Indeed, patients with MS who have head trauma may show an increase in cytokines and new plaques [136].

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Stress and its effect on the brain have always been of extreme importance in relation to MS. Indeed, emotional stress “antedating the onset or relapse of disseminated sclerosis was considered even (in itself) to be occasionally of significance”… “A significant number of patients with disseminated sclerosis stated that an emotional stress, often of a specific nature, led invariably within the lapse of a minute to an exacerbation of symptoms due to a pre-existing lesion” [137]. How stress might cause far-flung physiologic and pathologic changes in the nervous system has been the subject of many international conferences and meetings— for instance, “New Frontiers in Stress Research: Modulation of Brain Function” [117]. In this book distinguished scientists report that the effect of stress is farreaching. Whereas years ago clinicians could only conjecture from clinical findings that stress could aggravate diseases, now we have cumulative scientific evidence to show, using modern molecular biologic techniques, that after acute stress significant chemical changes take place in the brain, with BBB breakdown and even the alteration in a bidirectional way of gene expression controlling central neurotransmitters [117]. These data have been further analyzed in recent papers that are pertinent in this regard [116,117]. In addition, the effect of stress has been analyzed in humans and in animals for its neurobiologic and pathologic effects. In a seminal study, soldiers in the Gulf War exposed to stress had a leaky BBB; drugs given to them that normally did not cross the BBB were found to do so after stress [138]. This work highlighted the fact that BBB breakdown under stress would allow substances within the intravascular space to enter the brain and cause abnormal brain function [139]. A very important series of experiments were carried out in 1996 in which mole­ cular biologic changes in the brain of animals subjected to varying degrees of stress were studied [115]. The stress was minimal to moderate (animals were made to swim in cold water) and the subtle but definite changes in the nervous system were measured. They were found to have alterations in the level of neuronal c-fos oncogene expression and in the messenger RNA levels for acetylcholine, one of the most important neurotransmitters of the brain. Further studies showed that after acute traumatic stress, a robust cholinergic response triggered rapid induction of the gene in coding the transcription factor c-fos. “This protein then mediates selective regulatory effects on the long-lasting activities of genes involved in acetylcholine metabolism” [115]. Here, in the central nervous system of small animals one could see as a result of stress not only BBB breakdown but also changes in gene expression for neurotransmitters. Similar changes in the BBB due to stress have been demonstrated in humans and even in patients with MS in which serial MRI and CSF cytokine studies were carried out before and after head injury [136]. Further studies by Otte et al. using single protein emission computed tomography (SPECT) scans demonstrated that there were significant metabolic changes in the brain following whiplash injury [140,141]. Studies done on patients with whiplash injury again have a particular significance since they show that after whiplash injury, changes occur not only at the spinal cord but also in the rest of the brain. One such study demonstrated that six of seven patients with whiplash injury had significant metabolic changes in the brain involving parieto-occipital hypoperfusion [137,142]. The hypoperfusion and hypometabolism was localized by Single Protein Emission Computed Tomography (SPECT) scanning to watershed zones

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between the territories of large cerebral arteries. Additional studies showed that hypoperfusion and hypometabolism occurred in the posterior part of the brain secondary to whiplash injury [139,140]. These data demonstrate quite clearly that the lesions in the cervical cord after whiplash injury are often associated with more extensive lesions throughout the brain, and this may explain why lesions are not confined to the cord after whiplash injury but may extend to other parts of the brain [139,140].

Studies Showing that the Site of the BBB Breakdown is related to the Formation of New Demyelinating Plaques One feature that MS shares with EAE is that demyelinating lesions may be made to occur at a particular specific site. A possible pathogenic relationship between trauma and demyelination was demonstrated in 1955 and 1957 after investigators placed electrolytic lesions in the cerebral cortex and white matter of guinea pigs and rabbits several days a week following injection of spinal cord suspension and adjuvant to produce EAE [143]. In the animals which later developed EAE, lesions were found to occur adjacent to such electrolytic lesions, demonstrating that these lesions served as adequate stimuli for a local exacerbation [143,144]. These experiments were elaborated on by Levine, who produced in 1968 the same results by placing a heated electric soldering iron on the exposed but intact skull of the experimental animals; the EAE lesions were noted to cluster around the zone of coagulation necrosis [145]. This area corresponded to a BBB alteration, and this is extremely important since we now know that trauma is one of the causes of such BBB alteration. In 1972 Lumsden [146] had proposed that MS shares with EAE, in terms of their respective pathogenesis, one major characteristic: an initial BBB alteration. This shared effect of BBB breakdown needs to be looked at carefully. Since EAE is a cellmediated immune disorder, increased permeability will occur at the site of injury as part of the normal reaction (i.e., physiologic), when sensitized cells come in contact with the putative antigen. Simply because the BBB is broken down at focal points in MS does not mean that such breakdown occurs for the same pathogenic reason as in EAE. Indeed, failure to grasp this important point may lead researchers to group the two reactions as identical when, in fact, the BBB breakdown in EAE is a direct explicit example of an immunologic reaction, while in MS the exact mechanism of breakdown is not known. It most probably does not even reflect an immunologic reaction and is likely to be mediated via an astrocyte-metabolic process [147]. The accumulating data suggest that the BBB breakdown is secondary to metabolic change in astrocytes that induce an increased oxidative metabolic demand. This increased metabolic demand with impaired substrate supply causes further breakdown of myelin, particularly if that myelin is genetically abnormal [148]. Rather, as is universally claimed, than some factor diffusing out from the damaged blood vessel and causing demyelination, it may simply be more likely that oxygen and other metabolites that are needed are impaired from getting to the region required, and hence demyelination occurs in a metabolically compromised area.

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The molecular assembly of the BBB is a very complex structure and its mode of action is not fully understood. Clearly the endothelial cells and their tight junctions are enormously important, but likewise there is a most important contribution from astrocytes, so that the idiopathic BBB breakdown that occurs in MS could be mediated via either cell. Irrespective of the precise cause of breakdown of the barrier, once this barrier is broken down in a patient with a diathesis to develop MS, the breakdown initiates formation of new plaques. It is most important to look carefully at the whole concept of the BBB. We know that in mammals, including humans, the permeability of the small vessels in the brain is different from that of similar vessels throughout the body. In the case of the brain, it is extremely important that substances traversing and passing through the brain in its blood vessels do not get out through these blood vessels, since many substances already in the blood would damage the sensitive metabolism of neurons and other nerve cells. Substances such as oxygen, glucose, and certain ions are allowed free passage, but other substances, including toxins, large molecules, bacteria, and viruses, are actively excluded. The blood vessels of the brain are different from the blood vessels in the rest of the body by having complex junctions between the endothelial cells (i.e., the flat cells that line the blood vessels throughout the body and the brain). In the brain, where the cells join onto each other is an extremely complex area and what a few years ago was considered a simple apposition of cells to each other is now known to be a most complex biologic structure with many different proteins and structures, each having specific biologic functions. To see how this comes about, one must look at the BBB development. Initially, the serosal (i.e., endothelial) cells join each other and, due to a substance produced from adjacent astrocyte cells, where these cells join each other they develop a complex biologic structural assembly. Healing of the BBB would then be assumed to have a beneficial effect. This most likely occurs with natalizumab as one of its actions, but the “repair” to the barrier is likely to be incomplete and to hinder normal cellular traffic. This may help to explain the fatal complications that may occur in patients with MS, who develop progressive multifocal leukoencephalopathy after being treated with natalizumab, and may also explain the reported possible beneficial effect observed with beta-interferon [14,149]. The treatment for severe tremor occurring in MS has often in the past been stereotactic destruction of certain parts of the brain. These observers noted fresh MS lesions adjacent to and surrounding needle tracts. Since needle tracts had damaged the BBB, this had resulted in new lesions of MS (i.e., lesions induced by trauma). Such needle tracts were examined in other patients without MS (e.g., patients with Parkinson’s disease), but they did not develop lesions of MS, although their BBBs were broken. These observations have been confirmed by others and even by serial MRI studies in such patients [46,47]. Lord Brain noted that cervical spondylosis (i.e., arthritis of the neck bones) may be associated with MS. He suggested that “the effect of cervical spondylosis upon the spinal cord may be to make it more susceptible to the lesions of MS at that level” [48]. He and his colleagues found that when they looked at such patients who had died (i.e., patients who had both cervical spondylosis and MS), at postmortem there

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were often wildly disseminated plaques of demyelination (i.e., MS) throughout the brain and particularly in the spinal cord, where the changes were conspicuous in the cervical segments at the levels where spondylosis had occurred [48]. Lord Brain’s findings have been confirmed in other studies [150]. Oppenheimer made a study of this phenomenon [49]. He examined the spinal cords of 18 such patients with MS and found that the cervical portion had twice as many plaques as other parts of the spinal cord. He put forward a theory that this part of the cord under normal conditions had mild degrees of trauma inflicted during flexion and extension, but particularly in someone who has cervical spondylosis, the effects of this trauma would be increased. The degree of injury can vary from slight to severe, but the important aspect is that the mechanical stress is directed at the cervical and thoracic spinal cord [49]. He clearly demonstrated that the lateral portions of the spinal cord, which are close to the attachment of the dentate ligaments (ligaments that would be stretched in a whiplash injury), are the predictable sites of MS plaques, and postulated that it was the effect of trauma upon the permeability of the venous wall that led to the subsequent plaque formation [49]. MS plaques occur around small veins, and damage to such veins resulting in an increased permeability may precipitate MS in a predisposed person. Investigators have provided abundant evidence that such capillary hemorrhages can often be seen in brains after even minor trauma, which may result clinically only in mild concussion [109]. Since the BBB has been demonstrated conclusively to be damaged as one of the initial features of MS, breakdown of the BBB following specific focal trauma is very important. This breakdown has been shown conclusively to occur in a large number of studies both in humans and in animals, where the brains of animals after different types of even minor injury were examined [84,98,105,108]. Investigators found an extravasation of HRP into the vessel wall, and the surrounding brain tissue showing definite increased permeability. They further found localized destruction of the endothelium in close proximity to the marginal lines between endothelial cells. As Poser stated, “it would therefore appear reasonable to suggest that when the brain or spinal cord of a MS patient is injured, the area of injury becomes a potential nidus for new lesions; should such an injury involve an area in or near an existing plaque, the latter might be reactivated and/or enlarge and become symptomatic. Conversely, should a patient develop MS after an injury to the nervous system, plaques might be expected to develop in that vicinity. Even minor trauma may be important” [109]. Some of the most important observations on the relationship of BBB breakdown to the development of MS are those made by Gonsette et al. [44,45]. These researchers, in a paper discussing an experimental demyelination produced in guinea pigs by the injection of various concentrations of detergents, described the findings in three patients who had died after undergoing stereotactic surgery. The postmortem studies were carried out by the neuropathologist Dr. O. Perier. The authors employed stereotactic destruction of the thalamus to reduce the severe intention tremor of some patients with MS and Parkinson’s disease. The technique had been used particularly in France and was found to be generally satisfactory. Their observation that the patients all deteriorated clinically after this operation is extremely significant [44,45]. This is in accord with the experiences of Prof. Dorothy Russell, Kelly, and

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Oppenheimer [151–153]. It is important to stress that there was clinical deterioration in these patients after the procedure; as the authors state in reviewing the report of the neuropathologist, “His observations not only confirm our clinical impression of post-operative deterioration by reactivation or formation of plaques but introduce some particularly interesting ideas concerning the problem of the relationship between sclerotic plaques and lesions of the blood–brain barrier” [44]. Their neuropathologic examination was most thorough and detailed: “In each of our three cases, Perier finds sclerotic plaques in direct contact with the area of stereotactic destruction or in the path of the trocar. Knowing that this mechanical destruction of the cerebral parenchyma inevitably leads to the impairment of the blood–brain barrier, it is possible to suppose that the myelinotoxic substance circulating in the serum of these patients has penetrated the nervous tissue and exerted destructive effect. The change in the blood–brain barrier cannot by itself account for the demyelinisation, because the numerous anatomico-pathological tests on the Parkinson’s patients who have been operated on by stereotaxy, show very different and clearly less important attacks on the myelin. In no case did the lesions recall the typical images encountered in MS” [44]. The authors go on to state, “It would also be quite surprising that in each of our three cases, the trocar should end by chance in the centre of a sclerotic plaque” [44]. They claimed that their clinical observations permitted them to affirm that there was a lesion in the BBB in the area of the plaque, and they also put forward arguments favoring their hypothesis that changes in the permeability of the cerebral vessels precede the formation of demyelinating plaques. This determines both plaque formation and topography. In 1973 Riechert et al. [46] described two patients with MS in whom stereotactic treatments for motor and tremor disturbances were carried out. The first case was a woman who developed MS at age 25 and at age 33 had one of two stereotactic coagulations performed. The first procedure resulted “in the permanent improvement of the intention shaking and myoclonus on the right side.” However, 1 year later she required a further treatment on the other side, and this had the immediate effect of 80% relief. “One week later, however, an apraxia of swallowing and aphonia developed, and impulsion decreased. As a result, the patient could no longer be induced to sit up, to stand upright or to do physiotherapeutic, although the 80% relief of the intention tremor continued.” The patient developed pneumonia secondary to her neurologic deterioration and died [46]. A postmortem was performed by Prof. Pliess of the Pathological Institute, Nurnberg. He found that “the electrode track is through a multicystic focus of destruction of nervous elements with a scarred shrinkage surrounded by a complete demyelination with irregular borders … The fresh foci—only 2 weeks old—have damaged and functionally incapacitated the nerve cells of these structures and thereby disrupted the central regulation of circulation and respiration which led to the fatal pneumonia … However, the foci and the needle track are surrounded by even larger zones of demyelination than in the previously described left hemisphere … Because the clinical progression started approximately one week after the last operation, the very fresh encephalomyelitic alterations are also related to the second operation

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which led to death by aspiration pneumonia … The demyelinations on the margin or in the vicinity of the electrode track suggest that the penetration of the electrodes has induced new relapses of the encephalomyelitic demyelinating process. It is less probable that these relapses are spontaneous.” Like Gonsette et al., Riechert too observed the undisputed clinical deterioration in his patient [44–46]. Riechert’s second case was rather difficult, in that it concerned a 50-year-old man with a developmental lesion and only a possible diagnosis of MS. His “MS” was not diagnosed until autopsy; clinically and pathologically it is doubtful if this case was true MS. The patient also had Parkinson’s disease. In discussing the two cases, the authors stated, “The second has clearly shown that a stereotactic intervention does not necessarily precipitate new demyelination around the coagulation focus and electrode track. However, the first case reveals that this has indeed occurred” [46]. In case 5 of the pathological cases described in Dr. Oppenheimer’s MD thesis, he described the clinical effects of performing a brain biopsy in a patient with existing MS [153]. He stressed “more significantly, the biopsy site was the only place in the brain where intense, active demyelination was observed at autopsy” [153]. Surgery to obtain the biopsy was carried out some weeks prior to the patient’s death. In other words, the biopsy specimen that was removed was entirely normal, there being no MS in it, but as expected the operative site caused BBB breakdown and proved to be the site of new demyelination. This would explain that after an interval, fresh plaques occurred. These findings in this fatal case in whom a postmortem was carried out show that the procedure of cortical biopsy broke down the BBB, and in this patient with existing MS, it then caused activation there of her disease. These findings should be compared and contrasted to those of Gonsette and the confirmatory studies of Riechert in 1975 [44–46] and those cases where a similar procedure was examined by MRI [47]. Prof. Dorothy Russell mentioned a case in which she had performed a postmortem in the Radcliffe Infirmary at Oxford (i.e., RIPM No. 384-43 cited by Oppenheimer): “This was of a woman of 59, previously well, who after a not very severe head injury was grossly confused, and developed a spastic triplegia, with hemiballismus in the remaining arm. She died after five weeks and was found to have numerous plaques of MS. Microscopy showed these to be of long standing: in addition, there was widespread, diffuse presumably recent breakdown of myelin sheaths, not only in the vicinity of a contusion in the temporal lobe, but in the seemingly undamaged regions of the thalamus and the brain stem” [153]. Russell [151] commented, “such cases serve to show both that the disease is even commoner than is generally suspected and also that these subjects may react catastrophically to any interference within the central nervous system” (as after an operation). Sibley et al. [29] did not critically analyze another two patients in his own series who had had thalamotomies and were not regarded as exacerbations, although “both had progressive disease before, as well as after”. In relating his experience of such patients who had brain surgery, Kelly [152] suggested that those with MS tolerated major surgery on the brain very poorly “and everyone of the 14 personal patients who had been subjected to thalamotomy have had a severe exacerbation within three weeks of the operation”. Clearly the two patients in Sibley’s study are important in this regard. Kelly also, in a unique study of 980 cases, found 65 who had had a significant relapse or deterioration after trauma greater than that experienced in daily life.

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He further stated, “14 patients involved in road traffic accidents or falling downstairs or out of a tree or being mugged in the street similarly have experienced such a relapse” [152].

Effect of Electrical Injuries in the Precipitation or Worsening of MS Sibley et al. [29] noted in this study that attacks of clinical worsening of MS were often precipitated by electrical injuries. This occurred in 17 cases of their series and was found to be highly statistically significant. These findings, however, were not discussed in detail, nor was the pertinent literature on this important topic reviewed. As long ago as 1959 investigators began investigating the effect of convulsions on the BBB. Investigators at that time were interested in the fact that lipoid substances such as chloroform and alcohol penetrated the BBB rapidly, ions such as sodium and potassium passed more slowly, and large molecules such as dyes and proteins passed through the normal barrier extremely slowly. It was observed from previous studies that repeated convulsions would increase the BBB permeability, and clearly such data were significant when studying patients with epilepsy [154,155]. BBB permeability could be evaluated in animals by techniques measuring the rate of passage of radioactive iodinated human serum albumin (RIHSA) from plasma to CSF. Using this technique, hypoxia had been demonstrated to increase the permeability in dogs after giving them a drug (i.e., Metrazol) or applying an electroshock to induce seizures. BBB permeability was increased 25 times that of normal controls following such treatments. How seizures produce this particular BBB breakdown was then unknown, although some suggested that there was a definite decrease in oxygen saturation with seizures [154–157]. By the late 1970s an enormous amount of research data had accrued on the BBB breakdown that follows epileptic seizures. Techniques measuring the uptake of dye, cocaine, HRP, or radioactively labeled albumin were used, and all demonstrated that there was an increase in permeability following electrically induced seizures. Investigators then studied patients, particularly those with endogenous depression receiving electroconvulsive therapy (ECT). It had been demonstrated that the cerebral blood flow rose by as much as 120% during seizures, and this was confirmed looking at the cerebral blood flow and brain metabolism in human during ECT [154,155,157]. In 1961 Lee and Olszewski [158] demonstrated that BBB breakdown was specifically determined as a feature of the seizure activity itself, thereby excluding, as previously thought, the effects of hypoxia and anaerobic muscle metabolism. In other words, seizure activity from whatever cause increased brain vascular permeability. It became clear that a number of dynamic events were taking place during electrical seizures, namely increased cerebral blood flow, loss of autoregulation of vessel size, increased permeability of cerebral capillary vessels, and some changes in anaerobic metabolism and in oxygen utilization. This electrical activity in the brain increased the BBB permeability. The condition appeared to be reversible after a time interval, and this was demonstrable in both animals and humans [156,157].

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As might be expected, MRI, the most powerful tool for investigating pathologic changes of brain tissue, was applied to the study of ECT in humans. During T1 (longitudinal) and T2 (transverse) relaxation times both before and after ECT, it was possible to determine the increase in brain water and hence the BBB breakdown [158–160]. Two investigations demonstrated an acute reversible increase in the mean T1 of the brain after ECT; the authors interpreted this as a temporary BBB breakdown. This interpretation was clearly supported and based on the observations that (1) electroconvulsive shock and ECT causes BBB breakdown in animals, (2) BBB breakdown increases brain water content, and (3) brain water content correlates with such T1 findings. Similarly, the T2 relaxation time was positively correlated with BBB breakdown. “Electroconvulsive therapy therefore increases the brain water content secondary to breakdown of the blood–brain barrier” [160,161]. Since changes in affect (i.e., mood) are very common in patients with MS, and since MS patients often have serious clinical depression, it is evident that many MS patients would need to be treated with ECT. ECT has been carefully studied for its effect in such patients. “Several reports have now shown that patients with MS who are depressed may have an acute neurological deterioration following electroconvulsive therapy” [55,56,161]. There have been a large number of reports of ECT in patients with MS [55,56,161]. In one study patients were evaluated with gadolinium-contrast MRI scans prior to receiving ECT [56]. This allowed the investigators to measure the BBB status, since gadolinium in a paramagnetic contrast agent can cross the BBB when it is broken down [56,162]. This is the standard technique used for studying MS plaques to show the disease activity status, since in the early stages of plaque formation there is definite, undisputed BBB breakdown. Mattingly et al., in studying patients with MS receiving ECT, describes some who deteriorated following this treatment. One of their patients, patient 2, after the fifth treatment of ECT became disorientated and had impairment in his ability to concentrate and “His neurologic status markedly deteriorated. The patient began dragging his left foot and demonstrated weakness of his right medial rectus. He also became incontinent and an intravenous pyelogram revealed a large post-void residue” [56]. Because of the dramatic deterioration in his clinical status ECT treatment was stopped. At the 6-month follow-up he remained incontinent of urine and still had paresis of his left leg. A review of the 12 previous reported cases of ECT in patients with MS showed that two other patients developed severe neurologic deficits during treatment [56]. To date, there have been 15 reported cases of ECT given to patients with MS; 20% of them have developed neurologic deficits during treatment. According to the authors, “It seems unlikely that 20% of patients would develop such severe deficits during the limited time frame for a course of ECT” [56]. The authors went on to state, “Individuals with MS appear to have an increased risk of developing focal neurological deficits during the course of ECT, and patients with gadolinium-enhancing lesions may be at even greater risk” [56]. The finding by Sibley of 17 patients whose condition had deteriorated following electric shock and the many other reports of similar worsening makes, in view of these data, a very pertinent and important observation that cannot be dismissed as having no significance. Its importance is highlighted by the fact that here one has a good example of damage to the BBB, and it should be noted that there are various

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studies of both motor neuron disease and Parkinson’s disease where clinical deterioration occurs following acute electrical injuries [163,164], thus supporting the idea that MS is a neurodegenerative disease [20].

Therapeutic Trials of Breaking Down the BBB in Patients with MS Dr. Honor Smith and colleagues at Oxford University were particularly interested in the BBB in their treatment of patients with tuberculous meningitis. Their studies, carried out predominantly during the 1950s, were aimed at methods that would break down the BBB for the sole purpose of allowing drugs, mainly antituberculous ones, to gain access to the brain from the vascular space. To study the BBB they measured the bromide index—the amount of bromide per unit/volume of serum divided by the amount of bromide per unit/volume of CSF. In normal individuals with an intact BBB this ratio is 1.95–3.39, but inflammation of the leptomeninges (i.e., the membranes covering the brain) facilitated passage of many substances, including bromide, from the blood to the CSF, with a reduction of the index. They therefore induced a sterile meningitis by injecting tuberculin into the intrathecal space in patients who, by means of natural infection or by vaccination, were intensely sensitive and allergic to the tuberculin protein. This intrathecal injection of tuberculin in such sensitized patients, of course, caused massive breakdown in the BBB [50]. Basically, the idea was to take a normal person who previously had been exposed to tuberculosis and was as a result sensitive to tuberculin protein and to inject tuberculin into his intrathecal subarachnoid space. Over the next 48 h after the injection, patients would develop an intense allergic reaction of inflammation of the meninges to the injected tuberculin, with resultant BBB breakdown. This sterile meningitis caused an increase of white cells (i.e., marked CSF pleocytosis). The researchers found a two-wave cellular infiltrate reaction, initially an outpouring of polymorphonuclear leukocytes into the CSF and then a secondary reaction in which there was an increase in lymphocytes; overall there was a gradual increase in the protein content of the CSF secondary to a breakdown of the BBB. This caused a transudate of serum proteins from the blood into the CSF through the damaged barrier. This was an important reaction to study, and indeed Smith et al. considered that this reaction might have enormous importance in treating patients with meningitis or inflammation of the brain by allowing more specific drugs to cross the BBB and hence enter the brain tissue [50,51,165]. Their initial intention was to develop a method that could deliver drugs into the brain tissue more effectively. Furthermore, they were able to look at the effects of different substances, including antihistamines and steroids, for their healing properties on meningitis when the BBB was broken. By studying this reaction (i.e., producing a sterile meningitis), they were able to show that bromide and penicillin crossed the barrier more easily. These observations on the effect of natural meningitis and induced experimental meningitis on the passage of drugs from the blood to the brain due to the resultant BBB breakdown were

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of potential importance in treating patients with different types of meningitis, particularly tuberculous meningitis. Two years later, in 1957, she and colleagues reported their observations of carrying out this method in patients with MS [51]. These patients had become sensitized to tuberculin from natural infection and it was “inferred” that in MS, an alteration in formation and flow of the CSF and of the blood–CSF barrier, such as occurs in meningitis, might influence the course of the disease for the better [166]. They showed quite clearly that “the reactions markedly and constantly increase the permeability of the blood-CSF barrier” (i.e., as occurring after the injection of purified protein derivative [tuberculin]) intrathecally (i.e., into the CSF space). This reaction, in their words, not only increased the hyperemia of the leptomeninges but also affected the perforating vessels of the brain stem. “In their preliminary studies these researchers thought that some patients with MS having been given intrathecal tuberculin improved. Indeed, so impressed were they by the claims of a causal relationship between treatment and improvement that they carried out the study in over 280 patients. These Oxford investigators hinted at but did not give specific details of improvement” [166]. However, their suggestion that this technique might be important led other workers to repeat such experiments. Marshall and O’Grady studied 17 patients with MS, 9 patients with motor neuron disease, 3 with Huntington’s chorea without dementia, and 3 with Parkinson’s disease, all of whom were sensitive to tuberculin [167]. They recorded the clinical response as “this was characterised by headache, neck stiffness, fever, vomiting and in many cases drowsiness.” Curiously, these latter authors were interested in the immunologic and cellular response and gave little clinical details of how the patients behaved. They did, however, give some indication that the process was not without complication since “loss of sphincter control is especially common and in the patients with MS may persist for several weeks.” Kelly and Jellinek [168], having read the works of Smith, where they found “a hint of prolonged remission in disseminated sclerosis,” “felt obliged” to carry out further experiments to look for confirmation of this beneficial effect. They selected 20 patients with classic MS and noted, as had the previous workers, a febrile reaction with meningeal irritation in 19 of the 20 intrathecally injected subjects. Nine patients experienced “exacerbation of their symptoms and signs and six required catheterisation of the bladder.” One patient who had increased spasticity remained disabled and more spastic. Bladder infections occurred in six patients, and in four of those six it was in association with neurologic setbacks. “Episodes of further plaque demyelination occurred in five cases after the immediate clinical or meningeal reaction had subsided.” In describing some of these cases, it became clear that case 5, for example, became more ataxic, with weakness of his legs by the 12th day. Case 14 developed increased weakness of the upper arms and retention of urine and had not improved 3 months after discharge. Case 19 developed a complete left foot drop 10 days after the injection. “In two other cases the new episodes were alarming.” This included numbness of the trigeminal division of the right side of the face and motor and sensory signs in the left leg; the patient became “frankly suicidal” and required courses of ECT. She then went on to develop an ascending myelitis with intercostal paralysis, respiratory paralysis, rightsided facial paralysis, dysarthria, and left-sided deafness with bulbar palsy. Similarly, another patient, patient 20, lost control of the bladder and had spinothalamic lesions,

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total cord transection, weakness of the left limb, and blindness of the right eye. In following these patients these neurologists noted that “serial psychometric tests showed evidence of deterioration in 9 of the 16 trial cases during the year after the PPD was injected.” The trial had to be abandoned because of the severe reaction in these patients. The authors very cautiously stated, “Indeed, we are not satisfied that it may not lead to a reactivation of the condition during the month after the treatment” [167]. They even went on to state that “we hope that trials like ours and the similar results of Miller et al. will prevent the use of PPD as a placebo in a condition where there is great pressure for some active form of treatment” [167]. Miller et al. in 1961 had carried out a similar trial. Miller et al. were clearly puzzled as to the Oxford claims of clinical benefit since “although the reason for endowing this procedure with any possible therapeutic significance has never been clearly stated, we felt it important that the suggestion should be subjected to careful controlled assessment, and it is with the preliminary results of such a trial that the present paper is concerned” [169]. Their observations failed to reveal any influence of the injection of intrathecal tuberculin on the subsequent clinical course of MS. “In more than half the patients treated, the injection of tuberculin provoked an immediate clinical exacerbation of the chronic disease, with the reactivation of all symptoms of and provocation of new, but the present observations lend no support to the contention that the procedure influences the subsequent progression of the disease for good or ill once the immediate effects of treatment have subsided” [169]. They stopped their preliminary study and clearly noted the adverse effects of this form of treatment. These experiments, carried out for the best of reasons, clearly did not foresee that BBB breakdown in patients with MS brings about great deterioration and exacerbation of the disease, with the development of new symptoms and signs. The experience of Dr. Smith and others who repeated her work leaves no doubt whatsoever of the phenomenal clinical deterioration in a high proportion of the cases so treated. These experiments illustrate that BBB breakdown causes an exacerbation of the disease, and this strongly supports our contention that similar BBB breakdown, for whatever reason, particularly as outlined in our series of cases with cervical spinal cord trauma, brings about rapid clinical deterioration. Oppenheimer, in his MD thesis, studied three patients who had received the intra­ thecal injection of purified protein derivative (PPD) [153]. Two of these cases are recorded in detail, one dying 11 months and another 2 years later. The first of these had severe subacute lesions, while the second case was complicated in that the patient had fallen 9 months before her death and suffered a head injury. This latter patient had a large brain stem demyelinating plaque, which Oppenheimer attributed to the fall. Both of these cases had clinical relapse after PPD, but the third case died some 4 years after the PPD treatment, and the lesions were as one would expect in a standard case of MS [153].

Effects of Radiation on the BBB Experiments involving passive cellular transfer of EAE showed that if the recipient animals were irradiated prior to receiving the cellular transfer, they showed more virulent disease than their nonradiated littermates. Total body irradiation clearly

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allowed the transferred cells to induce a more rapid and severe encephalomyelitis [170]. Irradiating the donor rat reduced the severity of EAE, but giving specific focal spinal cord irradiation enhanced not only the severity of the disease but also caused it to develop earlier [170]. Such data are entirely consistent with the facts that irradiation damages the BBB. BBB damage has been shown in innumerable experiments, both in animals actively immunized to develop EAE and in those acting as recipients for passive cellular transfer, to enhance the severity of the disease, and the site and location of the disease could be predetermined. Levine and Hoenig showed that in such passive cellular transfer experiments in Lewis rats, a localized focal breakdown of the BBB artificially induced in recipients allowed them to develop the illness literally within hours of the cellular transfer [145]. Acute radiation therapy, particularly at tumoricidal doses, disrupts the BBB and can even cause an encephalopathy [31]. Patients with MS who are irradiated would be expected to do badly since BBB disruption would promote further demyelination. Indeed, cerebral demyelinating lesions of MS may appear as single or multiple contrast-enhancing lesions on MRI scans and can be mistaken clinically and radiologically for primary or metastatic brain tumors. Investigators reported five such patients who received radiation therapy [31]. Four of the patients received radiation in full tumoricidal doses and had extremely poor clinical outcomes. MRI scans of patients who have deteriorated after radiation show new lesions, which have been verified pathologically as acute new demyelinating lesions. At autopsy in such cases, profound conspicuous and aggressive demyelination has been found [31]. Aarli et al. reported the case of a man with MS who had a glioblastoma multiforme and was treated with radiation. He died 2 months later, and the autopsy showed no infiltrating tumor but instead diffuse demyelinating disease of the white matter [171]. Tourtellotte et al. studied 20 patients with MS who had received craniospinal radiotherapy and measured the levels of immunoglobulin G in their spinal fluid. Five patients had a transient decline in IgG synthesis, which lasted 3–6 weeks, 10 had totally inconsistent responses, and the remaining five, who were given steroids in addition to radiation, showed a decline in their IgG synthesis rate. None of these patients showed any clinical improvement [172]. Persisting with the idea that immune suppression may help, MS patients were given total lymphoid irradiation to induce immunocompetence [173]. A number of deaths occurred with this treatment and no clinical benefit was noted [12,173–175]. There was a definite increase in the severity of the disease and the development of infections. The worsening of demyelinating disease would appear to be the effect of radiation on blood vessels enhancing vascular permeability [31].

Bee and Wasp Sting Encephalopathy Reactions with Breakdown of the BBB It is well recognized, although rare, that an edematous encephalopathy may follow the sting of a wasp (Hymenoptera) or a bee (Apis mellafica); this edema of the brain

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is secondary to a damaged BBB [176]. Studies have confirmed in similar fatal cases that edema and increased capillary permeability occurs [177]. While these cases are rare, they tend to strongly support our theory. MS and optic neuritis have been reported following such stings [178].

Congophilic Amyloid Encephalopathy with Breakdown of the BBB Several patients have been described with the rare disorder cerebrovascular amyloid angiopathy who developed MS. In amyloid angiopathy, a condition that may occur sporadically or genetically, the small cerebral blood vessels deposit amyloid material in their vessel walls. Most of these cases are without any clinical event, but some are known, usually after minor head injuries, to develop intracerebral hemorrhages. The patients are usually elderly, and segments of a single vessel may be involved; in that segment the BBB is broken down (i.e., there is increased permeability). The vessels involved with the deposition of amyloid have an increase in their permeability; when this increase occurs in patients who have concomitant MS, the plaques are centered around such diseased portions of the vessels. Students of this disease have found that “amyloid accumulated massively in and around blood vessels, usually in the immediate vicinity of the plaques” [179].

Neurologic Disorders that may be Precipitated by Trauma Many neurologic diseases are known to be precipitated by trauma. Indeed, some of these disorders for the most part occur after head injury; the injury in some way initiates the illness but does not cause it.

Childhood Ataxia with Central Hypomyelination In this recently described condition in childhood there is massive demyelination of the brain. The first account was given in 1993 by Hanefeld et al. [180]. A variety of other scholars have described this condition, which typically occurs in childhood but can occur in adulthood. The patients usually present with cerebellar ataxia and stiffness of their legs and go on to develop optic atrophy and epileptic seizures. The condition is considered to be genetic in origin but interestingly is precipitated by minor head trauma [180– 182]. The important point about this leukoencephalopathy with massive demyelination is that it is an inherited, genetic disorder, and the clinical deterioration usually occurs following head injury. How the head injury initiates the illness is unknown [182–184] .

Familial Hemiplegic Migraine Familial hemiplegic migraine is considered an autosomal dominant subtype of migraine in which patients have with their aura recurring episodes of hemiparesis or hemiplegia during the acute phases of the headache.

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Some Forms of Common Migraine Prof. Matthews at Oxford described how even common migraine may be precipitated by head injury and gave as a classic example many patients he had studied who had developed migraine following head injury suffered during football [18]. Indeed, the term “footballers’ migraine” has been used by neurologists.

Motor Neuron Disease The association between trauma and motor neuron disease has long been proposed, and one of the injuries highlighted as precipitating motor neuron disease is electric shock [185]. Initially the studies were small, but when good case-controlled studies were done in which the sample size was larger, appropriate controls were used, and ascertainment bias and unclear recordings were avoided, these trials strongly suggested a definite association between trauma and the development of motor neuron disease. “One well defined study, which eliminated recall bias by examining the military records of men in whom ALS later developed compared to match controls, recorded a statistically significant excess of trauma in cases before and during military service” [186].

Alzheimer’s Disease Alzheimer’s disease has been described as occurring following head trauma [187].

Parkinson’s Disease Parkinson’s disease has also been described in some patients following head trauma, and also there is a curious history of Parkinson’s disease following electrical injuries [164].

Guillain–Barré Syndrome Although this is a demyelinating disease of peripheral nerves, many patients have developed Guillain–Barré following trauma [188]. While these conditions may occur after head injury, the relationship to trauma is particularly apt and noticeable in childhood ataxia with central hypomyelination and in familial hemiplegic migraine. The other illnesses are mentioned entirely for completion.

Conclusions This review highlights the clinical features noted by distinguished physicians of the occurrence of clinical MS or its exacerbation following trauma, particularly of the cervical cord. The arguments denying such an association are flawed and based on

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unproven tenets—that MS is an autoimmune disease and that T cells are involved in demyelination. All the patients in Sibley’s study already suffered from MS, which means that this would exclude any patient who had symptoms first appearing following the incident of trauma, and secondly lesions already present might be very well obscured, signaling the effect of new lesions. The authors did not discuss the crucial significance of BBB breakdown in the pathogenesis of the disease; where they referred to it, they assumed the veracity of the autoimmune theory. The authors further did not examine critically the effects of electrical injuries, which their own study found to be highly significant. Strangely, it is stated that “it is most unlikely that trauma is a primary cause of MS, but it could be a triggering factor” [29]. Patients we described [113] had the specific whiplash type injury with clear-cut symptoms of spinal cord involvement [113]. In our large series of over 100 cases, several of the patients who developed MS following trauma were over 60 years of age, a time when one would not expect the occurrence of new disease. It is clear that trauma has a relationship in the development of MS, and further studies of these cases may not only help elucidate mechanisms but may also serve as a basis for certain forms of therapy and better clinical management.

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[157] Brodersen P, Paulson O, Bolwig T, Rogon Z, Rafaelsen O, Lassen N. Arch Neurol 1973;28:334. [158] Lee J, Olszewski J. Neurology 1961;11:515. [159] Bolwig T. Acta Psychiatr Scand Suppl 1988;345:15. [160] Mander A, Whitfield A, Kean D, Smith M, Douglas R, Kendell R. Br J Psychiatry 1987;151:69. [161] Coffey C, Weiner W, Mccall W, Heinz E. J ECT 1987;3:137. [162] Grossman R, Joseph P, Wolf G, Biery D, Mcgrath J, Kundel H, et  al. Radiology 1985;155:649. [163] Gallagher J, Sanders M. Acta Neurol Scand 1987;75:145. [164] Quinn N, Maraganore D. Mov Disord 2000;15:587. [165] Bourdillon R, Fischer-Williams M, Smith H, Taylor K. J Neurol Neurosurg Psychiatr 1957;20:79. [166] Smith H, Hughes I, Hunter G. Br Med J 1961;24:101. [167] Marshall J, O'Grady F. J Neurol Neurosurg Psychiatr 1959;22:277. [168] Kelly R, Jellinek E. Br Med J 1961;2:421. [169] Miller H, Newell DJ, Ridley A, Schapira K. J Neurol Neurosurg Psychiatry 1961;24:118–20. [170] Oldendorf WHMD, Cornpord EMPD. J Neuropathol Exp Neurol 1977;36:50–61. [171] Aarli J, Mørk S, Myrseth E, Larsen J. Eur Neurol 1989;29:312–6. [172] Tourtellotte W, Potvin A, Baumhefner R, Potvin J, Ma B, Syndulko K, et  al. Arch Neurol 1980;37:620. [173] Cook S, Troiano R, Zito G, Lavenhar M, Devereux C, Hafstein M, et  al. Lancet 1986;327:1405–9. [174] Cook S, Devereux C, Troiano R, Zito G, Hafstein M, Labenhar M, et al. Ann Neurol 1987;22:634–8. [175] Devereux C, Vidaver R, Hafstein M, Zito G, Troiano R, Dowling P, et al. Int J Radiat Oncol Biol Phys 1988;14:197–203. [176] Means E, Barron K, Van Dyne B. Neurology 1973;23:881. [177] Remes-Troche J, Téllez-Zenteno J, Rojas-Serrano J, Senties-Madrid H, Vega-Boada F, García-Ramos G. Eur Neurol 2003;49:188. [178] Exacerbations of multiple sclerosis following wasp stings (Letter) Mayo Clinical Proceedings. Mar. 2000. 75(3):317–318. [179] Heffner Jr R, Porro R, Olson M, Earle K. Arch Neurol 1976;33:501. [180] Hanefeld F, Holzbach U, Kruse B, Wilichowski E, Christen H, Frahm J. Neuropediatrics 1993;24:244. [181] Prass K, Brück W, Schröder N, Bender A, Prass M, Wolf T, et  al. Ann Neurol 2001;50:665–8. [182] Knaap MS, Leegwater PAJ, Könst AAM, Visser A, Naidu S, Oudejans CBM, et  al. Ann Neurol 2002;51:264–70. [183] Van der Knaap M, Barth P, Gabreels F, Franzoni E, Begeer J, Stroink H, et  al. Neurology 1997;48:845–55. [184] Leegwater PAJ, Vermeulen G, Könst AAM, Naidu S, Mulders J, Visser A, et  al. Nat Genet 2001;29:383–8. [185] Jafari H, Couratier P, Camu W. J Neurol Neurosurg Psychiatr 2001;71:265. [186] Kurtzke J, Beebe G. Neurology 1980;30:453. [187] Nicoll JA, Roberts GW, Graham DI. Ann N Y Acad Sci 1996;777:271–5. [188] Arnason B, Asbury A. Arch Neurol 1968;18:500. [189] Kinnier Wilson SA. Aetiology In: Neurology. 2nd ed, Vol. 1, Butterworth: London; 1954, p.175.

8 Clinical Development and

Benefit–Risk Profile of Natalizumab Eileen M. O’Connor, Rae Ann Maxwell Medical Science Liaison, Biogen Idec Neurology, US Medical Affairs

Introduction Natalizumab (Tysabri™) is a humanized anti-integrin monoclonal antibody used for treatment of multiple sclerosis (MS) [1,57] and Crohn’s disease [2]. Natalizumab is a selective adhesion molecule (SAM) inhibitor therapy targeting the α-4 component of α-integrins. Integrins are one group of adhesion molecules that are involved in adhesion of activated leukocytes to underlying inflamed endothelium and consist of leukocytes function-associated integrin type-1 (VLA-4, α4β1, and α4β7).

Mechanism of Action During the inflammatory cascade of MS, and upon exposure to inflammatory cytokines such as tumor necrosis factor (TNF)-α, the expression of adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and intercellular adhesion molecule 1 (ICAM-1) is upregulated on the surface of cerebral endothelial cells [3,4]. The ligands for these adhesion molecules, which exist on the surface of activated leukocytes, are VLA-4 and ICAM-1/ICAM-2. Interaction between these adhesion molecules and their ligands plays a critical role in transendothelial migration of activated leukocytes. Since adhesion of leukocytes to endothelial cells is a required step for leukocyte migration, blocking this adhesion may suppress the inflammatory cascade significantly. The concept of using monoclonal antibodies against VLA-4 to block its interaction with VCAM-1 originated from the seminal experiment of Yednock et al. [5] on mice with experimental allergic encephalomyelitis (EAE). These investigators treated their EAE mice model with an anti–VLA-4 monoclonal antibody and observed that such treatment was associated with a dramatic decrease in accumulation of activated leukocytes within the central nervous system (CNS). Various anti–α-4-integrin monoclonal antibodies have been tested in animals with various immune-mediated inflammatory disorders [6], and of these only two have progressed to human clinical trials: anti-α4β1 monoclonal antibody for MS and antiα4β7 antibody for Crohn’s disease. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00008-3 © 2011 Elsevier Inc. All rights reserved.

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Mean concentration (mcg/mL)

60 50 40 30 20 10 0

0

1

2

3

4

5

6

7

8

9

Time (weeks)

Figure 8.1  Comparison of serum concentrations of natalizumab determined for 9 weeks after single IV infusion of 1 mg/kg () compared with 3 mg/kg ().

Pharmacokinetics and Pharmacodynamics A phase I, randomized, placebo-controlled, single intravenous (IV), five-dose escalation trial (0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg infused over 45 min) was conducted in 28 relapsing–remitting (RR) MS, secondary progressive (SP) MS patients to evaluate the safety and tolerability of natalizumab [7]. All doses were safe and well tolerated, with headache being the primary adverse event, followed by nausea, shakiness, and urticaria. All adverse events were self-limiting, with no patients withdrawing from the trial and no serious adverse events reported. Pharmacokinetic evaluation of serum natalizumab concentrations revealed a bi-exponential drug-concentration time curve, with a rapid distribution phase followed by a prolonged terminal phase. Doses of 0.03–3.0 mg/kg produced serum concentrations that rapidly fell below detectable limits of the enzyme-linked immunosorbent assay (ELISA) (50–2000 pg/mL) after the 45-min infusion. The 0.3-mg/kg dose produced sustainable natalizumab serum concentrations for 1 week, while the 1.0- and 3.0-mg/kg doses produced serum concentrations for 3–8 weeks [7] (Figure 8.1). The terminal half-life, based on the 3-mg/kg group, was 4–5 days [7]. Another placebo-controlled, dose escalation phase I trial, in 39 RRMS, SPMS patients, further evaluated higher doses, including 1, 3, and 6 mg/kg [8], with similar pharmacokinetic results. Since the pharmacokinetic and pharmacodynamic profiles of monoclonal antibodies may demonstrate substantial variability, evaluation of the α-4-integrin receptor saturation was further elucidated. Twenty-four hours after infusion of a single IV dose of natalizumab (1–6 mg/kg) has been found to produce maximal (defined as .80%) saturation of surface α-4 receptors on peripheral blood leukocytes [8]. Receptor saturation was maintained for 1, 3–4, and 6 weeks with respective natalizumab doses of 1, 3, and 6 mg/kg, with approximately 90% of the patients achieving maximal saturation after the two higher doses (3 and 6 mg/kg) [8]. The current dose of natalizumab that is commercially available for treatment is a single 300-mg dose administered IV once every 28 days.

Clinical Development and Benefit–Risk Profile of Natalizumab

169

GLANCE Study design Randomization (1:1) baseline

Extension protocol 1808

Treatment phase

Screening

GA 20 mg SC once daily + natalizumab 300 mg IV every 4 weeks

Open-label natalizumab

GA 20 mg SC once daily + placebo IV every 4 weeks

Week –4

0

• *

4

8

*

*

√ √ √ √ √

* MRI and antibody testing

12

• *

√ √ PK

16

20

*

*

24

• *

√

• EDSS

Figure 8.2  GLANCE Study design.

Clinical Development The phase II program for natalizumab began in 1999 with 213 patients enrolled from 26 centers in the United States, Canada, and the United Kingdom. Patients with RRMS and SPMS were eligible if they had experienced at least two relapses within the previous 2 years, had an Expanded Disability Status Scale (EDSS) score between 2 and 6.5, and had a minimum of three T2 lesions as determined by magnetic resonance imaging (MRI). This placebo-controlled dose-finding study used two weight-based regimens, 3 mg/kg or 6 mg/kg, which were infused IV every 28 days for 6 months. Patients were monitored for adverse events for an additional 6 months. Miller et al. [1] reported that the primary endpoint of the mean number of new gadolinium-enhancing lesions at 6 months was significantly different between the placebo and both natalizumab groups (P  ,  0.001). However, no significant difference was observed between the 3 and 6-mg/kg groups. Further, a decrease in the mean number of new lesions of approximately 90% was observed in both natalizumab groups compared to placebo, an effect that was observed after the first infusion and sustained throughout the 6-month treatment period. Adverse events reported were similar in all groups. GLANCE was a phase II, randomized, double-blind, placebo-controlled trial in patients who had been treated with glatiramer acetate (GA) for at least 1 year to determine the safety and tolerability of GA in combination with natalizumab over a 6-month period (Figure 8.2). Because the proposed mechanism of action of GA requires cellular entry into the brain, α-4-integrin blockade by natalizumab was hypothesized to impair, rather than enhance, the efficacy of GA. The combination of natalizumab and GA appeared to be safe and well tolerated [9]. The AFFIRM [10] and SENTINEL [11] trials were the pivotal phase III clinical trials that examined the efficacy of natalizumab in MS patients. AFFIRM and

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SENTINEL are two of the largest pivotal RRMS trials to date and were designed as randomized, double-blind, placebo-controlled and, parallel group trials. AFFIRM, the monotherapy trial, examined the effect of treatment with natalizumab 300 mg IV every 28 days versus placebo in terms of the annualized relapse rate (ARR) (primary endpoint year 1) and the cumulative probability of disability progression (primary endpoint year 2). SENTINEL, the add-on trial, was conducted to determine the benefit of natalizumab on the aforementioned primary endpoints when added to interferon beta-1a 30 µg (IFN-β1a) given intramuscularly (IM) once a week. A variety of MRI parameters were measured as secondary endpoints in both of the phase III trials: new or enlarging hyperintense T2 lesions, number of gadolinium-enhancing (Gd) lesions, number of new T1-hypointense lesions, as well as T1 and T2 lesion volume. AFFIRM and SENTINEL were the first phase III MS clinical trials to explore the effect of natalizumab on functional measurements that frequently affect MS patients. These prespecified tertiary endpoints included several measurements of quality of life (QoL) as well as visual function. Since the use of natalizumab in clinical practice is different from the population initially studied in AFFIRM, a post marketing phase IV study was designed to determine natalizumab’s efficacy in the MS population in which it is used. The Tysabri Observational Program (TOP) is an open-label, observational study in approximately 3000 patients naïve to natalizumab treatment. It is powered to determine the efficacy of natalizumab in clinical practice as measured by EDSS and ARR over 5 years [12]. Patients in the AFFIRM trial were randomly assigned in a 2:1 ratio to receive either natalizumab or placebo (Figure 8.3). Patients in the SENTINEL trial were randomly assigned in a 1:1 ratio to receive either natalizumab plus IFN-β1a or IFNβ1a plus placebo. Natalizumab was dosed as 300 mg IV every 4 weeks for up to 116 weeks in both the AFFIRM and SENTINEL trials. In the SENTINEL trial, patients received 30 μg IFN-β1a IM once weekly for up to 116 weeks. Inclusion and exclusion criteria for AFFIRM and SENTINEL are listed in Table 8.1.

Efficacy Traditionally, efficacy for MS therapies is measured by reduction in relapses over a year, various MRI indices (e.g., number of Gd, T2 lesions), and evaluation of disease progression as a change in EDSS by a one-point increase in score maintained for a specific time period (e.g., 12 or 24 weeks). In clinical trials with natalizumab, additional nontraditional measures such as QoL using a visual analog scale (VAS) and Short-Form 36 (SF-36), Multiple Sclerosis Functional Composite (MSFC), as well as visual changes were evaluated as either secondary or tertiary endpoints to further elucidate other clinical manifestations of the disease and further evaluate the impact of natalizumab on MS beyond the gold-standard measures (e.g., relapses, MRI). Based on the positive findings of AFFIRM, several additional post hoc analyses were performed using concepts from other immune-mediated disease states (e.g., rheumatoid arthritis) to further define treatment impact on MS patients.

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AFFIRM study design Randomization (2:1) baseline Extension protocol 1808

Treatment phase Screening

Natalizumab 300 mg IV infusion every 4 weeks to week 116

Open-label natalizumab

Placebo IV infusion every 4 weeks

Week –5 0

• * * √

24

*

• *

48

*

*

• √

* EDSS and MSFC ∆For

72

*

*

√ MRI

96

*

*

• √

120 Completion week 128∆

*

*

• MSQLI/SF-36

those not entering extension.

Figure 8.3  AFFIRM Study design. Table 8.1  AFFIRM and SENTINEL Inclusion and Exclusion Criteria Inclusion

Exclusion

18–50 years of age (AFFIRM) 18–55 years of age (SENTINEL)

Primary progressive MS, secondary progressive MS, or relapsing progressive MS

EDSS 0–5.0

Relapse within 50 days before randomization

MRI lesions consistent with MS

Treatment with interferon beta, cyclophosphamide, or mitoxantrone within the previous 12 months or GA, cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, or IV immune globulin within the previous 6 months

>1 medically documented relapse within the 12 months prior to study entry

Treatment with any other interferon product besides IFN-β1a IM in the prior 12 monthsa

Relapse while on interferon IFN-β1a IM for at least 12 monthsa a

SENTINEL only [10,11].

These additional measures include sustained improvement, defined as a reduction in EDSS score by one point sustained for 12 or 24 weeks, and a composite of several traditional measures (relapses, disease progression, MRI indices) to frame the concept of freedom of disease activity. In addition, several phase IV trials have been

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conducted to evaluate changes in cognition and fatigue, stemming from anecdotal reports of improvement in these areas by patients and their physicians. Various evaluations of subsets of patients were analyzed (e.g., disease activity at baseline [highly active], ethnicity, and other predefined demographic baseline parameters) to obtain a better understanding of what type of MS patient could most benefit from natalizumab therapy. All of these studies and analyses were done to gain better insight into how future therapies could work in MS patients, as well as to treat the entire patient and those various signs and symptoms she or he suffers from, which to date have been virtually left untreated or routinely monitored for improvement in the clinical setting. Natalizumab had a robust effect on all standard measures of MS treatment efficacy in both pivotal trials. In AFFIRM (monotherapy study), the primary endpoint at year 1 was ARR. The natalizumab patients experienced a relative reduction of 68% that was maintained over 2 years of the trial (Table 8.2) [10]—a first for any MS therapy. At 2 years, there was a 42% reduction in the risk of sustained progression of disability (P , 0.001) in the natalizumab group compared to placebo (Figure 8.4) [10]. Additional sensitivity analysis for risk of sustained progression at 6 months resulted in a 54% reduction compared to placebo (P , 0.001). In SENTINEL (addon study), the relative reduction in relapse rate was 54% (P , 0.001) at year 1, and it was maintained at year 2 in the add-on treatment group. In addition, the risk of sustained progression of disability was reduced by 24% (P    0.02) in the add-on treatment group [11]. Measurements of new or enlarging MRI lesions, Gd or T2 weighted, were reduced by 92% and 83%, respectively, compared to placebo. Interestingly, 97% and 57% of natalizumab-treated patients experienced no new or enlarging Gd or T2 lesions, respectively, at the end of the 2-year trial (see Table 8.2) [10]. Also, there was a 76% relative reduction in the mean number of T1-hypointense lesions over the 2 years with Tysabri (Tysabri 1.1 lesions versus placebo 4.6 lesions; P , 0.001) [13]. In SENTINEL, the add-on treatment group experienced an 83% reduction in T2 lesions (P , 0.001) and an 89% reduction in Gd lesions (P , 0.001) at 2 years compared to placebo [11].

Subgroup Analysis Data It is well accepted that MS is a heterogeneous disease and that patients may respond differently to various DMTs. Differences in response may be a factor of gender, ethnicity, or level of disease activity. To explore the effect of natalizumab in these populations, several prespecified analyses were conducted.

Highly Active Patients Patients with highly active relapsing MS experience more relapses and are more likely to become disabled sooner due to the accumulation of disease burden being

Table 8.2  Endpoints as Determined by Clinical Results and MRI Evaluationa 1 Year Natalizumab (n  627)

Placebo (n  315)

2 Years P-value

Natalizumab (n  627)

Placebo (n  315)

P-value

17

29

<0.001c

Clinical Primary endpoint at 2 years: cumulative probability — of sustained disability progression—%b

—

Primary endpoint at 1 year: annualized relapse rate—mean (95% CI)d  Preplanned interim analysis (after 900 patient-years)

0.26 (0.21–0.32)

0.81 (0.67–0.97)

,0.001

—

—

  Final analysis

0.27 (0.21–0.33)

0.78 (0.64–0.94)

,0.001

0.23 (0.19–0.28)

0.73 (0.62–0.87)

  0

501 (80)

189 (60)

454 (72)

146 (46)

  1

106 (17)

76 (24)

123 (20)

65 (21)

  2

17 (3)

36 (11)

36 (6)

63 (20)

  >3

3 (<1)

14 (4)

14 (2)

41 (13)

  Adjusted annualized relapse rate

—

—

0.24

0.75

,0.001

  Unadjusted annualized relapse rate

—

—

0.22

0.64

,0.001

—

—

0.22

0.67

,0.001

,0.001

Number of relapses—no. of patients (%)

Clinical Development and Benefit–Risk Profile of Natalizumab

Endpoint

Sensitivity analysise

f

  Mean relapse rate per patient MRI

0–1 Year

0–2 Years <0.001

,0.001

  0

382 (61)

72 (23)

360 (57)

46 (15)

  1

112 (18)

41 (13)

106 (17)

32 (10)

173

Number of new or enlarging T2-hyperintense lesions—no. of patients (%)

Table 8.2  (Continued) 1 Year Natalizumab (n  627)

Placebo (n  315)

  2

40 (6)

  >3

93 (15)

2 Years Natalizumab (n  627)

Placebo (n  315)

23 (7)

48 (8)

24 (8)

179 (57)

113 (18)

213 (68)

P-value

Number of new or enlarging T2-hyperintense lesions   Mean 1.24.7   Median 0

6.19.0

1.99.2

11.015.7

3.0

0

5.0

  Minimum, maximum

0, 77

0, 196

0, 91

0, 98 At 1 year

P-value

At 2 years

Number of gadolinium-enhancing lesions—no. of patients (%)   0 605 (96)

213 (68)

608 (97)

227 (72)

  1

17 (3)

42 (13)

12 (2)

39 (12)

  2

3 (,1)

15 (5)

1 (,1)

9 (3)

  >3

2 (<,1)

45 (14)

6 (,1)

40 (13)

,0.001

,0.001

0.11.3

1.33.2

0.11.4

1.23.9

  Median

0

0

0

0

  Minimum, maximum

0, 32

0, 33

0, 32

0, 48

 Plus–minus values are meanSD; CI, confidence interval.  Sustained disability progression was defined as an increase of 1.0 point or more in scores on the EDSS from baseline score of 1.0 or more or an increase of 1.5 points or more from a baseline score of 0 that was sustained for 12 weeks. c  The hazard ratio for sustained progression of disability in the natalizumab group as compared with the placebo group was 0.58 (95% CI, 0.43–0.77). d  Relapses that occurred after sustained progression of disability was reached and rescue treatment was initiated (per protocol) were censored. e  Analysis includes relapses that occurred after sustained progression was reached and rescue treatment was initiated (per protocol). f  The mean relapse rate per patient is the number of relapses for each patient divided by the total of number of years of follow-up. b

Neuroinflammation

Number of gadolinium-enhancing lesions   Mean

a

174

Endpoint

Clinical Development and Benefit–Risk Profile of Natalizumab

175

Proportion of patients with sustained progression of disability

0.4 P < 0.001 0.3

Placebo

0.2

Natalizumab

0.1

0.0

No. at risk Placebo Natalizumab

0

12

24

36

48

315 627

296 601

283 582

264 567

248 546

60 72 Weeks 240 525

229 517

84

96

108

120

216 503

208 490

200 478

199 473

Figure 8.4  Kaplan–Meier plots of the time to sustained progression of disability among patients receiving natalizumab, as compared with placebo. Natalizumab reduced the risk of sustained progression of disability by 42% over 2 years (hazard ratio, 0.58; 95% confidence interval, 0.43–0.77). The cumulative probability of progression was 17% in the natalizumab group and 29% in the placebo group.

greater than those with typical relapsing MS. A subgroup analysis was conducted in those patients from the AFFIRM and SENTINEL trials who had highly active disease, as defined by two or more relapses and one or more Gd lesions at baseline [14]. A total of 209 patients from AFFIRM (n  148 natalizumab, n  61 placebo) were evaluated for reduction in relapse rate and disability progression. Natalizumab reduced the 2-year ARR by 81% compared to placebo (0.28 versus 1.46, respectively; P  ,  0.001) and reduced the risk of disability progression sustained for 12 and 24 weeks by 53% and 64%, respectively, compared to placebo. These data suggest that natalizumab has a robust effect on patients who may have a more insidious course of disease. For SENTINEL, 169 patients met the criteria for highly active disease (n  74 INF-β1a plus natalizumab, n  95 INF-β1a alone). The addition of natalizumab to INF-β1a produced a 76% reduction in the 2-year ARR compared to INF-β1a alone. Add-on therapy reduced the risk of disability progression for 12 and 24 weeks by 61% and 58%, respectively.

Baseline Demographic Subgroup Hutchinson et al. [15] published the prespecified subgroup analyses from both of the phase III trials, which examined the effect of natalizumab, compared to placebo,

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according to baseline characteristics: relapse history 1 year prior to randomization (1, 2, or >3), EDSS score (<3.5 or .3.5), number of T2 lesions (,9 or >9), presence of Gd lesions (0 or >1), age (40 or >40), and gender, in addition to patients with highly active disease (>2 relapses in the year prior to study entry and >1Gdlesion at study entry). The ARR was reduced in all subgroups in both AFFIRM and SENTINEL, with the exception of the patients who had less than nine T2 lesions at baseline, likely due to the small number of patients in this subgroup. The risk of sustained disability progression was significantly reduced in most prespecified subgroups in AFFIRM and in SENTINEL in patients with nine or more T2 lesions at baseline, at least one Gd lesions at baseline, females, and patients under 40 years of age [15]. These data confirmed the original findings: natalizumab demonstrated consistent efficacy across all subgroups, including patients who had active disease at time of treatment initiation. Considering natalizumab’s mechanism of action, these results may indicate that natalizumab may be useful early in the disease, when inflammation is the critical component to disability progression.

African Americans There are data to suggest that patients of African descent with relapsing MS may not respond to interferon therapy with the same magnitude as Caucasian patients, in addition to sustaining more disease activity and more rapid disease progression [16]. A post hoc analysis was conducted on data from AFFIRM (monotherapy) and SENTINEL (add-on), including the placebo arms of both trials (combined groups) of patients who indicated “Black” on their screening form [55]. There were a total of 49 patients who met these criteria for further analysis, 10 from AFFIRM (4 in the natalizumab group, 6 in the placebo group) and 39 from SENTINEL (17 in the group taking INF-β1a plus natalizumab, 22 taking INF-β1a alone). Despite the small number of patients, there was a trend toward a lower number of relapses in the natalizumab group compared to placebo (1.38  0.67 versus 1.82  0.82). Also, over the 2 years of the trials, the ARR was reduced by 60% in the natalizumab group versus placebo. The mean number of new or enlarging T2 lesions and Gd lesions was significantly reduced by 90% and 79%, respectively, in the natalizumab group over the 2 years of the trial.

Novel Measures of Efficacy Multiple Sclerosis Functional Composite The MSFC was examined as a secondary endpoint to evaluate a new method of measuring disability. Most clinicians agree that the EDSS is limited because it relies predominantly on assessing lower limb ambulation disability. The MSFC incorporates quantitative measures of three components expressed as a composite Z-score.

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Ambulation was measured using the timed 25-foot walk (T25-FW); the nine-hole peg test (9-HPT) was used to measure upper extremity function; and the 3-second paced auditory serial addition test (PASAT-3) was used to measure cognition. Natalizumab-treated patients demonstrated a statistically significant improvement in MSFC scores at each time point over 2 years (P  ,  0.001). Each component of the MSFC was also statistically significantly different from placebo (T25-FW, P , 0.001; 9-HPT, P , 0.001; PASAT-3, P , 0.005) [17]. Rudick et al. [18] separately examined MSFC progression as defined by worsening from baseline on scores of at least one MSFC component by 20% (MSFC Progression-20) or 15% (MSFC Progression-15) for at least 3 months [18]. In these analyses, a greater proportion of placebo patients exhibited progression as measured by the MSFC Progression-15 and MSFC Progression-20 sustained for at least 3 months over 2 years of the trial than did natalizumab-treated patients. MSFC progression was most commonly driven by T25-FW in both the placebo and natalizumab groups.

Health-Related QoL A variety of health-related QoL measures were included in both AFFIRM and SENTINEL as tertiary endpoints to further elucidate other signs and symptoms that may affect patients with MS, beyond ambulation. These included the Multiple Sclerosis Quality of Life Index (MSQLI), developed by the Consortium of Multiple Sclerosis Centers’ Health Service Research Subcommittee, and VAS [19]. One component of the MSQLI includes the SF-36, one of the most widely used generic health status measures; it is used in a variety of chronic disease states [20]. The SF-36 contains 36 items that assess patients’ health status, and its impact on their daily lives and consists of two components: physical (PCS) and mental (MCS) [21]. The VAS was used to confirm the SF-36 results. The VAS consists of a single-item subjective global assessment of wellbeing that has a scale labeled “poor” (0) on one end and “excellent” (100) at the other; the patient draws a vertical line on the scale to indicate how he or she feels at that time [19]. Additional factors that may affect QoL were also evaluated, including changes in vision, pain, and cognition as measured by the PASAT portion of the MSFC. The primary objective of this analysis was to determine the effect from natalizumab monotherapy (AFFIRM) or add-on therapy with IFN-β1a (SENTINEL) on health-related QoL over 2 years [19]. Assessments were completed at baseline and at 24, 52, and 104 weeks. All health-related QoL outcomes were reported as mean scores and mean change from baseline for both SF-36 scales and VAS. Results were calculated for all time points. At baseline, the combined mean PCS and MCS scores were 43.20.4 and 47.00.5 across both studies. PCS and MCS scores for MS patients were significantly lower than the general US population: 50 is considered normal for both domains [21]. This illustrates that MS is associated with a significant QoL burden due to the disease. Higher EDSS scores were associated with lower PCS scores at baseline. PCS scores for all EDSS scores of 2.0 or more were significantly less in comparison with PCS scores at an EDSS of 0.0 (P , 0.005). By week 104, patients treated with natalizumab had significantly more improvement from

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baseline in both PCS and MCS than the placebo group. The PCS was significantly higher at 24 weeks and remained so through 2 years (P , 0.05) in the natalizumab group, while scores worsened over time for placebo. A clinically important change on the PCS and MCS was defined as at least a 0.5-SD change from baseline to week 104. A higher percentage of natalizumab-treated patients achieved a clinically important change than placebo patients for both PCS (24.9% versus 16.8%) and MCS (28.5% versus 21.6%). It is not surprising that the PCS showed a more robust effect with treatment than the MCS, since substantial physical impairment is the hallmark of MS. The VAS showed similar results: placebo-treated patients showed a worsening (6.2% change from baseline) and natalizumab appeared to keep patients feeling well (0.2% change from baseline) at 104 weeks. Further analyses of these data were conducted by Kieseier et al. [22] to evaluate the effect of natalizumab on health-related QoL of the highly active (HA) patients (n    209; 148 in the natalizumab group and 61 in the placebo group) previously identified in the AFFIRM trial versus the non-HA patients (n    733; 479 in the natalizumab group and 254 in the placebo group). No significant differences were found at baseline between the two subgroups regarding PCS, MCS, or VAS scores. In the HA subgroup, natalizumab significantly improved mean PCS, MCS, and VAS change from baseline to 2 years compared to placebo, and a greater percentage of patients experienced a clinical meaningful improvement with natalizumab over placebo (approximately 30% versus 20%, respectively).

Vision Another facet that may affect an MS patient’s QoL is change in vision over the course of disease. Visual data were collected in both AFFIRM and SENTINEL as assessed by the low-contrast Sloan letter chart (Precision Vision, LaSalle, IL). This testing captures the minimum size at which individuals can perceive letters of a particular contrast level (shade of gray on a white background). Sloan chart testing is the clinical measure that best identifies visual dysfunction in heterogeneous MS cohorts [23], with scores reflecting both vision-specific and overall health-related QoL in MS [24]. The primary evaluation was the change in visual function from baseline to 2 years. Clinically significant visual loss was defined as a two-line worsening of acuity sustained over 12 weeks. In AFFIRM, natalizumab reduced visual loss from baseline to 2 years by 47% (P , 0.001) at the 2.5% contrast level and by 35% (P  0.008) at the lowest contrast level, 1.25%. High contrast acuity (100%) was used to measure visual acuity as a descriptor of study cohorts [25] and was not a sensitive measure of change of visual loss. There were no differences among the treatment groups using 100% contrast letter charts (P  0.29). In SENTINEL, the add-on treatment group experienced a 28% reduction in visual loss as measured by the 1.25% contrast level.

Pain Another aspect of health-related QoL in MS patients is pain that can often interfere with daily routine tasks. One of the tertiary endpoints of AFFIRM and SENTINEL

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trials was measuring and evaluating pain using the Medical Outcomes Study Pain Effects Scale (PES), which is part of the MSQLI. The main objective was to assess the effects of natalizumab on patients’ assessment of pain and to determine if there was a relationship between disease activity and pain in patients with RRMS [26]. Pain was evaluated at four time points during the trials: baseline, 6 months, and 1 and 2 years. Only a subset of patients were available due to limi­tations of valid translations of the assessment tools. A total of 358 patients from AFFIRM and 721 from SENTINEL completed the PES. Higher baseline scores on the PES, indicating worse pain, were associated with higher baseline EDSS scores (more disability). Mean baseline PES scores were significantly higher in patients with an EDSS score of at least 2.0 (AFFIRM) or at least 2.5 (SENTINEL) compared to patients with an EDSS score of 0 (P < 0.017 in both trials). In AFFIRM, mean PES scores improved from baseline in patients treated with natalizumab and worsened in the placebo group over the 2 years (0.53 versus 0.42, P  0.332). Baseline PES scores and EDSS scores were significantly associated with change in PES score over 2 years (P , 0.001). Patients with at least two relapses or disability progression had significantly higher mean PES scores compared to patients with no relapses or disability progression over 2 years. Patients in SENTINEL had mean PES scores that improved from baseline when treated with natalizumab plus IFNβ-1a and worsened in those treated with IFN-β1a alone (0.85 versus 0.26, P , 0.001). Baseline PES score, EDSS score, and patient age were significantly associated with change in PES score over 2 years (P < 0.037). Natalizumab treatment resulted in significant improvements in PES scores in patients with higher levels of pain and disability at baseline. These data complement the findings from the generic SF-36 and VAS in which patients treated with natalizumab felt better physically and had a better sense of well-being, which in part could be attributed to having less pain.

Fatigue A common and disabling symptom of MS that has lacked improvement with current therapies is fatigue. A variety of ancillary medications have been prescribed to patients with MS to help reduce fatigue, such as modafinil and methylphenidate in conjunction with platform therapies, with limited improvement. Two trials with natalizumab have been conducted to determine if it has an effect on fatigue. The first, by Putzki et al. [27], evaluated 42 patients in a prospective, open-label, uncontrolled, observational study. Fatigue was measured using two scales. In the 21-question Modified Fatigue Impact Scale (MFIS) (score range 0–84), lower total scores indicate lower impact of fatigue. The Fatigue Severity Scale (FSS), another validated scale, contains nine statements concerning the severity, impact, and frequency of fatigue on daily living, with a scale of 1–7; higher scores indicate more impact of fatigue. Both scales were administered prior to starting natalizumab and 3 and 6 months after therapy. The VAS was also administered. The baseline total score for MFIS, 45.8    17.5, decreased to 40.1    18.0 (P  ,  0.01) after the sixth infusion. The mean FSS at baseline, 5.2    1.4, decreased to 4.6    1.9 (P    0.01). These results suggested that natalizumab had an effect on fatigue in patients who had failed

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to respond to platform therapies, and that it was measurable 6 months after initiation of therapy. The ENER-G trial [28] is an ongoing multicenter, open-label, 12-month study that evaluated fatigue and cognition at baseline and at 4, 8, 12, 24, and 48 weeks after initiation of natalizumab therapy. Scales and tools used are the VAS-F (a visual analog scale for fatigue), MFIS, FSS, and an Automated Neuropsychology Assessment Metrics (ANAM) to measure cognition, which is a tertiary endpoint. The trial was designed to further evaluate the impact of natalizumab on fatigue and cognition. Preliminary data (44/89 completed patients) indicate that patients show improvement by week 12 on the primary endpoint, change in VAS-F scores from baseline (P , 0.0001), as well as improvement on FSS and total MFIS scores from baseline (P , 0.0001).

Post hoc Analyses Due to the consistent and highly efficacious results that were found in the natalizumab phase III clinical trials, several post hoc analyses were designed and conducted using novel endpoints, such as sustained improvement of physical disability and freedom from disease activity, that heretofore have not been a possibility with previous MS DMTs. Munschauer et al. [29] explored the effect of natalizumab monotherapy on sustained improvement of physical disability, as measured by EDSS, and Havrdova et al. [30] investigated whether disease remission may be an attainable goal in patients treated with natalizumab.

Sustained Improvement The idea to define sustained improvement was derived from anecdotal reports of patients who previously walked with a cane and were able to walk distances without one after being treated with natalizumab. How could you measure improvement in a disease where the standard is to measure disease progression? The same principles were applied: there had to be a sustained improvement of 1 point in EDSS for 12 or 24 weeks in patients who had an EDSS of at least 2. Analyses revealed that natalizumab significantly increased the cumulative probability of a 1-point change in disability sustained for 12 weeks by 69% relative to placebo (P  0.006) (Figure 8.5) [31]. This effect was even more robust in HA patients (at least two relapses in the year prior to study entry and at least one Gd lesion at study entry): natalizumab increased this same measurement by 143% relative to placebo (P  0.045) [29].

Disease-Free The concept of “disease-free” or “freedom of new disease activity” is derived from literature of other immune-mediated disease states such as rheumatoid arthritis, psoriasis, and Crohn’s disease, where the goal is to have full remission of disease activity in patients. Based on the data from AFFIRM, investigators speculated that natalizumab may actually be the first MS therapy to achieve such results. A critical factor

Clinical Development and Benefit–Risk Profile of Natalizumab 0.50

Cumulative probability of sustained improvement

0.45

181

Adjusted HR = 1.69 (95% Cl: 1.16, 2.45) P < 0.006

0.40 Natalizumab 29.6%

0.35 0.30 0.25

Placebo 18.7%

0.20 0.15 0.10 0.05 0.00

0 No. of patients at risk Placebo Natalizumab

12

203 417

24

36

186 362

48 60 72 Weeks from baseline

84

166 317

156 297

96

108

120

145 279

Cl = confidence interval; EDSS = Expanded Disability Status Scale; HR = hazard ratio

Figure 8.5  Cumulative probability of a 1.0-point decrease in EDSS score sustained for 12 weeks in patients with a baseline EDSS score of >2.0. CI, confidence interval; EDSS, Expanded Disability Status Scale; HR, hazard ratio.

was building the paradigm using not only the standard efficacy measures used in pivo­ tal clinical trials, but also those used routinely by clinics. The objective was to produce a tool that could be integrated into clinical practice to evaluate patients and their response to prescribed therapy, and subsequently signal when a change was potentially warranted. Havrdova et al. [30] determined the effects of natalizumab on the proportion of patients who were free of MS disease activity over 2 years compared to placebo from AFFIRM. Absence of disease activity was defined as no activity on clinical measures (no relapses and no sustained disability progression), radiologic measures (no Gd lesions and no new or enlarging T2-hyperintense lesions on cranial MRI), or a more stringent definition, a composite of the two [30]. Of the randomized patients in AFFIRM, 917 (206 HA, as defined as at least two relapses and at least one Gd lesion at baseline, 711 non-HA) had data available for the analysis of disease activity. A total of 64% of patients taking natalizumab and 39% of placebo patients were free of clinical disease (P , 0.0001), 58% versus 14% were free of radiologic disease activity (P , 0.0001), and 37% versus 7% met the most stringent criteria, free of both clinical and radiologic disease activity over 2 years (P , 0.0001) (Table 8.3) [30]. The proportion of patients who were free of disease activity on the composite of clinical and radiologic measures in the natalizumab group was greater in the second year than the first year (68% versus 47%), but was similar in the placebo group (13% versus 15%), suggesting that natalizumab is not only effective during early treatment, but that its efficacy may also increase over time [30].

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Table 8.3  Proportions of Patients Without Disease Activity over 2 Years Placebo

Natalizumab

Absolute Difference (95% CI)

No relapse

130/300 (43%)

422/598 (71%)

27.3% (20.6–34.0%)

No progression

205/286 (72%)

486/581 (84%)

120% (5.9–17.9%)

No relapse and no progression

117/301(39%)

383/596 (64%)

25.4% (18.7–32.1%)

No gadoliniumenhancing lesions

164/290 (57%)

553/583 (95%)

38.3% (32.3–44.3%)

No new or enlarged T2 lesions

44/296 (15%)

346/593 (58%)

43.4% (37.7–49.1%)

No gadoliniumenhancing lesions and no new or enlarged T2 lesions

42/296 (14%)

342/593 (58%)

43.5% (37.9–49.1%)

No gadoliniumenhancing lesions, no new or enlarged T2 lesions, and no relapse

25/303 (8%)

244/600 (41%)

32.4% (27.4–37.4%)

No radiologic or clinical activity

22/304 (7%)

220/600 (37%)

29.5% (24.7–34.3%)

P , 0.0001, natalizumab versus placebo, for all individual and combined disease measures.

Real-World Data The question of how a therapy used in a controlled clinical trial setting will perform once available to the general public is a longstanding one in the medical community, and not only subject to MS treatments. However, natalizumab, due to the voluntary withdrawal and indication label revision, has initiated a more critical evaluation. Within AFFIRM, 92% of the patients had not been exposed to a DMT, while the label currently states that natalizumab is generally recommended for patients who are intolerant to a DMT. Due to the risk of progressive multifocal leukoencephalopathy (PML), in most cases natalizumab is initiated after a minimum trial of one interferon product or GA. The question then becomes: Does the level of efficacy seen in the phase III trials translate to the population treated in clinical practice? Several registries and prospective efficacy trials have shown that even in a population that is older, had a longer duration of MS disease, and had a higher EDSS at treatment initiation with natalizumab, the benefit of natalizumab is still robust and consistent with data from the original AFFIRM analysis [32–35]. These data are summarized in Tables 8.4 and 8.5.

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Table 8.4  Demographics of Prospective Efficacy Trials Versus AFFIRM Demographic

AFFIRM [10]

Oturai et al. [33]

Putzki et al. [34,35]

Outteryck et al. [32]

Patients (n)

627

234

97

384

Age (meanSD)

35.68.5

39.5a

36.54.7

38.059.4

Female, n (%)

449 (72)

NA

67 (69.1)

276 (72)

9.5330.8

7a

94

93.8

94.4

2.31.2

4.0a (0–8)c

3.60.8

3.531.68

12, 24

11.3

19.36.1

8.34.6

Duration of disease (meanSD years)

5.0

8

Patients receiving prior MS therapies (%)

8

EDSS (meanSD) No. of infusions (meanSD)

a

b

References [10,32–35]. a Median data. b Months. c Range.

Safety Natalizumab is a humanized monoclonal antibody with a novel mechanism of action. Both of these factors would be expected to influence the safety profile of this product. Subsequently, infusion/hypersensitivity reactions, immunogenicity, rates of infection, malignancies, and impact on germane cell subsets were carefully examined in the phase III and post marketing clinical trials. In the natalizumab phase III clinical program, three cases of PML occurred—two in the MS trials and one in the Crohn’s trial [36]. Details of these cases have been published elsewhere [37–39]. The unexpected occurrence of PML led to the design of several additional clinical studies (dose-suspension study, STRATA and TYGRIS [TYABRI Global Observation Program in Safety]) to study the safety of natalizumab in the post marketing setting after the removal and return of natalizumab to the commercial setting. As a result of the risk of PML, a risk map was also developed and implemented in the United States to ensure restricted distribution of natalizumab to registered prescribers, infusion centers, and pharmacies associated with infusion centers: the TOUCH™ (Tysabri Outreach: Unified Commitment to Health) Prescribing Program. Only patients who meet all of the conditions outlined in TOUCH are enrolled and administered natalizumab. The TOUCH system is designed to minimize morbidity and mortality due to PML via early detection through clinical vigilance. The dose-suspension safety study was conducted to determine whether PML had developed in any other patients who had been treated with natalizumab commercially or in any of the clinical trials for MS, Crohn’s disease, or rheumatoid arthritis [40]. An expert panel evaluated the patient’s clinical history, physical examination,

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Neuroinflammation

Table 8.5  Summary of Relapse and EDSS Data of Prospective Efficacy Trials Versus AFFIRM Measure

AFFIRM [10]

Oturai et al. [33]

Putzki et al. [34,35]

Outteryck et al. [32]

ARR pre-Tysabri (mean)

1.53

2.53

2.3

2.19

ARR post-Tysabri (mean)

0.22

0.68

0.17

0.59

% Reduction

85.6

73.0

92.6

73.0

Patients who are relapsefree (%)

80 at 1 year 72 at 2 years

NA

80.4

60

EDSS pre-Tysabri (mean)

2.0

4.0a

3.6

3.53

EDSS post-Tysabri (mean)

NA

NA

3.2

3.02

References [10,32–35]. a Median data.

brain MRI, and cerebrospinal fluid tested for JC virus DNA to confirm possible cases of PML. STRATA is an open-label, multinational study that enrolled a subset of over 1000 patients who had completed AFFIRM, SENTINEL, or GLANCE (feeder studies), as well as the dose-suspension safety study, to determine the long-term safety and efficacy of natalizumab. The US patients were treated for 24–48 weeks with natalizumab and then transitioned into the TOUCH system; some patients were also enrolled in TYGRIS. In other countries, patients were treated for 48 weeks with natalizumab and then transitioned into a 4-year extension study to assess the safety and efficacy of natalizumab. TYGRIS, a global observational study, is the largest long-term safety study in MS conducted to date. TYGRIS will follow approximately 5000 patients over 5 years to evaluate the long-term safety of natalizumab in clinical practice as well as identify any unexpected safety issues. Consistent with natalizumab’s mechanism of action, elevations in lymphocytes, monocytes, eosinophils, and basophils were observed in peripheral blood, with levels returning to baseline levels approximately 16 weeks after treatment discontinuation in the phase III trials. Neutrophil counts were not affected [10,11]. Some level of immunogenicity is to be expected with monoclonal antibody biologics and was minimally observed in natalizumab-treated patients [7,8,41]. Binding antibodies against natalizumab were assessed with the use of ELISA. Positive samples (0.5 μg/mL) were further tested in a flow-cytometry assay to determine whether these antibodies interfered with the binding of natalizumab to α-4 integrin. Results of the functional assay were highly correlated with the results obtained with ELISA. Nine percent of natalizumab patients experienced transient anti-natalizumab antibodies (detectable antibodies at one time point) and 6% experienced persistent anti-natalizumab antibodies (detectable antibodies at two or more time points at least 6 weeks apart) over the 2 years of the trial. Patients with anti-natalizumab antibodies were more likely to

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Table 8.6  Adverse Events (AEs) and Serious Adverse Events (SAEs) from Natalizumab Clinical Trials Event AE/SAE

AFFIRM

STRATA

TYGRIS

TOP

Headache

38%

11%

–

–

Fatigue

27%

8%

–

–

Arthralgia

19%

–

–

–

Nasopharyngitis

32%

19%

–

–

Hypersensitivity

,1%

,1%

,1%

,1%

Infection/infestations

3.2%

2%

1.5%

1.1

  Upper respiratory infections

13%

13%

,1%

–

  Urinary tract infections

20%

11%

,1%

–

Neoplasms

,1%

1%

,1%

,1%

PML

No

Yes

Yes

Yes

AEs from the SENTINEL trial were not included since it did not contain a natalizumab-only group and AEs from IFN-b1a would confound the results reported. SAEs reported in SENTINEL were similar to those reported in AFFIRM. Two cases of PML were reported in SENTINEL [10,43–46].

have infusion-related reactions (defined as any reaction that occurred within 2 h of the infusion) and loss of efficacy (as measured by disease progression [P  <  0.05], relapse rate [P    0.009], and MRI [P  <  0.05], compared to antibody-negative patients) [41]. Infusion reactions and hypersensitivity reactions are expected events with monoclonal antibodies. In the phase III studies, infusion reactions were defined as any event that occurred within 2 h of the start of the infusion. Hypersensitivity reactions were defined as hypersensitivity, allergic reactions, anaphylactic or anaphylactoid reactions, as well as urticaria, hives, or allergic reaction, as reported by the investigator [10]. Interestingly, data from the STRATA study have shown that infusion reactions, hypersensitivity reactions, and persistent antibody positivity occurred most frequently in patients who had received one or two natalizumab infusions prior to re-dosing in STRATA [42]. The most common adverse and serious adverse events from these trials are listed in Table 8.6. In AFFIRM, natalizumab was well tolerated, with the majority of adverse events and serious adverse events similarly reported in both treatment groups. These data from STRATA, TYGRIS, and TOP are consistent with the current safety profile of natalizumab. With the development of new compounds to treat MS comes the possibility of unknown effects on the immune system and the associated risks and sequelae. This was the case with natalizumab and the unexpected serious adverse event of PML. PML is a rare lytic, opportunistic infection of oligodendrocytes and astrocytes, hypothesized to be caused by the reactivation of JC virus, a polyomavirus that typically infects individuals during childhood and adolescence [47]. PML results from a

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Neuroinflammation

convergence of multiple viral, genetic, and immunologic risk factors, many of which are unknown [48–50]. Typically, PML has occurred in individuals who are immunocompromised, predominantly HIV and transplant patients, and it is generally fatal [51]. There are no known interventions that can reliably prevent or adequately treat PML if it occurs [36]. Symptoms at presentation may include cortical symptoms and signs, behavioral and neuropsychological alterations, retrochiasmal visual effects, and hemiparesis that progresses over time. Lesions are generally diffuse and asymmetric, extending homogenously confined to white matter tracks, and they show continuous progression. The incidence of PML in natalizumab-treated patients has been estimated to be 1:1000 from the initial clinical trials [40,52]. Clinical vigilance for new neurologic symptoms suggestive of PML, and new lesions as measured by MRI FLAIR and T2, assist in early detection and possibly improved outcomes. In the post marketing setting, additional cases of PML have been reported, and it has been determined that the risk of PML increases with longer treatment duration. The risk in patients who have received 24–36 doses is generally similar to the rates seen in the clinical trials [56]. There is limited experience beyond 3 years [36,52]. In STRATA, the risk of PML appeared to increase with treatment duration, similar to the other post marketing cases [43]. In addition to duration of natalizumab treatment, the risk of PML is also increased in patients who have been treated with an immunosuppressant (e.g. mitoxantrone, methotrexate, cytoxan, azothiaprine, myclophenolate mofetil, cladribine, rituiximab) prior to receiving natalizumab. This increased risk appears to be independent of natalizumab treatment duration [36]. At first suspicion, holding natalizumab therapy is recommended while PML is ruled out using cerebrospinal fluid JC virus DNA and comparative MRIs, if available. Careful evaluation of the post marketing PML cases has elucidated the following: neurologic deficits evolve over several weeks and can include changes in cognition, behavior, and personality; speech abnormalities, hemiparesis, monoclinic seizures, or visual disturbances may also appear as presenting symptoms. Lesions are often hyperintense on T2-weighted and FLAIR images; they are typically without mass effect or edema, and they generally occur in areas not previously affected by MS [56]. In contrast to PML seen with HIV, 40–50% of natalizumab PML cases had Gd lesions at diagnosis. This finding suggests that inflammatory responses might be present in PML lesions during natalizumab therapy and implies that new contrast-enhancing lesions cannot be assumed to be exacerbations of MS in this setting [56]. Confirmation of PML requires a progressive course, clinically and/ or radiologically, as well as positive cerebrospinal fluid JC virus DNA using quantitative polymerase chain reaction (PCR) analysis. If the initial evaluations for PML are negative but clinical suspicion for PML remains, the physician should continue to hold natalizumab and repeat evaluations [36]. A brain biopsy is considered a definitive diagnosis and was only required in one case [56]. Many of the post marketing PML cases had very low JC virus DNA copies at diagnosis; therefore, using an ultrasensitive quantitative real-time PCR assay is recommended (50 copies/mL or less). Undetectable JC virus by PCR testing does not rule out PML. Early diagnosis is important in limiting the degree of permanent brain damage before immune reconstitution can be accomplished. Accelerating removal of natalizumab using immunoadsorption or plasma exchange (PLEX) has occurred in the majority of cases [56]. The goal is to allow reconstitution

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of the immune system through immune surveillance of the CNS to combat JC virus and decrease JC virus pathology [52]. While PLEX has not been studied in natalizumabtreated PML patients, three sessions of PLEX over 5–8 days were shown to accelerate natalizumab clearance in a study of 12 patients with MS who did not have PML, although in the majority of patients α-4 integrin receptor binding remained high [53]. Generally, natalizumab PML patients have experienced immune reconstitution inflammatory syndrome (IRIS) within days to several weeks after natalizumab is removed from the circulation [56]. IRIS is a consequence of reconstituted leukocyte trafficking into the CNS following the decline of serum concentrations of natalizumab in the setting of PML. IRIS is characterized by a worsening of the patient’s neurologic status after the return of immune function. This clinical decline can lead to serious neurologic complications and/or death if not appropriately monitored and treated [54]. Development of enlarging MRI lesions or increased Gd enhancement on MRI scans supports the diagnosis of IRIS, but these scan changes are not necessary for diagnosis because inflammatory brain responses occur before MRI changes can be detected, and enhancement might be suppressed by corticosteroid use. When natalizumab is removed, resulting in renewed immune surveillance, the inflammatory response is robust. High-dose corticosteroid therapy, typically 1 g IV methylprednisolone daily for 5 days, followed by tapered doses of oral steroids, has been consistently used to treat IRIS and often results in clinical improvement. Since the duration of IRIS is uncertain, repeated courses of IV steroids may be required. The duration and timing of corticosteroid use remains to be refined [52]. Symptoms of CNS inflammation, signs of increased intracranial pressure, gadolinium enhancement surrounding the PML lesion, and new neurologic symptoms should alert health care professionals to the onset of IRIS. Since the immune system is generally intact in patients with MS instead of severely impaired, as in HIV patients, IRIS may be more robust in patients treated with natalizumab [52]. As of the date of this publication, the survival rate of natalizumab-treated PML patients is greater than 75%, considerably different from the survival rate of 30–50% reported in HIV patients [51,52]. Many of the MS PML patients who have survived, however, have severe morbidity and substantial and permanent disability [56]. Factors that may predict mortality include lesion size and location, age at diagnosis, and longer time from first symptoms of PML to diagnosis. Currently there are insufficient data to predict survival outcomes in patients treated with natalizumab who develop PML. Careful assessment, including genetic analyses of virus and host, and long-term monitoring of survivors might help further characterize risks [52].

Summary In November 2004, the treatment landscape for MS changed with the introduction of natalizumab, the first-in-class SAM inhibitor approved for MS. Natalizumab offers patients an alternative treatment option to the gold-standard DMTs currently available: IFN-β1b, IFN-β1a, and GA. Results from all clinical trials evaluating natalizumab have shown a consistently high level of efficacy as measured by traditional

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Neuroinflammation

clinical and radiologic measures (EDSS, ARR, T2 and Gd lesions) as well as novel treatment assessments (visual acuity, MSFC, freedom from disease activity, and sustained disability improvement). The strong and diverse efficacy profile of natalizumab to date has not been shown by other classes of agents used in the treatment of MS; however, the safety issue of PML remains a concern and an active area of research. PML associated with natalizumab treatment appears to be different in presentation and outcome than PML associated with HIV [52]. This could be due to the under­lying intact immune system of MS patients and/or could be a function of natalizumab’s mechanism of action. Research of JC virus and PML continues with the hope of providing insight into risk stratification of PML, determining influential genetic factors, optimizing treatment of IRIS, and identifying factors that may predict PML susceptibility [56]. As new treatment modalities are developed and potentially further alter the immune system via their mechanism of action, the benefit–risk ratios will have to be continually evaluated for each patient. With more therapeutic options becoming available for MS, treatment paradigms will emerge, with natalizumab remaining an integral part in managing the unmet needs of MS patients.

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[29] Munschauer F, Giovannoni G, Lublin F, O’Connor PW, Phillips JT, Polman CH, et al., Sustained improvement in physical disability with natalizumab in patients with relapsing multiple sclerosis. Presented at the 61st Annual Meeting of the American Academy of Neurologists. Seattle, WA; April 30, 2009. [30] Havrdova E, Galleta S, Hutchinson M, Stefoski D, Bates D, Polman CH, et al. Effect of natalizumab on clinical and radiological disease activity in multiple sclerosis: a retrospective analysis of the natalizumab safety and efficacy in relapsing-remitting multiple sclerosis AFFIRM) study. Lancet Neurol 2009;8:254–60. [31] Coyle PK, Jeffery DR. Clinical efficacy and benefit of natalizumab. Mult Scler 2009;15:S7–S15. [32] Outteryck O, Ongagna JC, Zepher H, Fleury MC, Lacour A, Blanc F, et  al. Demographic and clinical characteristics of French patients treated with natalizumab in clinical practice. J Neurol 2009;257:207–11. [33] Oturai AB, Koch-Henriksen N, Petersen T, Jensen PEH, Sellebjerg F, Sorensen PS. Efficacy of natalizumab in multiple sclerosis patients with high disease activity: a Danish nationwide study. Eur J Neurol 2009;16:420–3. [34] Putzki N, Kollia K, Woods S, Igwe E, Diener HC, Limmroth V. Natalizumab is effective as second line therapy in the treatment of relapsing remitting multiple sclerosis. Eur J Neurol 2009;16:424–6. [35] Putzki N, Yaldizli O, Maurer M, Cursiefen S, Kuckert S, Klawe C, et  al. Efficacy of natalizumab in second line therapy of relapsing-remitting multiple sclerosis: results from a multi-center study in German speaking countries. Eur J Neurol 2010;17:31–7. [36] Tysabri™ Prescribing Information. Biogen Idec, Inc. Version 10. July 2010. [37] Langer-Gould A, Atlas S, Bollen A, Pelletier D. Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. N Engl J Med 2005;353:375–81. [38] Kleinschmidt-DeMasters BK, Tyler KL. Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med 2005;353:369–74. [39] Van Assche G, Van Ranst M, Sciot R, Dubois B, Vermeire S, Noman M, et  al. Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn’s disease. N Engl J Med 2005;353:362–8. [40] Yousry TA, Habil M, Major E, Ryschkewitsch C, Fahle G, Fischer S, et al. Evaluation of patients treated with natalizumab for progressive multifocal leukoencephalopathy. N Engl J Med 2006;354:924–33. [41] Calabresi PA, Giovannoni G, Confavreux C, Galetta SL, Havrdova E, Hutchinson M, et  al. The incidence and significance of anti-natalizumab antibodies results from AFFIRM and SENTINEL. Neurology 2007;69:1391–403. [42] Polman CH, Goodman AD, Kappos L, Lublin FD, O’Connor PW, Rudick RA, et al. Safety and effectiveness of natalizumab re-dosing and treatment in the STRATA Study. Presented at the World Congress on Treatment and Research in Multiple Sclerosis. Montreal, Canada; September 18, 2008. [43] Polman CH, Goodman AD, Kappos L, Lublin FD, O’Connor PW, Rudick RA, et al. Efficacy and safety of natalizumab in the STRATA Study. Presented at the 62nd Annual Meeting of the American Academy of Neurology. Toronto, Ontario, Canada; April 15, 2010. [44] O’Connor PW, Polman CH, Goodman AD, Kappos L, Lublin FD, Rudick RA, et al. Efficacy and safety of natalizumab in the STRATA Study. Presented at the 61st Annual Meeting of American Academy of Neurology. Seattle, WA; April 30, 2009.

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9 Remyelination in Multiple Sclerosis Martin Stangel, Refik Pul, Thomas Skripuletz, Corinna Trebst, Elke Voss, Viktoria Gudi Department of Neurology, Hannover Medical School, Hannover, Germany

Introduction Since multiple sclerosis (MS) has a peak onset of disease in early adulthood and therefore contributes to the most significant cause of nontraumatic disability among young adults [1], repair of damaged tissue is warranted. The histopathologic hallmarks of MS include demyelination, inflammation, gliosis, and axonal damage [2] with demyelination being the most prominent feature. Remyelination is the natural repair mechanism of demyelination, and it is proposed that remyelination protects against progressive axonal injury [3] and consequently also diminishes long-term disability in MS patients. Therefore, therapeutic efforts that are aimed at supporting endogenous repair/remyelination mechanisms are clearly needed. Animal models demonstrate that remyelination can be substantial [4]. It is thought that remyelination recapitulates many steps also occurring during physiologic myelin development [5,6]. Although many factors have been identified that drive this process, it is still not well understood why remyelination fails in MS [7]. Remyelination is the result of migration, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) that contact with the axon and finally build myelin sheaths. During development oligodendrocytes express different molecules that can be considered as a marker for different developmental stages of oligodendrocytes (Figure 9.1). The different stages need to be orchestrated by many signals, and failure at any stage probably leads to remyelination failure.

Remyelination in MS Based on postmortem histopathologic studies it is known that remyelination of demyelinated plaques occurs in individuals with MS. Remyelination is particularly associated with early stages of the disease and acute phases, but it can also occur in later and chronic stages of the disease [8,9]. Remyelination can be quite extensive in a subset of patients, but there is a profound diversity in the amount of remyelination between cases. In some cases virtually all lesions showed signs of remyelination, whereas in others only small amounts of remyelination were found [8]. This diverse capacity to form remyelinated plaques did not correlate with clinical subtype, age of disease onset, Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00009-5 © 2011 Elsevier Inc. All rights reserved.

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Precursor

Olig2 PDGFa-R Nestin PSANCAM

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A2B5 PDGFa-R GD3 NG2

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Immature Oligodendrocyte

O4 GD3 NG2

O1 O4 GalC CNP

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O1 O4 GalC CNP MBP PLP MAG MOG

Migration Proliferation Differentiation Myelination

Figure 9.1  Stages and markers characteristic in the development of oligodendroglial lineage cells.

disease course, or gender [8,10]. In the majority of patients, a heterogeneous pattern of remyelination capacity of individual lesions was observed. Also, the location of a lesion influences the likelihood of remyelination, with subcortical lesions showing more signs of remyelination than periventricular or cerebellar lesions. Overall, the extent of remyelination in chronic lesions is less than in active lesions [9]. Recently, the presence of lesions in the gray matter has increasingly been recognized. The amount of cortical lesions is extensive and is also thought to contribute to disease progression and disability in MS patients. Cortical lesions are able to remyelinate and showed in one study even a higher capacity to remyelinate than white matter lesions [11]. Interestingly, in all studies complete absence of remyelination was rare and was observed in only a small subset of patients, indicating that remyelination is a natural and frequent process after demyelination in the majority of patients and lesions. Consequently, the challenge is to identify the factors that lead to the failure of remyelination of MS lesions. This is likely to be a failure of multiple factors. The essential processes for remyelination are recruitment of oligodendrocyte progenitors, differentiation, axon–oligodendrocyte interaction, and myelination. These processes could clearly be affected by changes in lesion environment and increasing axonal

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compromise, as suggested by the studies on remyelination in MS autopsy and biopsy material.

The Role of Animal Models for the Study of Remyelination Animal models are a helpful tool in exploring the underlying mechanisms of central nervous system (CNS) remyelination after experimentally induced demyelination. However, since demyelination is induced in an artificial way, all models only partly mimic the complex processes of remyelination in MS. Every model has its advantages and disadvantages, which can be subdivided into those specific for the species under investigation (e.g., mice, rats, guinea pigs) and others dealing with the induction and monitoring of the disease in the animal. Nevertheless, rodent models are needed to study basic mechanisms of successful remyelination, which is not possible in humans. Since no animal model covers the complex pathomechanisms of human MS, the various animal models should be used to selectively analyze remyelination processes dependent on the hypothesis investigated. Ideally, the findings in one model should be confirmed and supplemented in another model of demyelination. To induce CNS demyelination, different protocols and a variety of myelin insults, such as toxins, inflammatory reactions, viruses, and genetic myelin mutations, can be used.

Toxin-Induced Demyelination Toxic animal models are commonly used to study remyelination after experimentally induced demyelination since the processes are synchronized and the lesion location is known. Injections of demyelinating toxins such as lysolecithin or ethidium bromide into CNS tissue lead to focal myelin loss in the selected CNS region of interest [12]. Spontaneous remyelination is regularly observed after focal toxic demyelination. Remyelination can be stopped by irradiation of the animals to prevent proliferation of glial cell progenitors. Such an experimental design is mainly used for transplantation studies to differentiate between endogenous remyelination and remyelination achieved by the transplanted cells. Besides the advantage of a predefined demyelinating area, toxin injections induce a small traumatic injury of the blood– brain barrier (BBB) and brain, leading to recruitment of peripheral inflammatory cells such as macrophages and T cells. In addition to the injection of toxins into the CNS, feeding of cuprizone is another widely accepted toxic model of demyelination in rodents [4]. In this model predominantly young adult mice are fed with the copper chelator cuprizone (bis-cyclohexanone oxaldihydrazone), which leads to a reproducible demyelination within 5–6 weeks. After removal of the toxin from the diet, spontaneous remyelination occurs. Cuprizone feeding has been used mainly to study experimental demyelination and remyelination in the cerebral white matter tract corpus callosum. However, it was recently shown that other areas, like the hippocampus and the cerebral and cerebellar cortex, are also affected [13–15]. Since the time course of demyelination and the severity of

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glial reactions are different in the white and gray matter, the cuprizone model offers the opportunity to study the different pathomechanisms of gray and white matter demyelination and remyelination. The cuprizone model offers consistent and anatomically reproducible demyelination and remyelination processes that are easy to detect. In contrast to models of autoimmune encephalomyelitis or models of focal induced demyelination in the cuprizone model, no BBB breakdown occurs. In this context, the pathomechanisms of remyelination can be analyzed, bypassing possible interferences of peripheral immune cells.

Inflammation-Induced Demyelination Experimental autoimmune encephalomyelitis (EAE) is the animal model most frequently used to analyze inflammation in the CNS. EAE can be induced actively in susceptible animal species (mice, rats, rabbits, guinea pigs, nonhuman primates) by injection of CNS tissue or antigens (e.g., MBP, MOG, PLP, MOBP, MAG, OSP, Nogo-A, CNPase, α-B-cystallin, S-100) [16,17]. As a consequence of antigen injection, reactive T lymphocytes arise, migrate into the CNS, recognize their target antigen, and initiate an autoimmune response leading to CNS tissue damage. EAE can also be induced by adoptive transfer of activated myelin-reactive T lymphocytes by intravenous injection into naïve animals. The inflammatory CNS reaction and demyelination are dependent on the different immunization protocols and the species of the animal and genetic background [16,17]. Lewis rats, for example, are highly responsive to sensitization against MBP but barely respond to MOG. However, in C57BL/6 mice, reactivity of the two myelin proteins is opposite. Furthermore, anti-MOG antibodies seem to be crucial for demyelination. In MBP-induced EAE in which inflammation is high but only minor demyelination is present, infusion of anti-MOG antibodies led to a severe demyelination [18]. Although it is an excellent model to study CNS inflammation, assessment of remyelination is especially complicated in EAE, in which demyelination and remyelination can occur concurrently. This makes it difficult to distinguish an area that is not completely demyelinated from an area where remyelination begins. Thus, the interpretation of effects of remyelination-enhancing factors might be limited. Another disadvantage of EAE is its unpredictable localization in the spinal cord. Lesion locations cannot be predicted exactly as lesions can occur throughout the spinal cord.

Viral-Induced Demyelination Virus-induced infections of the CNS serve as another inflammation-mediated model of demyelination in animals. Demyelination can be initiated by infection with a diverse group of viruses in susceptible rodents. Furthermore, the viral models demonstrate how viruses enter the brain, spread, persist, and interact with immune responses. The Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination model has been extensively proven for demonstrating demyelination and remyelination [19]. Theiler’s virus is a natural pathogen of susceptible mice such as SJL mice that causes a persistent infection of the CNS and inflammation-mediated demyelination. Intracranial inoculation of the virus induces a biphasic disease consisting

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of early acute disease (encephalomyelitis) within 3–12 days, followed by late chronic demyelinating disease, which develops after 30–40 days in the spinal cord. Resistant mice, such as C57BL/6, develop only the early acute disease, clear the virus completely within 3 weeks, and do not develop the late chronic demyelinating disease. In SJL mice the late chronic demyelinating disease is associated with severe meningeal, perivascular, and parenchymal infiltration by T cells and monocytes/macrophages and leads to progressive spinal cord atrophy and axonal loss, with subsequent neurologic deficits. In demyelinated lesions in this model remyelination is present in variable degrees, allowing the investigation of remyelination-enhancing factors. The mouse hepatitis virus (MHV) is another virus used to induce experimental encephalomyelitis with subsequent CNS demyelination, when applied intracranially or intranasally [20]. However, it is still not known how MHV induces demyelination and to what extent the immune system plays a role in this model. It was suggested that some demyelination may be due to direct virus infection and damage of oligodendrocytes, but the majority is rather mediated through inflammatory responses [21]. The Semliki Forest virus (SFV)-induced CNS demyelination can be investigated following intraperitoneal infection of immunocompetent adult mice [21]. Twentyfour hours after infection the virus is detectable in the brain. In this model the effects of demyelination can be analyzed after a natural infection without a local neural lesion, allowing studies of the BBB. Demyelination in SFV-infected mice seems to be T-cell-mediated via damage of infected oligodendrocytes rather than through a direct viral effect. Interestingly, this model also presents changes in optic nerves.

Genetic Myelin Mutants In recent years, an increasing number of genetic animal models with a disturbance of CNS myelination have been characterized. The myelin-deficient (md) rat and mouse mutants jimpy have a mutation in the gene encoding for PLP that results in central myelination deficits. Both mutants serve as animal models for the human X-chromosome-linked Pelizaeus–Merzbacher disease, which is genetically related to a defect in the PLP gene [22]. The shiverer (shi) mouse is another myelin mutant model that has a defect in the MBP gene and causes abnormal myelination [23]. The advantage of myelin mutants is the consistency in myelination defects and known area of damage, allowing transplanted cells to be identified more clearly. Furthermore, genetic mutants serve as excellent models to study human dysmyelinating diseases. The disadvantage of genetic models is that they are poorly myelinating or nonmyelinating models rather than demyelinating lesion models. Furthermore, demyelinated areas in the CNS are noninflammatory. Thus, genetic models are less suitable for studies of remyelination in MS.

Growth Factors in CNS Remyelination Growth factors (GFs) are known to orchestrate migration, proliferation, differentiation, and survival of a variety of cell types, including neuronal and glial cells in the

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CNTF NRG 1 NT-3

CNTF NRG1 NT-3

CNTF NRG1/GGF2 PDGFA NT-3 FGF-2 BDNF PDGFA FGF-2

CNTF NRG1 NT-3

GDNF NGF

+

IGF-1 TGF1β BDNF CNTF IGF-1

CNTF LIF

Figure 9.2  Role of GF for oligodendrocyte lineage cells. Summarized scheme.

CNS. Together with chemokines and hormones, GFs not only regulate the development, specification, and maintenance of CNS structures, but also modulate its plasticity and repair, including generation of new glial and neuronal cells. This includes the maturation and myelination of newly arisen axons or remyelination of demyelinated axons in adult organisms. Remyelination is common in MS but is not always complete and abundant, despite OPCs being present in demyelinating lesions. It is thus thought that OPCs may not be capable of differentiating into myelinating oligodendrocytes and that GFs could be therapeutic agents to improve remyelination [6,24–29]. Different immune and glial cells infiltrate in the demyelinating lesions, producing a myriad of GFs and cytokines. There is strong evidence that inflammation and in particular mononuclear phagocytes are beneficial for remyelination [30–33]. Understanding and being able to control inflammatory events means being able to stop the loss of oligodendrocytes and/or to improve the remyelination capacity of the CNS. All glial cells and neurons are able to express various GFs and their receptors. Here we will concentrate on the role of GFs on oligodendrocytic cell lineage as a provider of myelin and a key factor in remyelination of axons. GFs influence all developmental stages (Figure 9.2) and support the survival of oligodendrocyte lineage. The largest body of evidence is provided by in vitro and in vivo research on rodent oligodendrocytes. Animal models and in vitro studies offer great insight into the development of pathologic conditions that affect oligodendrocyte function as well as the complex interactions between neuronal, immune, and glial cells. However, the findings in different species, different animal models, or in vitro studies are complex and cannot always be easily extrapolated to human

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oligodendrocyte lineage cells. Also, multiple factors can act in concert to initialize or regulate some events in oligodendrocyte life cycles.

Influence of GFs on the Fate of Progenitor Cells and Promotion of Generation of Oligodendrocytes In the mammalian CNS, oligodendrocytes arise from multipotent proliferating cells of the subventricular and ventricular zones [34]. Stem cells isolated from these areas can be maintained in vitro in the presence of epidermal growth factor (EGF) [35]. EGF has been shown to stimulate the proliferation and migration of precursor cells [36,37]. It has been reported that EGF-stimulated progenitors can migrate into demyelinating lesions and differentiate particularly into oligodendroglial cells [38]. Also, after EGF-stimulated progenitors are injected into a myelin-deficient environment, these cells differentiate into myelinating oligodendrocytes [39]. Neurotrophins are known to influence the final determination of multipotential precursor cells. Consequently, treatment of neurosphere cultures from embryonic striatum with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) led to the appearance of neuronal cells, whereas treatment with neurotrophin-3 (NT-3) resulted in bipolar neuronal cells and oligodendrocytes [40]. Neuregulin-1 (NRG1) and its signaling were also shown to play an important role in the generation of new oligodendrocytes. Murine embryonic striatal neuronal precursors express multiple NRG1 transcripts and proteins as well as their specific receptors, ErbB2 and ErbB4, but not ErbB3. Using soluble ErbB3 receptor to inhibit NRG1 function, it has been demonstrated that NRG1 controls the growth of precursors, increasing their proliferation and survival. In turn, soluble ErbB3 pretreatment of growing neurospheres specifically increased the number of oligodendrocytes differentiating from precursors migrating from the spheres [41] in vitro. In vivo, spinal cord explant cultures from NRG1 knockout mice fail to generate oligodendrocytes unless exogenous NRG1 is added, suggesting an important role for NRG1 in oligodendrocyte lineage specification [42].

GFs in Proliferation and Migration of OPCs The influence of basic fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) as potent mitogens for OPCs has been studied in vitro and in a number of animal models [43–49]. PDGFR-α is present on OPCs and disappears at the O4 stage of oligodendrocyte maturation [50,51]. FGF-2 can enhance expression of PDGFR-α on OPCs and block differentiation of OPCs [43]. In the cuprizone-induced toxic demyelination model the absence of FGF-2 promotes oligodendrocyte repopulation [52,53], whereas PDGF overexpression increases a number of OPCs following demyelination [54]. Also, PDGF is known to promote the migration of OPCs [55]. Hepatocyte growth factor (HGF) can also induce chemotaxis of OPCs and promote the proliferation of rodent OPCs in vitro and in vivo [56,57]. It has been reported that a functional HGF/c-Met system, which can influence the proliferation, development, and cytoskeletal organization, is present

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in oligodendrocytes [58]. Also, ciliary neurotrophic factor (CNTF) has been shown to enhance the proliferation of OPCs in vivo and in vitro [59]. NRG1/GGF2 can promote the proliferation and inhibit the differentiation of rodent OPCs [60] and can induce differentiated oligodendrocytes to undergo a phenotypic reversion characterized by loss of MBP expression, re-expression of the intermediate filament protein nestin, reorganization of the actin cytoskeleton, and a dramatic reduction in the number of processes per cell [61]. NT-3 has also mitogenic effects on rodent OPCs in vitro and in vivo. Enhanced proliferation of rat OPCs has been reported after treatment with NT-3 in vitro [62,63]. In vivo, NT-3 has been reported to promote proliferation after intracranial injection into the rat brain and after transplantation of genetically modified fibroblasts into spinal cord lesions [64,65]. BDNF also shows a beneficial effect on the proliferation of OPCs and subsequent myelination in the CNS [65]. Often the mitogenic effects of NT-3 are seen only when NT-3 has been administrated in combination with either another GF or insulin [66,67]. Analysis of mice lacking NT-3 and its receptor TrkC demonstrate a decline in PDGFαR-positive cells and also mature oligodendrocytes within the CNS [68].

Influence of GFs on Differentiation of OPC and Promotion of Myelination and Remyelination NT-3 also seems to accompany the further determination of oligodendrocytic lineage. OPCs require exogenous NT-3 or transfection with NT-3 gene for differentiation into oligodendrocytes [63,69]. NGF and NT-3 show beneficial effects on differentiation of cortical OPCs [70]. In chemical-induced demyelination, a direct application of NT-3 led to an increased number of mature oligodendrocytes and enhanced remyelination [71]. Also, BDNF shows a beneficial effect on differentiation of OPCs and subsequent myelination in the CNS [65,72]. In EAE, BDNF delivery reduces demyelination and increases remyelination [73]. It is also possible that BDNF controls myelination of the optic nerve through direct modulation of retinal ganglion cell (RGC) axon diameter [74]. Insulin-like growth factor 1 (IGF-1) was reported to play an important role in the differentiation of oligodendrocytes and to promote myelination [75–78]. IGF-1 overexpressing mice show a significant increase of the number of myelinated axons and of the myelin thickness [79]. IGF-1 knockout mice exhibit a decreased number of oligodendrocytes and myelinated axons in the corpus callosum and anterior commissure [80]. Beneficial effects of IGF-1 on remyelination have been studied in various experimental demyelination animal models. For instance, EAE treatment with IGF-1 has been associated with enhanced remyelination [81], although the positive effect has been observed only transiently in the acute phase and is not notable in the chronic phase [82]. In conditional mouse mutants, in which the expression of the Igf1r gene is ablated by Cre-mediated recombination, no accumulation of OPCs, a severe decrease of oligodendrocyte regeneration, and no remyelination after cuprizone-induced demyelination has been observed [83]. The upregulation of IGF-1 in cuprizone-induced

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demyelination has been previously described as well [84]. In lysolecithin-induced demyelination, IGF-1 and transforming growth factor-beta (TGF-β1) mRNAs were evident within the spinal cord by 5 days after lesion induction [48]. TGF-β1 inhibits the proliferation of OPCs, promotes oligodendrocyte development [85], and enhances myelination [86]. TGF-β1 can prevent EAE and suppress disease [87–89]. TGF-β2, another member of the TGF protein family, reduces demyelination in the viral model of MS via immunomodulatory mechanisms [90]. Despite the presence of IGF-1 and TGF-β1, which are considered to be the key regulators of oligodendrocyte differentiation, CNTF and leukemia inhibitory factor (LIF) also seem to be strongly involved in these processes. Both molecules act via gp130 receptors and could enhance the generation of oligodendrocytes in cultures of dividing O-2A progenitors and promote oligodendrocyte maturation, as determined by expression of myelin basic protein (MBP) [91]. CNTF seems to promote oligodendroglial differentiation/maturation but does not affect cell fate decision of adult hippocampus-derived neuronal precursor cells and also oligodendroglial progenitors derived from early postnatal cortex [92]. CNTF can prevent the death of oligodendrocytes under pro-inflammatory conditions in vitro [93]. It can also promote the survival and differentiation of adult spinal cord-derived OPCs in vitro but fails to promote remyelination in vivo [94]. However, in vitro CNTF has been shown to enhance myelin formation directly [95]. This pro-myelinating effect of CNTF is proposed to be mediated through the JAK/Stat pathway [95]. In EAE, CNTF–/– mice suffer from a significantly earlier onset and more severe progression of disease [96]. Poor recovery is accompanied by decreased proliferation and generation of oligodendrocytes and an increased apoptosis rate. LIF has been also suggested to act directly on oligodendrocytes. LIF receptor β (LIFRβ) is expressed on oligodendrocytes and is activated in affected tissue [97]. LIF supports the survival of mature oligodendrocyte and can prevent interferon-γ-induced apoptosis of oligodendrocytes [98]. There are also reports that LIF is involved in the differentiation of oligodendrocytes. Acting in one defined time window, LIF is, however, not an essential factor for this process. Only female LIF-deficient mice exhibit lower levels of MBP [99]. Developmental myelination of the optic nerve from LIFdeficient mice was delayed due to impaired maturation of OPCs [100]. In cuprizone-induced demyelination, LIF knockout mice display more severe demyelination and impaired remyelination, although oligodendrocyte replenishment is not significantly compromised [101]. Application of exogenous LIF upon demyelination limits myelin loss, eventually due to LIF-promoted survival of oligodendrocytes. However, treatment upon remyelination fails to reveal any significant effect [101]. In the EAE LIF receptor signaling limits the severity of inflammatory demyelination [97]. This study showed that LIF directly prevents oligodendrocyte death in EAE. Moreover, in another study anti-LIF neutralizing antibodies can potentiate demyelination and oligodendrocyte loss [102]. LIF has been also shown to play an important role in the modulation of immune cells during EAE [103]. Subsequent to oligodendrocyte generation, NRG1 may also play a role in promoting oligodendrocyte differentiation as well. Transgenic mice expressing a dominant-negative erbB2 receptor showed hypomyelination, a decrease in mature oligodendrocytes, and an

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increase in the number of progenitor cells [104]. The impact of NRG1 on remyelination has been studied in several animal models. Systemic delivery of NRG1 to mice exposed to EAE delayed signs of the disease, decreased the severity, and resulted in significant reductions in relapse rate [105,106]. Moreover, NRG1-treated groups exhibited more remyelination in CNS lesions than controls. In contrast to studies in the EAE model, application of NRG1 into demyelinated areas does not improve remyelination in toxic demyelination [107]. Further studies are required to determine the precise mechanism of NRG1 action on oligodendrocyte and on immunomodulation.

Effect of GFs on the Interactions Between Oligodendrocyte and Axon The key moment in successful remyelination is the interaction between the axon and the oligodendrocyte. NGF is involved in myelinating cell–axon interaction and promotes myelination of TrkA-expressing dorsal root ganglion (DRG) neurons by Schwann cells and inhibits oligodendrocytes in vitro [108]. In this context, in DRG neurons, NGF can induce via TrkA the axonal expression of LINGO-1, a known inhibitor of oligodendrocytes [109,110]. The myelination-promoting effects of glial cell-derived neurotrophic factor (GDNF) have been predominantly demonstrated in spinal cord injury animal models, in the peripheral nervous system, as well as in vitro by acting on Schwann cells and on neurons and modulating Schwann cell–axon interaction [111–113].

The Role of GFs in MS In MS lesions, BDNF is present on T cells, macrophages/microglia, and reactive astrocytes. TrkB, the full-length receptor for BDNF, has been found on neurons and reactive astrocytes [114]. Also, TGF-β1 has been found overexpressed by reactive astrocytes within MS lesions [115]. CNTF-receptor complex members, CNTFRα, LIFRβ, and GP130, were increased in MS cortical neurons. CNTF was increased and also expressed by neurons. Phosphorylated STAT3 and the anti-apoptotic molecule, Bcl2, known downstream products of CNTF signaling, were also increased in MS cortical neurons, suggesting a response to the chronic insult or stress in the pathogenesis of MS with upregulation of a CNTF-mediated neuroprotective signaling pathway [116]. PDGFR-α-expressing OPCs have been found within demyelinating lesions [27]. This suggests that PDGF is likely to play a role in the orchestration of remyelination.

Transcription Factors in Remyelination As mentioned, remyelination requires a series of highly orchestrated events that coordinate OPCs to proliferate, migrate toward the axons to be myelinated, and undergo terminal differentiation into myelinating oligodendrocytes [5,117]. Accordingly, adult OPCs stepped into the spotlight because of their potential for recruitment into demyelinated lesions [118–126]. They are distributed throughout the CNS and represent an

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endogenous source of progenitor cells, making up 2–9% of the CNS cell population [124]. In recent years, a great deal of research has been devoted to transcription factors that regulate the development and differentiation of these cells. Especially the identification of the Olig genes led to a valuable insight into myelinogenesis [127–130]. Olig1/2 are basic helix-loop-helix (bHLH) transcription factors that play multiple roles in determining the oligodendroglial lineage and are expressed in mature oligodendrocytes as well as in both developmental and adult OPCs [131,132]. During development of the CNS, Olig1/2 are localized in the nucleus of OPCs. In the adult CNS Olig2 remains in the nucleus of OPCs; however, Olig1 is found to be cytoplasmic [133]. Interestingly, in a murine demyelination model and within tissue from MS patients, Olig1 has been shown to be translocated into the nucleus of OPCs as in the embryonic development, suggesting that Olig1 initiates oligodendrocyte differentiation and, thus, remyelination [133,134]. The first Olig1 knockout mouse revealed a normal development of myelin but lack of remyelination after experimental demyelination [133]. In contrast, Xin et al. [135] reported a lethal development defect in differentiation of OPCs and myelination using a different Olig1 null mouse. An explanation for this discrepancy could be the fact that in the transgene construct used in the first model, Olig2 may have been enhanced. Olig2 is required for the development of oligodendrocytes at early stages as well as motor neurons, whereas Olig1 functions later in oligodendrocyte development [130,135,136]. The homeodomain transcription factors Nkx2.2, MASH1 (ASCL1), and Olig2 are elevated in OPCs prior to remyelination [137–139]. Their co-expression is already known to induce oligodendrocyte differentiation in embryonic development [140– 142]. MASH1 is another bHLH transcription factor required for oligodendrocyte development. In MASH1 null mutants fewer OPCs are generated and expression of myelin genes is distinctly diminished [143,144]. Nkx2.2 is required for the normal differentiation of oligodendrocytes, especially for the generation of MBP- and PLPpositive ones [145]. Moreover, Olig1/2 can form heterodimers with the inhibitor of differentiation (ID) gene products, especially ID2 and ID4, which may cause a downstream of bone morphogenetic protein (BMP) signaling. BMPs are a family of secreted transforming growth factor-A signaling factors acting to suppress OPC differentiation. In several demyelination models, including chronic EAE, upregulation of BMPs was observed [146–148]. BMPs are thought to promote gliosis and inhibit remyelination [148]. ID2 and ID4 mimic the effects of BMPs as their overexpression in cultured OPCs prevents differentiation and induces an astrotype phenotype [149–151]. Transcription factors using the Wnt pathway have recently been shown to be specific to remyelinating white matter, and this pathway including β-catenin has been shown to be activated in a pathologic context [152]. The transcription factor Tcf7l2 inhibits the Wnt signaling pathway through its ability to bind to β-catenin [153]. It promotes oligodendrocyte differentiation and, probably, remyelination [152]. Myelin transcription factor 1 (MYT1) is a zinc-finger protein that can bind to the promoter region of the PLP gene. In MS lesions increased Myt1 expression was reported [154]. Overexpression of Myt1 leads to inhibition of oligodendrocyte differentiation [155].

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The Notch1/Jagged1 signaling is a further pathway involved in remyelination. Notch receptors are transmembrane proteins expressed on developmental and adult OPCs [156]. Following ligand binding, they are cleaved by a metalloproteinase and a γ-secretase, generating Notch intracellular domain (NICD), which then translocates to the nucleus [156]. Notch1 is thought to act through Hes5, a bHLH transcription factor, which suppresses transcription of myelin genes [157]. In chronic active MS lesions an increased expression of Jagged1 by reactive astrocytes, as well as Notch1 and HES5 in OPCs, has been reported [158]. In conclusion, transcription factors in remyelination constitute a complex network with either suppressive or promoting functions on oligodendrocyte development. Recent work shows their involvement in remyelination and uncovers a diversity of involved signaling pathways. Yet it remains uncertain which of these factors and pathways are the most important for remyelination and which mechanisms sustain suppressive signaling in remyelination failure.

Epigenetic Control of Oligodendrocytes Ineffective remyelination has been associated with aging [159]. This age-related effect is probably due to an impairment of OPC differentiation and recruitment [160]. Differentiation of OPCs seems to be a more important process than their recruitment because increasing the availability of OPCs did not enhance remyelination [54]. As described earlier, OPC differentiation is controlled by a complex network of transcription factors. The selective expression or silencing of genes during this differentiation process is also controlled by epigenetic regulatory mechanisms. This crosstalk between transcription factors and modulators of gene expression, including posttranslational modifications of nucleosomal histones, changes in histone variants, chromatin remodeling enzymes, DNA methylation, and microRNAs (miRNAs), provides a dynamic interaction between genetic and epigenetic programs. Epigenetic modulators control gene expression by regulating transcript levels independently of changes in DNA sequence [161,162]. Histone deacetylases (HDACs) remove the acetyl groups from histone lysine residues and render chromatin less accessible to transcription factors [163]. HDACs seem to be required for the morphologic and functional differentiation of oligodendrocytes from progenitor cells [164,165]. The reconversion to a progenitor cell is prevented by blocking transcription factors, for example Sox2 [166], while HDAC inhibitors reduce the number of oligodendrocytes due to a larger population of astrocytes and neurons [167–169]. Recently, Yin Yang 1, a transcription factor, has been shown to facilitate the recruitment of an HDAC to the promoters of some transcriptional inhibitors, allowing expression of myelin-specific genes [167]. Closely associated with the state of histone acetylation is histone methylation of lysine residues, which plays a role in the selection of the oligodendrocyte lineage by preventing a neuronal or astrocytic cell fate choice [170]. The most prominent histone methyltransferase is Ezh2, which methylates lysine 27 on histone H3 [170].

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Modification of histone arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), generating irreversible protein structural modifications. High nuclear levels of these enzymes have been observed in experimental models of demyelination [171,172] and in normal-appearing white matter isolated from patients with MS, suggesting a contribution to the pathogenic process [173]. Another important epigenetic mechanism is DNA methylation at the CPG-rich 5' promotor region of genes, which decreases gene expression. In brain tissue of MS patients, higher DNA demethylase activity has been reported [174]. This increased activity is assumed to be associated with a higher rate of citrullination as the promoter regions for PADs contain CPG motifs. Demethylation of these regions leads to a reactivation of PADs [172]. Apart from these mechanisms operating at the transcriptional level, microRNAs (miRNA), which are small noncoding RNAs, modulate mRNA translation. Bound to argonaute proteins, they mediate suppression of specific target mRNAs by imperfect base pairing at the 3' untranslated region. This may result either in translational repression or in destabilizing and degradation of the target mRNA. miRNAs are present at very high copy numbers per cell and are thought to have a profound impact on gene expression regulation [175]. They are involved in various cancer types [176], differentiation or maintenance of cell lineages [177], and synaptic [178] and cardiovascular development [179]. In oligodendrocytes the expression of miRNA has just recently been reported [180–182]. During the transition from progenitor to premyelinating cell, changes in the expression levels of miRNA have been shown, suggesting that miRNAs are regulators of oligodendrocyte differentiation and, probably, myelination [181]. Several miRNAs (i.e., miR-219, miR-138, and miR-338) have been proposed to be involved in this process [183]. miR-219 seems to play a more important role as it represses the expression of PDGFRα, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. In another study miR-206 has been shown to downregulate the tubulin polymerization-promoting protein (TPPP), resulting in inhibition of oligodendrocyte differentiation [182]. TPPP is expressed mainly in myelinating oligodendrocytes of the CNS [184]. Laminin B1 is considered an important protein for myelin maintenance, but its excessive expression results may lead to premature arrest of oligodendrocyte differentiation. Lin et al. showed that miR-23 partially downregulates the gene expression of this protein, suggesting a beneficial role in CNS myelination. Moreover, deletion of Dicer1 disrupts miRNA processing and results in failure of OPC differentiation and CNS myelination [183]. Dicer1 is a cytoplasmic RNA polymerase that detruncates primary miRNA to its short mature form [175]. In conclusion, alterations to amino acid residues of nucleosomal histones modulating gene expression in oligodendrocytes include deacetylation, methylation, and citrullination, which in turn establish a cell-type-specific histone code. Histone changes determine the structure of chromatin and thus enable or disable transcription of genes. miRNAs in turn modulate at the posttranslational level by suppression of mRNA. Their involvement and biology in oligodendrocyte differentiation is unclear, but recent work points to critical regulation mechanisms.

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Clinical Trials for Remyelination in MS The findings that remyelination is a regular process after experimentally induced demyelination have demonstrated that repair is possible. Despite very limited knowledge why remyelination fails in MS [7], the search for a remyelination-supporting treatment has been one of the major tasks in research during the past two decades. In general, two strategies could be followed: (1) promotion of endogenous repair mechanisms and (2) exogenous support by the transplantation of myelin-forming cells. The endogenous promotion of remyelination could again be achieved by several mechanisms: (1) application of substances (e.g., GFs, antibodies) that directly influence OPC proliferation, migration, or differentiation; (2) indirect creation of a repair-promoting microenvironment in the CNS (e.g., via immune cells [protective autoimmunity [185]]); (3) removal of myelin formation inhibitory factors [186]; or (4) protection of myelin-forming cells. Most approaches have been successfully tested in animal experiments, but there are only few trials in patients with MS.

Enhancement of Endogenous Repair Mechanisms The important role of GFs for remyelination has been described earlier. Despite this presumed importance there has been only one small, not controlled trial with IFG-1 in patients with MS [187]. In this open, crossover study 50 μg IGF-1 was administered twice daily for 6 months after a baseline period of 6 months. The primary outcome was the number of gadolinium-enhancing lesions on monthly magnetic resonance imaging (MRI). There were no differences between the baseline and the treatment period, including secondary outcome parameters with clinical endpoints. Given the small sample size, the uncontrolled nature of the study, and the short study duration, it is not surprising that this trial failed. Furthermore, IGF-1, like other GFs, may influence OPCs and thus myelination differentially at different stages of OPC and oligodendrocyte development. Considering the complex orchestration of remyelination by a huge number of signals, it seems unlikely that administration of a single GF given over several weeks or months will lead to sustained remyelination. The timing of such a treatment may be essential, and a sequence of various GFs may be required. Creation of a remyelination-supportive environment in the lesion is another approach. It is well known that, for example, suppression of microglial phagocytosing activity impairs remyelination [33,188]. Minocycline, a substance that has been proposed to have neuroprotective properties and that may modulate microglia, was shown in animal models to be beneficial on demyelination and inflammation in the CNS [189,190]. In a small open clinical trial CNS inflammation was suppressed in five MS patients as measured by gadolinium-enhancing lesions on MRI [191]. In a follow-up of these patients and five additional patients who were treated with 100 mg minocycline twice daily, a reduction in relapse rate was observed and it was attributed to changes in metalloproteinase and cytokines [192]. Although these results are promising, controlled trials are necessary to prove the efficacy of minocycline in MS. A series of experiments mainly in the TMEV model have demonstrated that natural antibodies directed against oligodendrocytes are capable of inducing remyelination

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[193–195]. Although not as effective as some monoclonal antibodies, there was also the promotion of remyelination by human polyclonal intravenous immunoglobulins (IVIGs) in TMEV [194]. This has led to the design of clinical trials aimed at investigating the remyelination capacity of IVIGs, which are used successfully in inflammatory demyelinating diseases of the peripheral nervous system [196]. Unfortunately, none of the three trials in MS patients could demonstrate remyelination on clinical or electrophysiologic grounds [197–199]. Several reasons for this failure have been speculated, like the wrong timing (IVIGs were administered in all trials months after the last relapse, which may be too late to induce remyelination) or the wrong antibodies (all trials used IgG preparations, while the most potent monoclonal antibodies are IgM) [200]. Although human monoclonal IgM antibodies with remyelinating properties in the animal model have been described [201], there are no published clinical trials in MS patients. An antibody against LINGO-1, a component of the Nogo receptor complex exclusively expressed on oligodendrocytes in the CNS [109], has recently been identified as a new candidate to promote remyelination. The antibody has been tested successfully in several animal models [202] and a clinical phase I study is ongoing. The results of clinical trials are needed before the potential for MS patients can be evaluated.

Transplantation of Myelin-Forming Cells In many experimental approaches and different animal models, the transplantation of different myelin-forming cells has been proven to restore function [203–205]. In general, different sources of myelin-forming cells can be used, namely OPCs, Schwann cells, olfactory ensheathing cells (OECs), stem cells (embryonic, adult neuronal, and bone marrow derived), and theoretically also xenotransplants [28,205]. Besides the ethical problems in particular with the generation of embryonic stem cells, there are several other complicating issues associated with cell transplantation in MS patients. First, MS is a multilocular disease with lesions disseminated all over the CNS, and it is not possible to transplant all these lesions. Transplantation of potentially strategic lesions (that are difficult to define) may also bear the risk of transplantation-associated complications in these strategic brain regions. Second, it is not clear what happens to the transplanted cells. Can they reactivate the autoimmune process, and will they be destroyed by the ongoing inflammatory disease? Third, in case of axonal damage these axons may not be receptive for remyelination, or even in the case of successful remyelination there may be no functional gain. Fourth, what is the best time to transplant cells? It is probably shortly after the demyelinating attack; however, often clinicians wait if there is clinical improvement after application of corticosteroid or plasma exchange. Fifth, which is the best cell type to transplant? In view of these questions, one small trial to transplant autologous Schwann cells into MS lesions has been performed. Unfortunately this trial has never been published in a peer-reviewed journal, and the results have been only temporarily available at www.myelin.org. The advantage of Schwann cells is that an autologous transplantation is possible, the cells can be expanded in vitro and

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cryopreserved [206], and they may escape the autoimmune reaction. The trial was designed to investigate safety aspects of Schwann cell transplantation in MS with increasing cell numbers to be transplanted into a right frontal MS lesion. The trial was terminated after three patients. Although there seemed to be no safety problems, a planned biopsy after 6 months of the transplantation did not show any transplanted Schwann cells in the lesion. In view of these results, the concept of transplantation as a regenerative treatment for MS patients needs to be reconsidered. Experimental results from systemic transplantation of neuronal stem cells (including human cells in a nonhuman primate model) suggest that these effects may not be mediated by direct remyelination but rather by creation of a repair-supporting microenvironment [207,208].

Neuroprotective Treatment Approaches Although the term “neuroprotection” is usually applied to axons and neurons, the protection of oligodendrocytes or their precursors from damage may also contribute to remyelination. However, trials investigating this specific aspect are not available (although the currently available immunomodulatory treatments for MS indirectly protect myelin sheaths from being attacked by the autoimmune reaction). There are a few small clinical trials investigating neuroprotective effects in MS patients for drugs that are being used for other indications. Riluzole, an established treatment for amyotrophic lateral sclerosis (ALS), has been investigated in an open trial in 16 patients with primary progressive MS [209]. The primary endpoint was spinal cord atrophy as measured by MRI. Compared to the pretreatment period of 1 year with progressive spinal cord atrophy, there was a stabilization during the treatment period of 1 year. However, this result has never been reproduced or confirmed in a randomized, placebo-controlled trial. Erythropoietin (EPO) has been found to be part of a neuroprotective pathway [210] in the CNS, and thus a small study with EPO was carried out in eight patients with chronic progressive MS [211]. Five patients were treated with 48,000 IU weekly for 12 weeks, followed by biweekly administration for another 12 weeks, and a followup observational period for 24 weeks. There was an improvement in motor function and cognitive performance in the high-dose group, while there was no improvement in the low-dose group and in two patients with Parkinson’s disease as controls. These preliminary data may support the design of a randomized, placebo-controlled trial but cannot prove a neuroprotective or regenerative effect of EPO in MS. Since axonal injury (e.g., by nitric oxide) leads to a high intracellular sodium concentration via persistent activation of sodium channels and inhibition of mitochondrial Na-K ATPase, there is an exchange of intra-axonal sodium ions for extracellular calcium that in turn injures axons. Therefore, sodium channel blockers, well-established drugs in the treatment of epilepsy, were investigated for a neuroprotective effect [212]. Lamotrigine was used in a phase II placebo-controlled clinical trial involving 120 patients with secondary progressive MS [213]. Preliminary results could not demonstrate a positive effect in central cerebral volume as measured by MRI, the primary outcome parameter for cerebral atrophy. Deterioration in

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the 25-foot walk was slower in the lamotrigine group; however, this needs confirmation in a larger trial. Overall, this randomized controlled trial could unfortunately not demonstrate a neuroprotective effect.

Imaging of Remyelination in Clinical Trials In most clinical MS trials, disease course and treatment response are monitored by progression of clinical symptoms with the Expanded Disability Status Scale (EDSS) and by disease activity on MRI. For treatment outcome measures in clinical trials as well as for the diagnosis of MS, standard MRI protocols have been established that include conventional T2-weighted images, T1-weighted images with and without gadolinium (Gd), and fluid-attenuated inversion recovery (FLAIR) sequences. However, the correlation of these standard measures with the clinical disease manifestations and course is limited because the conventional MRI sequences represent the pathophysiologic processes incompletely and they fail to differentiate between demyelinated and remyelinated lesions [214]. Demyelinated as well as partially remyelinated lesions appear hyperintense on T2-weighted images and hypointense on T1-weighted images, whereas fully remyelinated lesions may appear less T1-hypointense as shadow plaques. Histologically the T2-hyperintense lesions represent a heterogeneous spectrum of myelination ranging from extensive demyelination to full remyelination [215]. Advanced nonconventional MRI imaging techniques like magnetization transfer imaging (MTI) and diffusion tensor imaging (DTI), high-field MRI, and positron emission tomography (PET) are promising techniques to visualize neuronal injury and repair [216]. The principle of MTI is based on the interaction and exchange of unbound protons in free water with those bound to macromolecules like protons present in myelin. Damage to myelin during demyelination results in a reduced exchange of protons or magnetization transfer ratio (MTR), whereas increased proton exchange or MTR reveals remyelination [217,218]. The MTR of remyelinated lesions differs from that of normal-appearing white matter and demyelinated lesions. In brain autopsy material a correlation of myelin content and MTR in lesions and normal tissue was observed [219]. There is also evidence that MTR changes are associated with clinical disability and recovery [220,221]. DTI measures the motion of water molecules in tissue and provides information about tissue microstructure and architecture [222]. Furthermore, it serves as a basis for fiber tractography [223]. Alterations in the tissue morphology during demyelination and remyelination may change the diffusivity values. In the cuprizone model decreased axial diffusivity was associated with axonal degeneration, while increased radial diffusivity reflects demyelination in the corpus callosum [224]. Further studies are ongoing to verify whether the DTI technique is capable of differentiating between degenerating and regenerating fibers in MS. With increasing field strength of MRI from 1.5 to 3 T, the sensitivity for the detection of T1-weighted Gd-enhancing lesions and T2-weighted lesions is significantly increased [225]. But to improve the detection of structural and myelin changes,

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ultrahigh-field MRI studies are required. Recently Schmierer et al. [226] demonstrated, in a postmortem study of MS brains with a 9.4 T MRI, distinct changes of T2-weighted images in demyelinated and remyelinated lesions. On the other hand, these ultrahigh-field MRIs need long scanning times and are so far restricted to specialized imaging centers. Besides the advanced MRI techniques, promising developments to detect myelin with molecular imaging techniques like PET have been made. The Congo red derivate 1,4-bis-(4-aminostyryl)-2-methoxy benzene (BMB) selectively binds to myelin and has been proven to distinguish dysmyelination in myelin mutants and demyelinated lesions in EAE. Staining of MS brain tissue with BMB can discriminate between demyelinated and remyelinated lesions or normal-appearing white matter [227]. However, it is unclear when this new PET technique will be available for clinical routine use. During the past decade several modern imaging techniques have been discovered to improve the visualization of the pathophysiologic process of demyelination and remyelination during MS. It is likely that in the next years more potential neuroprotective strategies will go into clinical development, but as of now no established imaging protocols to monitor neuroprotection or remyelination are available. Combining conventional with nonconventional MRI techniques seems to be a promising way for clinical trials to improve the monitoring of regeneration. Nonetheless, this requires the development of new standardized acquisition schemes and analysis procedures.

Obstacles for Regenerative Treatments in MS Although our knowledge of myelin and oligodendrocyte biology has vastly increased in the past decades, the exact molecular mechanisms that drive remyelination are still not fully understood. Due to the extremely complicated control of OPCs by hundreds of factors that differ in each developmental stage from stem cell to mature oligodendrocytes maintaining myelin sheaths, it seems unlikely that a single factor is responsible for remyelination failure. This in turn also implies that there is not a single therapeutic agent that will drive all the processes from OPC proliferation to migration, differentiation, and maintenance of myelin. The picture is even more complicated by the pathophysiologic heterogeneity of MS [228]. This would possibly mean that the molecular requirements for successful remyelination may differ according to each MS subtype. Although many treatment strategies have been successfully employed in animal models, one of the main obstacles for the design of therapeutic strategies in human MS is our lack of knowledge of why remyelination fails. Although there have been several suggestions for molecular targets [158,229], none of these have been proven. Another difficulty for the design of clinical trial protocols to investigate remyelination is the lack of an accepted clinical or paraclinical marker. None of the neurophysiologic, MRI, or other imaging methods like PET can unambiguously demonstrate remyelination. For the patients’ benefit, clinical improvement should be the aim, no matter if this is achieved by remyelination or another mechanism.

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Although a regenerative and/or neuroprotective treatment for MS is urgently warranted, the results from the clinical trials conducted so far have all been very disappointing. Enhancement of the endogenous remyelination potential seems currently more promising compared to the many open questions associated with cell transplantation.

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10 Transverse Myelitis Amir-Hadi Maghzi1,2,3, Masoud Etemadifar1,2, Mohammad-Reza Savoj1, J. Steven Alexander 4, Eduardo Gonzalez-Toledo5,6,Alireza Minagar 6 1 

Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan, Iran 2  Department of Neurology, Isfahan University of Medical Sciences, Isfahan, Iran 3  Neuroimmunology Unit, Centre for Neuroscience and Trauma, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK 4  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 5  Department of Radiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 6  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Introduction Acute transverse myelitis (ATM) is an idiopathic inflammatory form of myelopathy that represents only one of the many causes of acute transverse myelopathies. A large and heterogeneous group of pathogenic disorders can cause transverse myelitis, including inflammatory demyelinating disorders like multiple sclerosis (MS), neuromyelitis optica (NMO), and ATM; infections such as herpes simplex and herpes zoster; para- and post-infectious conditions such as acute disseminated encephalomyelitis; vascular disorders; neoplastic and paraneoplastic conditions; collagen vascular diseases such as systemic lupus erythematosus (SLE); and other inflammatory conditions such as neurosarcoidosis. During the initial assessment of patients with acute myelopathy, neurologists must rule out compressive causes of myelopathy by immediately obtaining magnetic resonance imaging (MRI) of the whole spinal cord. Due to the existence of an enormous number of causes for acute myelopathy, highly detailed patient histories and meticulous neurologic examinations are necessary to exclude confounding diagnoses. Consequently, a more focused investigation with specific paraclinical tests is warranted to pinpoint the exact cause of acute myelopathy. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00010-1 © 2011 Elsevier Inc. All rights reserved.

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Clinical Manifestations Typically, transverse myelitis presents with spinal cord symptoms and signs that evolve over hours to 2 weeks, but it may present from minutes or recur over a month. In some cases, patients also experience nonspecific symptoms such as fever and muscle pain. Despite the term “transverse,” which refers to the presence of horizontal cord lesions, there is usually a longitudinally growing cord lesion, which expands both rostrally and caudally, and there may be greater involvement in the more central regions of the cord rather than the peripheral areas and with less involvement of the peripheral cord tracts [1–4]. Transverse myelitis may also be classified as either complete or partial. Patients with the complete form of transverse myelitis exhibit more severe symptoms, which indicates involvement of all spinal cord tracts and accompanying loss of corticospinal, sensory, and autonomic functions below the level of the lesion. This form of acute myelopathy is usually observed in the context of trauma or acute necrotizing viral myelitis. The partial form of acute myelitis manifests in several clinical pictures based on the involved tracts. Patients with partial myelitis may present with BrownSéquard hemicord syndrome, and these patients suffer from ipsilateral corticospinal dysfunction (an ipsilateral corticospinal form of weakness) and posterior columnar and contralateral spinothalamic dysfunction. Patients with compressive myelopathies or MS may also present with this form. Patients with occlusion of the anterior spinal artery often present with involvement of bilateral anterior horn cells/corticospinal tracts as well as spinothalamic and autonomic dysfunction. Lesions of the posterior cord secondary to vitamin B12 or copper deficiency present with bilateral loss of posterior column function, which translates into loss of light touch, vibration, and proprioception. Patients with central cord lesions due to NMO or syrinx suffer from dysfunction of crossing spinothalamic, corticospinal, and autonomic tracts. Clinically, these patients develop dissociated sensory loss, weakness, and autonomic dysfunction below these lesions. Lesions of the conus medullaris involve autonomic outflow and sacral spinal cord segments; clinically these patients present with sphincter dysfunction, sacral sensory loss, and mild weakness. Patients with cauda equina pathologies present with impairment of the spinal nerve roots of the cauda equina and suffer from asymmetric flaccid weakness and sensory loss of the lower extremities and autonomic dysfunction. Acute cytomegalovirus polyradiculitis is an example of cauda equina pathology. Selective tract involvement such as selective corticospinal or posterior column tractopathy occurs in metabolic or degenerative myelopathies such as vitamin B12 deficiencies or paraneoplastic syndromes. Clinically, patients with ATM initially experience ascending paresthesia or low back pain associated with weakness of the lower extremities and loss of sphincter function. The initial paresthesia progresses toward loss of pain, temperature, and vibration, and later the patient develops a sensory level, usually at the thoracic cord. Occasionally, a region of hyperesthesia for two to three segments may be present rostral to the lesion. Some patients report a band-like tightness around their trunk. Weakness in patients with ATM may affect one or both of the lower extremities and can range from paresis to complete paralysis with spasticity. In many patients the

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weakness is initially flaccid (spinal shock). Since ATM affects the cervical cord less frequently, arm weakness presents less often than leg weakness (seen in nearly 25% of patients). Autonomic pathway involvement produces urinary retention, constipation, adynamic gastrointestinal ileus, attacks of myocardial ischemia and hypertension, impaired sweating below the lesion, and other autonomic dysfunctions [2,4,5]. In most patients with ATM, within a few days to weeks after the initial presentation some of these symptoms abate. Improvement of the clinical manifestations peaks by 3–6 months after the beginning of disease and may continue for a number of years. The degree of improvement ranges from complete to partial with persistent motor, sensory, and autonomic abnormalities [4]. Recurrent transverse myelitis without dissemination of lesions to the other sites of the central nervous system (CNS) (with negative MRI and cerebrospinal fluid [CSF] studies) is uncommon, and may not progress toward MS; however, it may be a component of NMO [6–10]. Uncommonly, infectious diseases such as brucellosis and hepatitis C are associated with recurrent transverse myelitis [10].

Epidemiology Most patients with ATM are young adults, typically in their mid-teens to the mid-40s. The annual incidence of ATM is one to four new patients per 1 million, with no gender or familial predisposition [5,11–13]. Patients of African-American or Eastern Asian background present more often with the NMO picture, with more myelitis and more severe disability compared to Caucasians with MS. ATM may be a manifestation of MS, and these patients show asymmetric clinical symptoms and more sensory than motor deficits; on spinal cord MRI, they exhibit lesions that expand over less than two spinal segments, with abnormal brain MRIs and oligoclonal bands in their CSF [2,14–18].

Pathogenesis The majority of cases of ATM in adults are idiopathic, while in pediatric populations most cases are secondary to other underlying disorders. Two-thirds of ATM cases in children and one-third of ATM cases in adults are associated with viral infections [1,11,19]. Some of the reported viruses include adenoviruses, Coxsackie viruses A and B, Epstein–Barr virus (EBV), herpes simplex virus (HSV-1 and -2), influenza, mumps, rubeola, herpes zoster, and cytomegalovirus [20,21]. ATM associated with viral infections manifests between 3 and 18 days after upper respiratory tract viral infection [22–25]. Less commonly, more protracted courses of transverse myelitis have been reported in patients with HIV and human T-lymphotropic virus (HTLV-1) infections, with a duration of several days to weeks. Nonviral infections such as Mycoplasma, tuberculosis, and borreliosis are occasionally associated with transverse myelitis. Rarely, ATM may occur within the 3 weeks following vaccination due to an immune-mediated reaction to the vaccine (examples include smallpox and rabies). Such reactions have been reported with other vaccines like hepatitis B, typhoid, influenza,

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rubella, and tetanus [23–28]. A number of mechanisms have been proposed to explain cases of ATM associated with viral infections, including (1) direct injury to the cord tissue (observed in subacute sclerosing panencephalitis) and HIV myelopathy; (2) immune responses to the viral infection that harm cord tissue as a “bystander phenomenon”; (3) cord injury due to release of cytokines; and (4) induction of an autoimmune reaction against the CNS antigen(s) due to discharge of injured myelin, which may be identified as non-self and processed by antigen-presenting cells due to cross-reactivity between viral and host antigen(s) (molecular mimicry). Post-infectious encephalomyelitis [29] and post-rabies vaccination myelitis are examples of this phenomenon [30].

Neuropathology Neuropathologic changes in ATM are restricted to the spinal cord and reveal differences with those observed in MS. In patients with ATM, neuropathologic changes include both axonal loss and demyelination, and in severe cases necrosis and cavitation with extensive tissue damage are present. In MS lesions, lesions are scattered throughout the spinal cord and many of the axons are still preserved. The spinal cord lesions in ATM are symmetrical and usually bilateral. In addition, in ATM spinal cord lesions are more obvious in central parts of the cord rather than the periphery. Conversely, in patients with MS, these lesions are more peripheral (sub-pial beginning) and typically contain demyelinating lesions with roughly spared axons. Microscopically, MS spinal cord lesions demonstrate perivascular infiltration of monocytes and focal infiltration of lymphocytes into the center of the spinal cord, while in cases of transverse myelitis polymorphonuclear leukocytes as well as lymphocytes infiltrate the meningeal membranes; macrophages are usually also present in the parenchyma. Certain forms of inflammatory myelopathies such as NMO, acute hemorrhagic leukoencephalopathy, and progressive necrotizing myelopathy also reveal a necrotizing component in their neuropathology.

Diagnosis Diagnostic criteria for transverse myelitis are presented in Table 10.1. A diagnosis of idiopathic ATM requires that all the inclusion and exclusion criteria in Table 10.1 be met. Since an extensive group of disorders with dissimilar neuropathologies can cause ATM, a multistep diagnostic workup is mandatory. The first step consists of obtaining a detailed clinical history and performing a general and neurologic examination to identify fever, confusion, meningismus, genital infections, rashes, arthralgia and arthritis, pleuritis, shortness of breath, hematuria, anemia, lymphadenopathy, orogenital ulcers, hepatic or splenic enlargement, uveitis, retinitis, livido reticularis, or immunosuppressed status. Then, the most significant step is to exclude spinal cord compression by obtaining an MRI of the spinal cord. Once compressive causes of myelopathy are excluded, other causes of myelopathy should be explored by choosing the appropriate paraclinical tests. Routine laboratory tests for common myelopathies include complete blood count (CBC) and differential, C-reactive protein (CRP),

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Table 10.1  Diagnostic Criteria for Idiopathic ATM Inclusion Criteria Development of sensory, motor, or autonomic dysfunction attributable to the spinal cord Bilateral signs and/or symptoms (though not necessarily symmetric) Clearly-defined sensory level Exclusion of extra-axial compressive etiology by neuroimaging (MRI or myelography; CT of spine not adequate) Inflammation within the spinal cord demonstrated by CSF pleocytosis or Elevated IgG index or gadolinium enhancement. If none of the inflammatory criteria is met at symptom onset, repeat MRI and lumbar puncture (LP) evaluation between 2–7 days following symptom onset meets criteria Progression to nadir between 4 h to 21 days following the onset of symptoms (if patient awakens with symptoms, symptoms must become more pronounced from point of awakening) Exclusion Criteria History of previous radiation to the spine within the past 10 years Clear arterial distribution clinical deficit consistent with thrombosis of the anterior spinal artery Abnormal flow voids on the surface of the spinal cord consistent with arteriovenous malformation *Serologic or clinical evidence of connective tissue disease (sarcoidosis, Behçet’s disease, Sjögren syndrome, SLE, mixed connective tissue disorder, etc.) *CNS manifestations of syphilis, Lyme disease, HIV, HTLV-1, mycoplasma, other viral infection (e.g., HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, enteroviruses) *Brain MRI abnormalities suggestive of MS *History of clinically apparent optic neuritis *Do not exclude disease-associated ATM Cited from Ref. [3], with permission from Lippincott Williams & Wilkins. AVM  arteriovenous malformation; SLE  systemic lupus erythematosus; HTLV-1  human T-cell lymphotropic virus-1; HSV  herpes simplex virus; VZV  varicella zoster virus; EBV  Epstein–Barr virus; CMV  cytomegalovirus; HHV  human herpes virus.

antinuclear antibody (ANA), serum B12 and folate levels, rapid plasma regain (RPR), NMO-IgG antibody, and serum HIV and HTLV-I/II to distinguish common causes of myelopathy from idiopathic ATM. Other more specific paraclinical blood tests include serum angiotensin-converting enzyme level (for sarcoidosis), autoantibodies ds-DNA, sjogren syndrome type A antigen (SS-A) (Ro), Anti-La (SS-B) (La), Smith antibody (Sm), and ribonucleoprotein (RNP), complement levels, and antiphospholipid antibodies. In certain patients, a computed tomography (CT) scan of the chest, Schirmer’s test, and liver/salivary gland biopsy should be performed. Brain MRI with and without gadolinium should also be done to determine the degree of CNS involvement, which can assist toward the correct diagnosis. MR images of the spinal cord of patients with idiopathic ATM demonstrate contrast enhancement on post-gadolinium T1-weighted views; they present as hyperintense lesions on T2-weighted (Figure 10.1) and short-tau inversion-recovery (STIR) images, which indicate swelling and inflammation of the spinal cord [19,21,31–33].

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Usually there are isointense or hypointense signals on T1-weighted images. Patients with myelopathy may also have a normal MR examination of the spinal cord. In addition, the extent of spinal cord involvement in MRI does not correlate with clinical severity [34]. Typically, the center of the cord within the thoracic region is involved, as shown by MRI, and several contiguous segments of the cord are affected; other cord regions can be involved, although less commonly [35,36]. Obvious cord swelling is seen in about half of patients with transverse myelitis, but this is not prominent in MS [12]. In patients with extensive MRI lesions, which show contrast enhancement, more residual disability may be observed [37,38]. Patients with heterogeneous cord lesions or simultaneous brain involvement in MRI are more likely to develop MS [39]. CSF analysis also plays a crucial role in the diagnostic workup of ATM, particularly if no structural abnormalities are found on spinal cord MRI. The opening pressure and glucose level are normal in nearly all patients with idiopathic transverse myelitis [2,5]. However, elevated CSF protein levels (around 100 mg/100 mL) and moderate pleocytosis are present in up to 50% of adults and up to 80% of children [1,11,12,19,32,40]. Other requested tests for analysis of CSF include myelin basic

Figure 10.1  (A) MRI. Sagittal T2-weighted view showing hyperintense focus in the spinal cord corresponding to a transverse myelitis. Compare (B) transverse T2-weighted image showing the white and butterfly-shaped gray matter with (C), an image through the area of myelitis.

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protein, IgG index and oligoclonal bands in CSF (and a concomitant peripheral blood specimen), cytologic analysis, viral studies (both cultures and other methods to detect DNA or RNA viruses), and routine bacteriologic and parasitic cultures and studies. Other diagnostic exams such as CT myelograms, evoked potentials, optical coherence tomography, and electrodiagnostic tests should be performed in appropriate clinical settings.

Differential Diagnosis Table 10.2 lists diseases with heterogeneous causes in the differential diagnosis of ATM. Table 10.2  Differential Diagnosis of ATM

Inflammatory Multiple sclerosis Neuromyelitis optica Systemic lupus erythematosus Primary Sjögren syndrome Mixed connective tissue disorder Neurosarcoidosis Behçet’s disease Systemic sclerosis Antiphospholipid antibody syndrome

Vascular Anterior spinal artery occlusion Posterior spinal artery occlusion Arteriovenous fistula Hematomyelia Fibrocartilaginous disk embolism

Infectious/Infection-Associated AIDS Human T-lymphotropic virus type I Herpes simplex virus Herpes zoster virus Human herpesvirus 6 Cytomegalovirus Enterovirus

Borrelia Burgdorferi (Lyme Disease) Hepatitis A, B, C

Treponema Pallidum Mycoplasma Pneumoniae Intramedullary or epidural abscesses of spinal cord (usually due to Staphylococcus aureus) (Continued)

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Table 10.2  Diagnosis TableDifferential 10.2  (Continued ) of ATM

Metabolic–Toxic Vitamin B12 deficiency Folate deficiency Copper deficiency Celiac disease Toxins (e.g., arsenic, clioquinol, ortho-cresyl-phosphate)

Paraneoplastic Myelopathies associated with paraneoplastic antibodies/cancers Small cell lung carcinoma Breast cancer Ovarian cancer Non-small cell lung cancer Lymphoma

Other Causes Decompression sickness Radiation-induced myelopathy Trauma Herniated disk Pathologic vertebral fracture Spondylolisthesis Epidermal lipomatosis

Course and Prognosis The natural history of disease differs among patients and is only loosely related to the severity of early disease. Roughly one-third of patients recover without any sequelae and the same fraction of patients are severely disabled. The remaining third have mild to moderate disability [1,2,4,11]. Generally, patients with partial cord involvement have better outcomes than ones with complete cord involvement [12,40]. Indicators of poorer outcome include severe early weakness, a more rapid progressive course, severe initial knife-like pain, early spinal shock, incontinence, no prominent recovery after 3 months from disease onset, and the presence of a cervical sensory level [41]. Conversely, patients who are younger, with more protracted courses of progression over days to weeks, with preserved deep tendon reflexes and posterior column functions, and with early recovery have more favorable outcomes [1,2]. CSF cell and protein abnormalities are not predictive of outcome [2,5]. The risk of developing MS varies between patients with complete versus partial ATM [42]. A 2–25% of patients with partial transverse myelitis and negative brain MRI will develop MS, but patients with positive brain MRIs have a 44–85% chance [42–44]. Features that increase the probability of future MS in partial cord disease are CSF positive for oligoclonal bands, brain MRI lesions compatible with MS, presence of gadolinium-enhancing cord lesions, positive HLA-DR2, abnormal somatosensory and visual-evoked potentials, and posterolateral cord involvement [42,43–45].

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Treatment The first step in treatment is to exclude treatable causes such as compressive lesions, infections, collagen vascular disease, paraneoplastic syndromes, local tumors, and vascular diseases affecting the spinal cord. Based on the underlying pathology the appropriate treatment should be initiated (e.g., surgery for a compressive lesion, antibiotics for infection, and immunosuppressants for collagen vascular diseases). Once the secondary causes of ATM have been excluded, patients should be treated with intravenous corticosteroids (methylprednisolone 1 g intravenously daily for 5 days). Treatment with intravenous corticosteroids or adrenocorticotropin hormone (ACTH) is usually associated with significant improvement of the clinical manifestations. Treatment with corticosteroids or ACTH expedites recovery but has no significant impact on the long-term prognosis. Other symptoms of ATM such as spasticity or pain require symptomatic management. For spasticity, stretching along with muscle relaxants such as baclofen, tizanidine, and dantrolene and a benzodiazepine should be used. Pain management includes medications such as gabapentin, carbamazepine, phenytoin, or simple anesthetics. Use of anticholinergic agents may reduce urinary frequency due to a spastic bladder. Early and late physical rehabilitation can accelerate recovery.

Prognosis The prognosis of ATM is variable; as noted earlier, usually one-third of patients completely recover, one-third have only partial recovery, and one-third will not improve [1,2,4,11]. Patients with partial transverse myelitis (similar to MS) have a more favorable prognosis than those with complete myelitis, and patients with idiopathic ATM recur less frequently than patients with transverse myelitis due to MS. Poor prognostic factors include a sudden catastrophic onset, profound weakness, sensory abnormalities at cervical levels, presence of spinal shock, incontinence, and no improvement after 3 months. Irani and Kerr [41] reported accumulation of 14-3-3 protein in the CSF of patients with ATM who showed minimal or no recovery and recommended that this marker be used as a prognostic factor. Favorable prognostic factors include subacute progression of sensory or motor manifestations over several days or weeks, younger age, early recovery, and retention of posterior column function [1,2].

References [1] Lipton HL, Teasdall RD. Acute transverse myelopathy in adults. A follow-up study. Arch Neurol 1973;28:252–7. [2] Ropper AH, Poskanzer DC. The prognosis of acute and subacute transverse myelopathy based on early signs and symptoms. Ann Neurol 1978;4:51–9. [3] Group TMCW (Transverse Myelitis Consortium Working Group) Proposed diagnostic criteria and nosology of acute transverse myelitis. Neurol 2002;59:499–505. [4] Dunne K, Hopkins IJ, Shield LK. Acute transverse myelopathy in childhood. Dev Med Child Neurol 1986;28:198–204.

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  [5] Altrocchi PH. Acute transverse myelopathy. Arch Neurol 1963;9:111–9.   [6] Garcia-Merino A, Blasco MR. Recurrent transverse myelitis with unusual long-standing Gd-DTPA enhancement. J Neurol 2000;247:550–1.   [7] Grewal AK, Lopes MB, Berg CL, Bennett AK, Alves VA, Trugman JM. Recurrent demyelinating myelitis associated with hepatitis C viral infection. J Neurol Sci 2004;224:101–6.   [8] Kim KK. Idiopathic recurrent transverse myelitis. Arch Neurol 2003;60:1290–4.   [9] Jacob A, Nicholas R, Das K, Boggild M. Exploring the relationship of relapsing myelitis with neuromyelitis optica (Devic’s disease) and multiple sclerosis. Ann Neurol 2004;56(Suppl. 8):S33. A88 [10] Chan KH, Tsang KL, Fong GCY, Ho SL, Cheung RTF, Mak W. Idiopathic inflammatory demyelinating disorders after acute transverse myelitis. Eur J Neurol 2006;13:862–8. [11] Berman M, Feldman S, Alter M, Zilber N, Kahana E. Acute transverse myelitis: incidence and etiologic considerations. Neurol 1981;31:966–71. [12] Jeffery DR, Mandler RN, Davis LE. Transverse myelitis. Retrospective analysis of 33 cases, with differentiation of cases associated with multiple sclerosis and parainfectious events. Arch Neurol 1993;50(5):532–5. [13] Christensen PB, Wermuth L, Hinge HH, Bomers K. Clinical course and long-term prognosis of acute transverse myelopathy. Acta Neurol Scand 1990;81(5):431–5. [14] de Seze J, Stojkovic T, Breteau G, Lucas C, Michon-Pasturel U, Gauvrit JY, et al. Acute myelopathies: clinical, laboratory and outcome profiles in 79 cases. Brain 2001;124(Pt 8): 1509–21. [15] Miller DH, Ormerod IE, Rudge P, Kendall BE, Moseley IF, McDonald WI. The early risk of multiple sclerosis following isolated acute syndromes of the brainstem and spinal cord. Ann Neurol 1989;26(5):635–9. [16] Ungurean A, Palfi S, Dibo G, Tiszlavicz L, Vecsei L. Chronic recurrent transverse myelitis or multiple sclerosis. Funct Neurol 1996;11(4):209–14. [17] Scott TF, Bhagavatula K, Snyder PJ, Chieffe C. Transverse myelitis. Comparison with spinal cord presentations of multiple sclerosis. Neurol 1998;50(2):429–33. [18] Bakshi R, Kinkel PR, Mechtler LL, Bates VE, Lindsay BD, Esposito SE, et  al. Magnetic resonance imaging findings in 22 cases of myelitis: comparison between patients with and without multiple sclerosis. Eur J Neurol 1998;5(1):35–48. [19] Miyazawa R, Ikeuchi Y, Tomomasa T, Ushiku H, Ogawa T, Morikawa A. Determinants of prognosis of acute transverse myelitis in children. Pediatr Int 2003;45:512–6. [20] Miller HG, Stanton JB, Gibbons JL. Para-infectious encephalomyelitis and related syndromes. Q J Med 1956;25:427–505. [21] Knebusch M, Strassburg HM, Reiners K. Acute transverse myelitis in childhood: nine cases and review of the literature. Dev Med Child Neurol 1998;40:631–9. [22] Paine RS, Byers RK. Transverse myelopathy in childhood. Am J Dis Child 1953;85:151–63. [23] Das RN, Jaykumar J. Acute transverse myelitis following typhoid vaccination. Ulster Med J 2007;76:39–40. [24] Cizman M, Pokorn M, Osredkar D. Re: transverse myelitis after measles and rubella vaccination. J Paediatr Child Health 2005;41:460. [25] Fonseca LF, Noce TR, Teixeira ML, Teixeira Jr AL, Lana-Peixoto MA. Early-onset acute transverse myelitis following hepatitis B vaccination and respiratory infection: case report. Arq Neuropsiquiatr 2003;61(2A):265–8.

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[26] Booss J, Davis LE. Smallpox and smallpox vaccination: neurological implications. Neurol 2003;60(8):1241–5. [27] Larner AJ, Farmer SF. Myelopathy following influenza vaccination in inflammatory CNS disorder treated with chronic immunosuppression. Eur J Neurol 2000;7(6):731–3. [28] Ahasan HA, Chowdhury MA, Azhar MA, Rafiqueuddin AK. Neuroparalytic complications after anti-rabies vaccine (inactivated nervous tissue vaccine). Trop Doct 1995;25(2):94. [29] Label LS, Batts DH. Transverse myelitis caused by duck embryo rabies vaccine. Arch Neurol 1982;39:426–30. [30] Tartaglino LM, Heiman-Patterson T, Friedman DP, Flanders AE. MR imaging in a case of postvaccination myelitis. AJNR Am J Neuroradiol 1995;16:581–2. [31] Miller DH, McDonald WI, Blumhardt LD, du Boulay GH, Halliday AM, Johnson G, et al. Magnetic resonance imaging in isolated noncompressive spinal cord syndromes. Ann Neurol 1987;22:714–23. [32] Austin SG, Zee CS, Waters C. The role of magnetic resonance imaging in acute transverse myelitis. Can J Neurol Sci 1992;19:508–11. [33] Kalita J, Misra UK, Mandal SK. Prognostic predictors of acute transverse myelitis. Acta Neurol Scand 1998;98:60–3. [34] Andronikou S, Albuquerque-Jonathan G, Wilmshurst J, Hewlett R. MRI findings in acute idiopathic transverse myelopathy in children. Pediatr Radiol 2003;33:624–9. [35] Barakos JA, Mark AS, Dillon WP, Norman D. MR imaging of acute transverse myelitis and AIDS myelopathy. J Comput Assist Tomogr 1990;14:45–50. [36] Choi KH, Lee KS, Chung SO, Park JM, Kim YJ, Kim HS, et al. Idiopathic transverse myelitis: MR characteristics. Am J Neuroradiol 1996;17(6):1151–60. [37] Sanders KA, Khandji AG, Mohr JP. Gadolinium-MRI in acute transverse myelopathy. Neurol 1990;40:1614–6. [38] Pardatscher K, Fiore DL, Lavano A. MR imaging of transverse myelitis using Gd-DTPA. J Neuroradiol 1992;19:63–7. [39] Simnad VI, Pisani DE, Rose JW. Multiple sclerosis presenting as transverse myelopathy: clinical and MRI features. Neurol 1997;48:65–73. [40] Tippett DS, Fishman PS, Panitch HS. Relapsing transverse myelitis. Neurol 1991;41:703–6. [41] Irani DN, Kerr DA. 14-3-3 protein in the cerebrospinal fluid of patients with acute transverse myelitis. Lancet 2000;355:901. [42] Scott TF, Kassab SL, Singh S. Acute partial transverse myelitis with normal cerebral magnetic resonance imaging: transition rate to clinically definite multiple sclerosis. Mult Scler 2005;11(4):373–7. [43] Perumal J, Zabad R, Caon C, MacKenzie M, Tselis A, Bao F, et  al. Acute transverse myelitis with normal brain MRK: long-term risks of multiple sclerosis. J Neurol 2008;255:89–93. [44] Sellner J, Luthi N, Buhler R, Gebhardt A, Findling O, Greeve I, et  al. Acute partial transverse myelitis: risk factors for conversion to multiple sclerosis. Eur J Neurol 2008;15:398–405. [45] Bashir K, Whitaker JN. Importance of paraclinical and CSF studies in the diagnosis of multiple sclerosis patients presenting with partial cervical transverse myelopathy: cranial MRI. Mult Scler 2000;6:312–6.

11 Neuromyelitis Optica Natalie Cornay1, Robin Davis1, Meghan Harris2, Brian Rabin2, Karen Small1, Edward Johnson1, Eduardo Gonzalez-Toledo2,3, Stephen Jaffe2, Alireza Minagar 2 1 

Louisiana State University School of Medicine, Shreveport, LA, USA Department of Neurology, Louisiana State University School of Medicine, Shreveport, LA, USA 3  Department of Radiology, Louisiana State University School of Medicine, Shreveport, LA, USA 2 

Definition and Epidemiology Neuromyelitis optica (NMO) is an inflammatory, immune-mediated disease that targets the optic nerves and spinal cord. Dr. Eugene Devic is credited with the original description of NMO. However, Sir Thomas Clifford Allbutt recognized the combination of optic neuritis (ON) and myelitis almost 20 years earlier [1–4]. NMO may present with either a relapsing–remitting course or a primary progressive course with fulminant disability [4]. Only 10–20% of patients have this monophasic course; approximately 80–90% have a relapsing–remitting course [5]. NMO is often misdiagnosed as multiple sclerosis (MS), since the two diseases can present clinically in a similar fashion. The prevalence and incidence of NMO have not been well established partly due to its original designation as a subtype of MS. Females outnumber males by 3:1, and females more often develop a relapsing–remitting course [4]. Both children and adults may be affected. Asian populations are affected more often than Caucasians; in fact, Asian optico-spinal MS appears to be identical to NMO [6].

Pathophysiology Neuropathologically, NMO lesions demonstrate perivascular and parenchymal leukocyte accumulation, hemorrhagic exudation, and finally fibrosis and tissue necrosis. The underlying pathophysiology of NMO is not completely understood; however, there is strong evidence to support a role for humoral immunity. The NMO-IgG antibody was discovered at the Mayo Clinic by Lennon et al. when a staining pattern in the subpial and Virchow-Robin spaces of rat brain sections being studied for paraneoplastic antibodies was found to be nearly identical to a stained brain section Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00011-3 © 2011 Elsevier Inc. All rights reserved.

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obtained from an NMO patient. Their work was verified when the same staining pattern was identified in 35 out of 45 Japanese and North American NMO patients, giving a sensitivity of 73% for antibody-positive patients. Additionally, brain slices from 14 patients who underwent a study concentrating on paraneoplastic antibodies also demonstrated this specific staining pattern [3,7]. Upon case review, the clinical history of these patients suggested a diagnosis of either NMO or transverse myelitis. Once the antibody was identified, it was shown that the aquaporin water channel, specifically aquaporin 4 (AQP4), was the binding target of the NMO-IgG. This was based on the staining patterns previously discussed, which localized the antibody to the pericapillary regions of astrocytes and the abluminal faces of cerebral microvessels [8,9]. Lennon et al. confirmed this in a series of experiments using AQP4 knockout mice, NMO-IgG KEK cell recognition, and immunoprecipitation [3,9]. Several pathologic studies have shown consistent loss of AQP4 reactivity in acute NMO lesions [10,11]. This occurs both with and without demyelination, suggesting that loss of AQP4 can occur independently of demyelination [10,12]. Importantly, NMOIgG antibody has not been detected in other neuropathologic conditions.

Clinical Manifestations Clinically, NMO is characterized by severe attacks of ON (Figure 11.1) and/or transverse myelitis (Figure 11.2). The clinical onset is often abrupt and may produce a prodrome that can include headache and low-grade fever. Upper respiratory symptoms

Figure 11.1  Coronal fat-suppressed MRI showing enhancement of the optic nerves after contrast administration, consistent with optic neuritis.

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or gastrointestinal symptoms often precede the neurologic events. Usually the first clinical manifestation of NMO is ON, which is seen in 56–76% of patients [13]. The attacks of ON can be recurrent or simultaneously bilateral [5]. Merle et al. studied ophthalmologic signs and symptoms in patients diagnosed with NMO and found that ON may present in one eye or more rarely both eyes, and it usually develops before myelitis. Loss of visual acuity to at least 20/200 occurred in one eye within 15–33 months of disease onset, while progression to bilateral optic nerve involvement occurred within 10–16 years of NMO onset. This significant visual loss was often seen after only two attacks. Overall, NMO has a much worse prognosis for residual visual acuity loss than in MS [14]. ON and myelitis occur simultaneously in 4–11% of patients. The time period for involvement of both spinal cord and optic nerve may vary from a few days to years [15]. A diagnosis of NMO may be easily missed if the time interval between the spinal cord and optic nerve involvement spans several years. Transverse myelitis in NMO by definition involves three or more segments of the spinal cord, and myelitis may be the presenting feature. These patients usually have a severe symmetric paraplegia and sensory loss below the level of the lesion. The most common site of myelitis is the thoracic cord (66%) followed by the cervical cord (24%). Bladder and anal sphincter disturbances occur in approximately 87% of

Figure 11.2  Sagittal STIR MRI sequence showing an extensive hyperintense lesion in the cervical spinal cord consistent with demyelination.

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patients, while respiratory failure affects 22% of patients [13]. Bladder dysfunction usually presents with retention, and most patients are not aware of their bladder difficulties. The clinical NMO episodes are usually more severe than those occurring in MS, and the likelihood of recovery is less [16]. Lhermitte’s sign, an “electric shock” sensation traveling down the back with neck flexion, is often a sign of cervical spinal cord involvement and can often be seen in patients with cervical myelitis [17]. These patients may also develop spasms of the limbs and trunk, which can be painful and debilitating. Lesions in the cervical spinal cord of NMO patients can extend into the brain stem, resulting in nausea, hiccups, and even acute respiratory failure and death. It is uncommon to see acute respiratory failure in MS patients, as the lesions in MS are typically partial and usually do not involve multiple vertebral segments [5]. Because of the risk of respiratory failure, an attack of NMO is often considered a medical emergency.

Diagnostic Criteria In 2006, Wingerchuk et al. introduced the most recent set of diagnostic criteria as a refinement of criteria proposed by the same group in 1999. The 2006 criteria have been simplified to include the presence of (1) ON and (2) acute myelitis with (3) at least two of the following: (a) a contiguous, longitudinal lesion at least three cord segments in length; (b) magnetic resonance imaging (MRI) of the brain that does not meet MS diagnostic criteria; and (c) NMO-IgG seropositivity [18]. The 2006 criteria have been put into regular practice since their proposal and have been determined to have a specificity of 83.3% and a sensitivity of 87.5%. While the current criteria are indeed more specific, the sensitivity of this criteria set has decreased from the 93.3% sensitivity of the 1999 set [19]. Recently, an imaging-based diagnostic approach has yielded a sensitivity of 94% and a specificity of 96%. An NMO diagnosis is based on patients having an MRI that does not fulfill MS diagnostic criteria and a spinal lesion measuring at least three vertebral segments [20]. Current thought has allowed for the assignment of an NMO diagnosis to patients meeting the 2006 criteria with slightly aberrant presentations such as unilaterally symptomatic ON, an indeterminate amount of time between the initial event of ON or myelitis followed by its counterpart, and relapses occurring an unspecified amount of time after the initial attack [16]. The spinal lesions of NMO are readily identified on T2-weighted or short inversion time inversion recovery (STIR) MRI (see Figure 11.2) as extensive longitudinal lesions over three or more vertebral segments that enhance with gadolinium on T1 sequences [18,21]. In the acute phase, ON lesions (see Figure 11.1) will also enhance, and no other cerebral lesions will be apparent [16]. This lack of additional lesions as a diagnostic criterion has been called into question by Wingerchuck et al., among others, who have considered allowing the existence of additional brain and spinal pathology as long as major criteria such as spinal lesion length are met [18]. This was supported by Pittock et al., who stated that although being a somewhat rare

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presentation, lesions of the deep white matter and the basal ganglia in NMO may be pathologically typical, especially in children [22].

NMO-IgG At the forefront of the development of NMO diagnostic criteria is the use of the autoantibody NMO-IgG. Recognition of the anti-AQP4, or NMO-IgG, antibody has allowed for deeper understanding of its predictive value in NMO patients. Since its discovery, its presence in an NMO patient has been linked to an increased number of attacks experienced by that patient, along with increased residual disability following resolution of the acute phase [23]. Weinshenker et al. found NMO-IgG to be predictive of which NMO patients would experience at least one relapse. In 38% of studied NMO patients with initial attacks of transverse myelitis identified as seropositive for NMO-IgG, 55% experienced a recurrence of disease within 1 year of the initial event. In contrast to this, no seronegative patients experienced recurrent symptoms within the year [24]. Another study indicated that of 22 patients diagnosed with MS, only 2 were NMO-IgG antibody seropositive, providing a specificity of 91% [3,7]. Emphasizing the diagnostic importance of the antibody, Matiello et al. showed that patients of nonCaucasian ancestry were more likely to be NMO-IgG seropositive. Moreover, seropositive patients were more likely to experience a more severe initial attack of ON, and patients who went on to develop myelitis after ON onset had significantly higher antibody titers than patients who did not experience myelitis during the study period. In their study, recurrent longitudinally extensive transverse myelitis (LETM) patients had a 52% seropositivity rate, while 40% of patients experiencing a single, isolated episode of LETM were seropositive [25]. Takahasi et al. confirmed suspicions regarding anti-AQP4 status and disease severity in a study that positively correlated persistent total blindness after acute ON, brain lesion severity, and the peak size of spinal lesions to anti-AQP4 titer levels [26]. The investigators also demonstrated that treatment with high doses of corticosteroids resulted in a significant drop in anti-AQP4 titers, with an apparent decrease in symptom severity [26]. Finally, the concordance of complement deposition and eosinophilic infiltration has been identified in areas of NMO-IgG deposition on brain biopsy, which provides both insight into the disease process as well as an additional, though nontraditional, route for diagnosis of NMO [27].

Visual Evoked Potentials and Ocular Coherence Tomography Current studies employ visual evoked potentials (VEPs) and optical coherence tomography (OCT) to support the diagnosis as well as the appropriate identification of patients with NMO. VEP, in particular, has shown promise linking NMO-IgG seropositivity to increased ON severity. VEP analysis of a group of NMO patients showed that a significantly greater number of NMO-IgG-positive patients lacked the VEP P100 component completely compared to control IgG-seronegative patients, while antibody-negative patients were significantly more likely to experience only a delayed latency of the P100 in both the acute and remission phases of the disease [28].

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OCT has been used to show the loss of retinal fiber layers associated with visual acuity changes occurring in NMO [29]. MS spectrum disorders and NMO both involve thinning of the retinal nerve fiber layer (RNFL) to some degree, but there was a significantly greater decrease in both the thickness of the mean RNFL and macular retinal ganglion cell volume in NMO patients compared to relapsing– remitting MS patients. Also, VEP latencies were longer, visual field defects were larger, and visual acuity showed a greater reduction in NMO patients. The first attack of NMO ON was responsible for the greatest decrease in RNFL thickness when compared to damage from subsequent ON episodes [29–31]. Ratchford et al. [31] have postulated that the decrease in RNFL change after the first ON episode may be due to the fact that a lower percentage of axons are available to be affected by subsequent attacks.

Differential Diagnosis The differential diagnosis of NMO is quite extensive given the number of physiologic etiologies that can lead to ON, transverse myelitis, or a combination of the two. In constructing a differential for NMO, one must first include diseases falling into the spectrum of inflammatory demyelinating diseases. These include isolated ON, isolated transverse myelitis, acute demyelinating encephalomyelitis, Balo’s concentric sclerosis, relapsing myelitis, and benign MS [32]. In considering patients at initial presentation with limited imaging and laboratory workup, the differential must be expanded to include all manner of viral, bacterial, and parasitic infections; toxic exposures; neoplastic and paraneoplastic processes; degenerative vascular disease; sarcoidosis; and autoantibody syndromes [33]. Early differentiation of NMO, and particularly distinction from MS, is critical as it allows for appropriate early treatment interventions specific for NMO while preventing the use of both unnecessary and inappropriate therapies [11]. Complicating and confusing the specific diagnosis of NMO is the relative frequency with which ON and myelitis occur in MS. Moreover, myelitis and ON are not infrequent presentations of Sjögren syndrome and systemic lupus erythematosus [16]. Further complicating the differential is the fact that autoantibodies such as ANA and SSA/SSB are frequently found in NMO patients, but their significance is uncertain since conflicting reports exist as to their prevalence in NMO-IgG-seropositive patients [34]. As MS and NMO are frequently confused diagnostically, careful attention must be given to distinguishing their clinical presentations. In contrast to MS, NMO ON patients may have mild papilledema, a central scotoma on visual field testing, color blindness, and bitemporal hemianopsia [33]. The severity of the ON episode is dramatically more pronounced and eventual visual recovery is markedly decreased compared to that of MS patients. Recurrent isolated ON without the evidence of MS brain plaques also serves as a hallmark of NMO, but it is a presentation pattern that is often overlooked during the diagnostic process [35]. NMO may also present with persistent hiccupping and respiratory failure, symptoms not usually seen in MS patients.

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Lhermitte’s sign and paroxysmal tonic spasms are not good distinguishing signs for the two disorders since each occurs with similar frequency in both disorders [35,36]. Diagnostic testing will ultimately serve to distinguish NMO from MS, specifically antibody testing, cerebrospinal fluid (CSF) analysis, and neuroimaging. Beyond the previously discussed NMO-IgG, acute NMO often has a helpful presentation in the CSF, with a mostly polymorphonuclear pleocytosis (>50 cells/mm3; 39%), infrequent oligoclonal bands (34%), and an elevated protein level (25%) [21]. The polymorphonuclear pleocytosis is more common in NMO than MS during an acute myelitis attack, and while the majority of MS patients possess oligoclonal banding, only 20–40% of NMO patients have such a banding pattern [21]. Lack of this banding pattern can only be interpreted as suggestive of NMO, however, as studies indicate that the sensitivity and specificity of oligoclonal band absence is quite low [18]. Differentiating the ocular signs and symptoms in MS and NMO may be difficult; but as previously discussed, OCT can be useful [37]. While both MS and NMO patients may present with visual impairment, the distinction between the diseases can be seen at the level of the optic nerve and the pathologic characteristics of the lesions themselves. NMO patients have optic nerves that appear softer and grayer in color, and the lesions may actually extend across the optic chiasm [38]. MS optic nerve lesions do not show the atrophy and necrosis seen in NMO [14]. Even the actual demyelination in NMO shows more of a preference for the optic nerve’s central area, and this may create a central hollowing [39]. From the neuroimaging viewpoint, the brain lesions of MS identified on MRI are often greater than 3 cm in length, ovoid, and located in the periventricular, cerebellar, and infratentorial regions [40]. On the other hand, MRI brain lesions, which are sometimes present in NMO, are seen in the brain stem and diencephalon [40]. In the spine, MS plaques are shorter and are regularly less than one segment in length [16]. However, the diagnosis of NMO cannot be excluded in a patient who has only a 1-cm spinal lesion, since NMO lesions may partially resolve and thus decrease in size, necessitating a review of prior spinal MRIs [16,41]. To add to the diagnostic confusion, as NMO lesions age, they may take on the radiographic appearance of MS lesions [18]. NMO-IgG seropositivity may also provide insight into the interpretation of imaging results, as there is a higher frequency of deep white matter lesions as well as a comparatively longer length of the spinal cord lesion in seropositive compared to seronegative NMO patients [34]. Finally, it may be difficult to assign the diagnosis of NMO during an initial attack, but it should always be remembered that recurrent attacks of ON and/or myelitis with no abnormalities otherwise on the brain MRI are highly suggestive of NMO, being a pathophysiologic hallmark of this disease [18].

Treatment Evidence for the efficacy of treatment options for NMO is limited mainly to case reports and small retrospective studies. Large randomized controlled trials are not practical because of the rarity of the disease and the ethical problem of placebo

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treatment of a disease that has a high morbidity and mortality and thus requires early treatment. Comparative studies of the therapeutic options are also limited. Because humoral immunity is implicated in the pathogenesis of NMO, therapy focuses on immunosuppression, especially modification of B-cell responses [42]. Therapy for NMO involves treatment of acute attacks and maintenance immunosuppression for prevention of relapses. A basic treatment plan for NMO proposed by Wingerchuk and Weinshenker designates corticosteroids for acute attacks, with plasmapheresis added to the steroid regimen in cases of steroid failure. For prevention of relapses in mild NMO, low-dose steroids and chemotherapeutic agents are recommended; mycophenolate mofetil should be reserved for moderate NMO and rituximab for severe NMO [43]. For acute attacks, intravenous corticosteroids are the first line of treatment. There are no studies specifically corroborating their use in NMO ON or myelitis attacks; however, studies in MS patients show benefit [44,45]. Intravenous methylprednisolone given at a dose of 1000 mg daily for five consecutive days with an optional oral prednisone taper is a common protocol [46]. In acute attacks, plasma exchange as an add-on to corticosteroid therapy is the accepted treatment for minimizing residual impairment. In a retrospective study of 43 patients with acute NMO and extensive transverse myelitis, daily plasma exchange for 5 days with concomitant steroid treatment was compared to steroid-only treatment [47]. Residual impairment, defined in this study as a change from baseline to a higher residual Expanded Disability Status Scale (EDSS), was lower in the group treated with plasma exchange and steroids than in the group treated with steroids alone. NMO-IgG seropositivity was found to be irrelevant to the efficacy of plasma exchange. There was no difference in the efficacy of plasma exchange in patients in whom exchange was initiated up to 41 days after attack onset. In patients with NMO, the progression of disability is attack-dependent; therefore, maintenance immunosuppression is an important component of therapy. Maintenance immunotherapy should be started when a patient has two or more attacks and should be considered with a first attack if there is longitudinally extensive myelitis and NMO-IgG seropositivity, as these characteristics are associated with a high risk of relapse [46]. Immunosuppressive drugs have been shown to be more effective than immunomodulatory drugs such as interferon-beta or glatiramer acetate (GA) for longterm therapy [48]. Unfortunately, comparative studies of the different immunosuppressants used to treat NMO do not exist. One of the first-line options in maintenance immunosuppression is azathioprine combined with corticosteroids. The current standard protocol used by many neurologists consists of azathioprine, 2–3 mg/kg daily, along with oral prednisone, 1 mg/kg daily, followed by a slow taper of the prednisone [43]. The results of a prospective uncontrolled study of a cohort of seven patients with NMO suggest there is benefit with this combination in improving EDSS, and preventing relapses for more than 18 months [49]. A course of high-dose intravenous methylprednisolone was given, 500 mg twice daily, as a 2-h infusion for five consecutive days, followed by oral prednisone (1 mg/kg/day) for 2 months. Oral azathioprine (2 mg/kg/day) was started at week 3 with a slow taper of prednisone at the end of the second month. None of the patients

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experienced serious side effects that necessitated discontinuation of therapy. The main side effects of azathioprine were gastrointestinal upset and hepatotoxicity [43]. Other chemotherapeutic drugs that have been used in combination with corticosteroids include cyclophosphamide and methotrexate. Cyclophosphamide can be given as monthly pulses of 0.5–1.5 g/m2 for 6–8 months. Methotrexate is given at a dose of 7.5–15 mg weekly with folate supplementation [50]. Monotherapy with low-dose corticosteroids has also been proposed and would have the benefit of avoiding the side effects of additive chemotherapeutic agents. In a retrospective study of nine patients with NMO, low-dose corticosteroid monotherapy was found to decrease the annual relapse rate [51]. The authors compared the relapse rate during the use of corticosteroids with that during corticosteroid-free periods and found the relapse rate to be significantly lower when patients were taking corticosteroids. Doses of greater than 10 mg/day were associated with a lower relapse rate than doses less than 10 mg. Studies comparing corticosteroid monotherapy to therapy combining corticosteroids with chemotherapeutic drugs are warranted. Two chemotherapeutic agents that have shown benefit for more severe NMO are mitoxantrone and mycophenolate mofetil. Both drugs have a cytostatic effect on both T and B lymphocytes [42]. In a small prospective 2-year study of five NMO patients, Weinstock-Guttman et al. explored the treatment of NMO with mitoxantrone. In four of the five patients, this drug had a beneficial effect based on reduction in relapse rates and stabilization or improvement of the EDSS and the MRI [52]. Mitoxantrone was given intravenously 12 mg/m2 monthly for 6 months followed by three additional treatments every third month until a maximum dose of 100 mg/m2 was reached, at which time azathioprine and methylprednisolone were given until the end of the 2-year study period. Mitoxantrone has a restricted cumulative life dose of 100 mg/m2 because of cardiotoxicity, and its use requires lifetime ejection fraction monitoring [52]. Use of mitoxantrone has also been associated with an increased risk of developing myeloid leukemia [42]. Mycophenolate mofetil reversibly inhibits inosine monophosphate dehydrogenase, a necessary enzyme in guanosine nucleotide synthesis, thereby blocking T- and B-cell proliferation [53]. Mycophenolate mofetil can be given alone or with pulse doses of steroids, intravenous immunoglobulin (IVIG), or plasmapheresis. Falcini et al. described a case of a 9-year-old girl with NMO who failed to respond to a trial of azathioprine but did respond to 2 g/day of mycophenolate mofetil with a 2-year relapse-free period and reduction in lesion load on MRI [54]. A retrospective analysis of 24 patients with NMO spectrum disorders showed that mycophenolate (median dose 2 g/day) reduced annualized relapse rates in 79% and stabilized or reduced disability as measured by EDSS in 91% of patients [55]. In one of the patients, low white blood cell counts necessitated discontinuation of the drug. Mycophenolate mofetil has also been associated with progressive multifocal leukoencephalopathy when used with other immunosuppressants in transplant and systemic lupus erythematosus patients [56]. Rituximab, a chimeric human-murine anti-CD20 monoclonal antibody that selectively depletes CD20 B cells, is a promising drug for maintenance immunosuppression in patients with NMO. In an open-label study of eight patients with NMO,

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patients received rituximab intravenously at a dose of 375 mg/m2 given once per week for 4 weeks, with bimonthly monitoring of B-cell counts [57]. If B-cell counts became detectable, patients were given the option of retreatment with rituximab. Two IV infusions of 1000 mg, 2 weeks apart, constituted retreatment. Six of eight patients remained attack-free for a median period of 12 months, with a median pretreatment attack rate of 2.6 attacks per patient per year decreasing to 0 attacks per patient per year. In a retrospective analysis of 25 NMO patients given infusions of rituximab, 80% of patients appeared to have a reduction in the frequency of relapses, and an improvement or stabilization of disability [58]. Patients were treated with one of two rituximab regimens: either 375 mg/m2 infused once per week for 4 weeks or 1000 mg infused twice with 2 weeks between infusions. Seventeen patients received rituximab retreatment. Rituximab use was associated with infection in 5 of 25 patients (20%) and included reactivation of herpes simplex virus, positive tuberculosis skin test conversion, recurrent C. difficile, cutaneous fungal infection, and a fatal urinary tract infection-related septicemia. Rituximab use has also been associated with progressive multifocal leukoencephalopathy in patients with systemic lupus erythematosus or systemic vasculitis, and lymphoma patients treated with rituximab in combination with other immunosuppressants [59]. In addition, rituximab is associated with a risk of infusion reactions, including fever, angioedema, and hypotension [46]. Rituximab treatment also has the disadvantage of being significantly more expensive than other immunosuppressants. A case report by Capobianco et al. [60] paints a less optimistic picture of the efficacy of rituximab in NMO. They described a patient with very severe NMO who was given rituximab (375 mg/m2 weekly for 4 weeks) after failing to respond to treatment with steroids and cyclophosphamide. Within 1 month of completion of treatment, the patient had nondetectable CD19 B cells but experienced a relapse with an increase in EDSS and a new spinal lesion on MRI. The same authors presented a case of another patient with NMO who was treated with rituximab and experienced initial clinical improvement, but then had a relapse and new spinal lesion on MRI when CD19 B cells became detectable 6 months after the completion of treatment. A second course of rituximab was then administered, with undetectable CD19 cells at 6 months and no further clinical relapses. Based on the correlation between CD19 levels and disease activity in the second patient, the authors suggested more frequent monitoring of CD19 levels than the bimonthly detection proposed by previous studies. Another proposed maintenance immunosuppressive regimen is monthly intravenous gamma globulin. Because antibodies are implicated in the pathogenesis of NMO, IVIG (blocking antibodies) seems a promising therapy [42]. Information on the efficacy of IVIG is limited. Bakker and Metz [61] reported two cases in which patients with NMO unresponsive to other immunosuppressive therapy were started on IVIG and experienced 5-year and 1-year relapse-free periods with improvement in neurologic status. In addition to its use in acute therapy, plasmapheresis may also be indicated for long-term therapy. Miyamoto and Kusunoki [62] proposed the use of intermittent plasmapheresis in combination with immunosuppressants to prevent attacks in patients in whom steroids and other maintenance immunosuppression have failed.

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They reported four patients in whom plasmapheresis—plasma exchange, double filtration plasmapheresis, or immunoadsorption plasmapheresis—was successful in preventing relapse and in achieving functional improvement. The patients received one to five plasmapheresis treatments over a 1- to 2-week period in addition to maintenance therapy with prednisolone and azathioprine or cyclophosphamide. Immunomodulating drugs, including GA and interferon-beta, are generally less effective for NMO than they are for MS [63]. GA likely works by inducing immunomodulatory Th2-like populations. Because activation of eosinophils with a resultant Th1 to Th2 shift and thus further activation of eosinophils is implicated in the pathophysiology of NMO, a drug that induces a Th1 to Th2 shift seems problematic. However, the proposed benefit of GA may be due to the fact that GA-specific Th2 cells produce brain-derived neurotrophic factor, which may promote neuronal survival and repair of myelin [64]. There have been many case reports of a decrease in relapse rate in NMO with the use of GA. Gartzen et al. [65] described a case of a 48-yearold woman with relapsing NMO who failed to respond to cyclophosphamide and steroid treatment but did respond to GA (20 mg subcutaneously daily) and monthly steroid pulses with a reduction in relapses from three in 11 months on cyclophosphamide to a relapse-free period of over 38 months on GA. MRI at 12 months of therapy showed previous cervical lesions to be in remission, with no new lesions. Bergamaschi et al. [66] reported a case of a patient with relapsing NMO whose relapse rate was reduced from 0.93/year to 0.25/year after starting GA. Despite lack of evidence for the efficacy of GA and suggestions that immunomodulation may not be as effective as immunosuppression, GA should not be discarded as a therapeutic option. It has a good safety profile and could be useful in patients who cannot tolerate immunosuppressive drugs. Interferon-beta, another immunomodulatory agent that has been shown to be effective in MS, has not been shown to be effective in NMO—and a few studies suggest it may even be harmful in NMO. In a retrospective study, Tanaka et al. analyzed the effects of IFNβ-1b treatment on disease exacerbation and disability progression in MS and NMO patients. The relapse numbers and annualized relapse rates in the MS patients significantly decreased within 1 year after interferon treatment (250 μg of interferon-beta subcutaneously every other day for 1 year), but there was no statistically significant decrease in the NMO group. NMO patients also had a greater change in EDSS 1 year after treatment initiation than MS patients. Tanaka et al. [67] concluded that interferon treatment was not effective in reducing either the relapse rate or the disability progression of NMO patients. Another study retrospectively reviewed therapies in 26 patients with NMO and compared the probability of relapse on interferon therapy with the probability of relapse on immunosuppressive therapy such as cyclophosphamide, mitoxantrone, or azathioprine. The probability of relapse was found to be significantly lower in the immunosuppressive drug group [48]. Shimizu et al. [68] described a case of a woman with NMO, and a case of a woman with recurrent LETM without ON. Both patients were positive for anti-AQP4 antibody. The patients received interferon and within 2 months of treatment initiation, both developed extensive tumefactive brain lesions that were not present before interferon therapy. The authors suggested that if the interferon administration was causative,

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it have been due to interferon’s transient upregulation of Th1 cytokines. Another study in Japan by Warabi et al. [69] retrospectively analyzed the effects of IFNβ-1b treatment on patients with NMO, MS, and borderline MS with NMO features. The study found that patients with NMO or MS with severe optic nerve and spinal cord demyelination mimicking NMO received no benefit from IFNβ-1b treatment and that the clinical status of such patients was actually exacerbated (increase in relapse rate and disease progression) during treatment. One important side effect of interferon treatment is skin ulceration at the injection site, which can be severe enough to necessitate surgical treatment [69]. Symptomatic therapies for NMO involve the use of anticonvulsants for paroxysmal tonic spasms, antispasticity drugs and botulinum toxin injections for spasticity, and tricyclic antidepressants or anticonvulsants for neuropathic pain [43]. There is no evidence that diet and lifestyle factors are beneficial, but physical therapy may be helpful. Interventions such as mechanical ventilation or ambulatory assistive devices may be temporarily required during attacks [43]. Research concerning therapy for NMO is currently under way. With the recent finding that NMO is associated with a specific autoantibody (anti-AQP4), new therapies will likely focus on this target—especially if the antibody is found to have a definite pathophysiologic relationship to NMO [70]. Controlled studies of these potential therapies will be necessary but difficult, as previously discussed.

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[51] Watanabe S, Misu T, Miyazawa I, Nakashima I, Shiga Y, Fujihara K, et  al. Low-dose corticosteroids reduce relapses in neuromyelitis optica: a retrospective analysis. Mult Scler 2007;13:128–32. [52] Weinstock-Guttman B, Ramanathan M, Lincoff N. Study of mitoxantrone for the treatment of recurrent neuromyelitis optica (Devic disease). Arch Neurol 2006;63:957–63. [53] Allison AC, Eugui E. The design and development of an immunosuppressive drug, mycophenolate mofetil. Springer Semin Immunopathol 1993;14(4):353–80. [54] Falcini F, Trapani S, Ricci L, Resti M, Simonini G, de Martino M. Rheumatology 2010;45(7):913–5. [55] Jacob A, Matiello M, Weinshenker B, Wingerchuk D, Lucchinetti C, Shuster E, et  al. Treatment of neuromyelitis optica with mycophenolate mofetil: retrospective analysis of 24 patients. Arch Neurol 2009;66(9):1128–33. [56] US Food and Drug Administration Communication about an ongoing safety review of CellCept (mycophenolate mofetil) and Myfortic (mycophenolic acid). Rockville, MD: US Food and Drug Administration; 2008. [57] Cree BA, Lamb S, Morgan K, Chen A, Waubant E, Genain C, et al. An open label study of the effects of rituximab in neuromyelitis optica. Neurology 2005;64(7):1270–2. [58] Jacob A, Weinshenker BG, Violich I, McLinskey N, Krupp L, Fox RJ, et al. Treatment of neuromyelitis optica with rituximab; retrospective analysis of 25 patients. Arch Neurol 2008;65(11):1443–8. [59] US Food and Drug Administration. Rituximab (marketed as Rituxan) information [FDA alert]. Rockville, MD: US Food and Drug Administration; 2006. [60] Capobianco M, Malucchi S, Di Sapio A, Gilli F, Sala A, Bottero R, et  al. Variable responses to rituximab in neuromyelitis optica (Devic’s disease). Neurol Sci 2007;28:209–11. [61] Bakker J, Metz L. Devic’s neuromyelitis optica treated with intravenous gamma globulin (IVIG). Can J Neurol Sci 2004;31:265–7. [62] Miyamoto K, Kusunoki S. Intermittent plasmapheresis prevents recurrence in neuromyelitis optica. Ther Apher Dial 2009;13:505–8. [63] Bergamaschi R. Glatiramer acetate treatment in Devi’s neuromyelitis optica. Brain 2003;126:e1. [64] Ziemssen TZ, Kumpfel T, Klinkert WEF, Neuhaus O, Hohlfeld R. Glatiramer acetatespecific T-helper 1- and 2-type cell lines produce BDNF: implications for multiple sclerosis therapy. Brain 2002;125:2381–91. [65] Gartzen K, Limmroth V, Putzki N. Relapsing neuromyelitis optica responsive to glatiramer acetate treatment. Eur J Neurol 2007;14:e12–13. [66] Bergamaschi R, Uggetti C, Tonietti S, Egitto MG, Cosi V. A case of relapsing neuromyelitis optica treated with glatiramer acetate. J Neurol 2003;250:359–61. [67] Tanaka M, Tanaka K, Komori M. Interferon-β1b treatment in neuromyelitis optica. Eur Neurol 2009;62:167–70. [68] Shimizu Y, Yokoyama K, Misu T, Takahashi T, Fujihara K, Kikuchi S, et  al. Development of extensive brain lesions following interferon beta therapy in relapsing neuromyelitis optica and longitudinally extensive myelitis. J Neurol 2008;255:305–7. [69] Warabi Y, Matsumoto Y, Hayashi H. Interferon beta-1b exacerbates multiple sclerosis with severe optic nerve and spinal cord demyelination. J Neurol Sci 2007;252(1):57–61. [70] Petzold A, Pittock S, Lennon V, Maggiore C, Weinshenker BG, Plant GT. Neuromyelitis optica-IgG (aquaporin-4) autoantibodies in immune mediated optic neuritis. J Neurol Neurosurg Psychiatry 2010;81(1):109–11.

12 Optic Neuritis:

Pathophysiology, Clinical Features, and Management Clare Fraser, Gordon T. Plant The National Hospital for Neurology and Neurosurgery, London, UK Moorfields Eye Hospital, London, UK St Thomas’ Hospital, London, UK Failure of sight limited to one eye, often accompanied by neuralgic pain about the temple and orbit and by pain in moving the eye; many recover but permanent damage and even total blindness may ensue; there is at first little, sometimes no, ophthalmoscopic change, but the disk often becomes more or less atrophic in a few weeks … The defect in vision is often described at first as a “gauze” or a “yellow mist” or a “dark patch” or a “spot” which covers the object looked at and gives an unnatural colour, the hand looking, for example, as if covered by a brown glove. Edward Nettleship, St Thomas’ Hospital, London, 1884 [1]

Introduction In the early medical literature the term “optic neuritis” (ON) was used to describe disk swelling from any cause that included papilledema, for which the most common cause was a brain tumor. Over the first half of the 19th century, Frick described a type of blindness due to “some immediate affection of the optic nerve” and the term “optic neuritis” began to be associated with a discrete disease of the optic nerves [2]. With the advent of the ophthalmoscope in 1851 descriptive accounts of ophthalmic disease were refined. In particular, the 1871 text On the Use of the Ophthalmoscope in Diseases of the Nervous System and of the Kidneys served to further define the clinical features and differential diagnosis of ON [3]. Edward Nettleship published what is described as the most comprehensive early account of ON in English in 1884 [4]. Since then, and despite ongoing advances in neurodiagnostic techniques, the medical history and examination of the afferent visual sensory system described remain at the core of the diagnosis of ON. The annual incidence of ON ranges from 1.4 to 6.4 new cases per 100,000 in a predominantly Caucasian population [5,6]. The incidence of ON is highest in populations located at higher latitudes, in the northern United States and western Europe, and is lowest in regions closer to the equator [7]. In Asian countries, however, the Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00012-5 © 2011 Elsevier Inc. All rights reserved.

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incidence of ON is much lower, at 0.83 per 100,000, with higher rates of papillitis and lower rates of multiple sclerosis (MS) than reported in the Optic Neuritis Treatment Trial (ONTT) [8]. Patients with ON have a good prognosis, but a minority of patients experience persistent visual loss. Patients with neuromyelitis optica (NMO) generally have a poorer recovery. When ON is associated with other central nervous system (CNS) diseases, the morbidity and mortality of those disorders contribute substantially to the final outcome.

Definition ON is an inflammatory disease of the optic nerve. The inflammation can be due to primary demyelination as part of disease spectrum that includes MS (typical ON) or inflammation and demyelination secondary to other causes (atypical ON). The typical ON cases are usually acute and unilateral and will recover spontaneously. Atypical ON patients require long-term treatment for recovery to be seen and maintained. The term “papillitis” can be used if there is optic disk swelling on clinical examination. Papillitis may be associated with flame-shaped hemorrhages and is the most common type of ON in children. If the optic disk appears normal on examination, then the term “retrobulbar optic neuritis” is used. This is the most frequent type seen in adults. Neuroretinitis is a variant of ON and is usually post-infectious. On examination there is papillitis in conjunction with a macular star composed of hard exudate [9]. In 1894, Eugene Devic described 16 patients with loss of vision in one or both eyes, who subsequently developed spastic weakness of the lower limbs [10]. The term “neuromyelitis optica” is now used in preference to Devic’s disease. NMO is a demyelinating disease that preferentially affects the optic nerves and spinal cord. Optic perineuritis is an uncommon variety of orbital inflammatory disease that involves the optic nerve sheath; most cases are isolated or idiopathic [11]. If an inflammatory lesion affects the optic chiasm, the terms “optic chiasmal neuritis” or “chiasmitis” are used. This is distinguishable from bilateral simultaneous ON by a preferential involvement of the bitemporal visual fields. Chiasmitis can be seen in both MS and NMO [12]. These two conditions will not be discussed further in this chapter. Most ophthalmologists use the term “optic neuritis” to describe idiopathic or demyelinating ON [13].

Pathophysiology Typical ON In cases of both typical MS-associated and isolated ON, the cause is presumed to be an inflammatory autoimmune reaction, resulting in demyelination of the optic nerve. The trigger for these autoimmune mechanisms is uncertain; however, attention is focused on an external factor (such as a virus) initiating events in susceptible individuals.

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The disease now known as MS was first described in 1868 by Jean-Martin Charcot as “la sclerose en plaques” [14]. MS is characterized by episodic neurologic dysfunction caused by localized damage to nerve fibers within the brain and spinal cord. A typical MS lesion causes the loss of myelin. This demyelination results in a slowing or complete blockage of the neuronal signals, thus resulting in the neurologic dysfunction. Early in the disease course remyelination occurs, and the neurologic function will often recover to some extent. This phenomenon was thought to be due to the preservation of the nerve fibers themselves. It has now been demonstrated that this process of demyelination causes damage to the underlying nerve axons and ultimately leads to brain atrophy and the accrual of progressive neurologic dysfunction [15]. Once the degenerative process starts, the changes are irreversible, and thus treatment is most effective when started early. The overall risk factors for MS in an individual are also well documented. Caucasians of northern European heritage are the most commonly affected ethnic group, with women 2–3 times more frequently affected than men. Living in a temperate climate causes a higher risk of MS, and those who migrate from a high-risk to a low-risk area after the age of 15 remain at high risk [16]. Several infectious agents have been postulated to be the environmental trigger for MS, with the current leading candidates being Epstein–Barr, herpes VI, and Chlamydia pneumoniae [17]. A recent review article concluded that it appears likely that hypovitaminosis D is one of the risk factors for MS. Vitamin D does significantly influence regulatory T-lymphocyte cells and would seem to have a role in several autoimmune diseases [18]. Other work has highlighted an increasing incidence of MS in previously low-risk areas such as Australia and California. It appears that the increased use of sunscreen, decreased sun exposure, and subsequent reduction in individual vitamin D levels in these areas may be contributing to a rise in the incidence of MS [19]. A genetic predisposition to MS has been identified, although the only significantly associated genotypes are the major histocompatibility complexes (HLA) DR and DQ [20]. On histologic examination of MS patients with ON, the demyelinating lesions along the optic nerve are similar to the MS plaques seen in the brain [21]. There is an inflammatory response marked by perivascular cuffing, T cells, and plasma cells. The key pathologic features are inflammation, demyelination, axonal loss, and gliosis [22]. There are reports that a heterogeneity in demyelinating lesions in the brain may suggest that several different mechanisms may be involved in MS pathogenesis [23]. Others, however, propose that there is a uniform pathogenic mechanism that underlies the formation of new plaques, and that disease evolution accounts for the different lesions described [24]. In general it is thought that T-helper (Th) cells (CD4) are activated in the periphery by an environmental factor and cross the blood–brain or blood–optic nerve barrier. Inside the CNS they encounter neural autoantigens, proliferate, and activate and recruit other inflammatory cells. This results in stimulation of the local immune and parenchymal cells such as microglia and astrocytes to produce pro-inflammatory cytokines. CD8 cells, B cells, and complement are also involved in the neural damage [25,26]. Free radical damage and glutamate excitotoxicity are thought to play an

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important role in the axonal and myelin damage and have been linked to mitochondrial dysfunction [27]. Another hypothesis challenges this paradigm by suggesting that oligodendrocyte apoptosis is the earliest change in newly forming MS lesions. A subsequent recruitment of the systemic immune response amplifies the tissue injury [28]. The signals for resolution of inflammation are not well known. The final functional recovery is through a combination of the resolution of inflammation, remyelination, and neural plasticity. Modern imaging techniques provide insight into pathologic processes in vivo. The loss of axons, neurons, and myelin can be assessed using quantitative magnetic resonance imaging (MRI) and optical coherence tomography (OCT) techniques [29–31].

Atypical ON Little is known about the pathology of isolated idiopathic ON. A biopsy specimen in one reported patient with isolated ON showed the presence of perivascular lymphocytic infiltration, multifocal demyelination, and reactive astrocytosis in the retrobulbar portion of the optic nerve. Abnormal intrathecal immunoglobulin G (IgG) synthesis, reflected by the presence of oligoclonal bands in the cerebrospinal fluid (CSF), is found in 60–70% of patients with isolated ON, suggesting an immunologic pathophysiology similar to MS [32]. ON can occur as part of a connective tissue disorder such as systemic lupus erythematosus (SLE) or Behçet’s disease [33], as well as sarcoidosis. Features specific to each underlying condition will be present. In patients with known SLE the usual accepted cause for the ON is vasculitis [34], although the optic nerve damage in this case is likely to be ischemic secondary to the vascular pathology. However, it may be that in some cases of SLE there is indeed a true autoimmune ON. Sarcoidosis is a granulomatous inflammatory disease, which on biopsy specimens is non-caseating. The granulomatous tissue can affect the optic nerve sheath or invade the optic nerve directly [35].

Neuromyelitis Optica NMO is a distinct inflammatory demyelinating disease consisting of ON in combination with longitudinally extensive transverse myelitis. Though this is classified as one of the “atypical” ON causes, it will be discussed separately. Patients may also experience a limited form of NMO with isolated recurrent bilateral ON or isolated transverse myelitis episodes; this is considered at one end of the spectrum toward definite NMO. There is no secondary progression, as can be seen in MS. Histopathologic features differ from those of typical ON. Early demyelinating lesions in NMO are associated with perivascular deposition of immunoglobulins, in particular IgM and IgG, local activation of the complement cascade, and a marked eosinophilic infiltrate. This ties in with the immunopathology of NMO being antibody-dependent and complement-mediated with the recruitment and degranulation

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of eosinophils. The presence of eosinophils and small numbers of CCR3-positive lymphocytes may indicate the active involvement of a Th-2 T cell. This combination of features is relatively specific for early NMO lesions. There are also accompanying immunopathologic changes in the CNS such as macrophage/microglia activation and axonal damage that are also ubiquitous in forms of MS [36,37]. Compared with MS there is excavation of the affected tissue, the formation of cavities is common, and gliosis is characteristically absent or minimal. On examination of the brain the cerebellum and subcortical cerebral arcuate fibers are rarely affected, whereas in MS these are often severely damaged [27,38]. A significant number of patients (~75%) with NMO have been found to have the presence of a specific serum, NMO-IgG autoantibody, which targets the water channel aquaporin-4 (AQP4) [39]. This is also found in those with bilateral ON alone. AQP4 is the predominant water channel in the human brain and plays a critical role in the regulation of water movement when expressed by astrocytes. It is associated with astrocyte membranes that closely appose endothelial cell basement membranes, which in turn maintain the blood–brain barrier (BBB). Of more relevance to the ON, the AQP4 on fibrous astrocytes might function to spare the optic nerve from volume fluctuation under normal physiologic conditions. The optic nerve is perhaps more sensitive to volume changes induced by AQP4 dysfunction than other areas of the CNS, with resultant ON [40]. The distribution of AQP4 in the CNS is shown in Table 12.1. Higher serum and CSF IL-6 levels are also found in NMO patients compared with other groups. CSF IL-6 levels correlated with anti-AQP4 levels and disease severity in the NMO patients. This suggests that IL-6 is also involved in NMO pathogenesis, presumably via anti-AQP4-associated mechanisms [41]. Pathologic findings of biopsy specimens from the limited form are identical to those of autopsy from the definite form, demonstrating extremely active demyelination and extensive loss of AQP4 immunoreactivity in plaques. Moreover, the definite form displays significantly higher amounts of IL-6 in CSF than the limited form and MS. NMO continuously displays a consistent homogeneity of immune mechanisms through terminal stages, whereas MS should be recognized as a heterogeneous disease that may switch from an inflammatory to a degenerative phase [42]. Table 12.1  The Distribution of AQP4 in the CNS [40] Tissue

Cell Type

Cellular Localization

Gray and white matter Ventricles (not choroid plexus) Pia mater Inner ear Posterior optic nerve Retina

Astrocytes Ependymal cells Meningeal cells Hensen’s cells Astrocytes Muller cell astrocytes

Perivascular end feet Basolateral Not determined Basolateral Perivascular end feet Vitreal end feet Perivascular end feet

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Generally other autoantibodies are found in the sera of NMO patients but the brain expresses high levels of AQP4, suggesting this to be the pathologic target. The other autoantibodies, including antinuclear antibodies, anti-myelin oligodendrocyte glycoprotein (MOG), and anti-myelin basic protein, most likely represent a response to neo-antigen liberated from dead cells [43]. Also released, due to glial and astrocyte damage, is glial fibrillary acidic protein (GFAP). This is found in higher levels in NMO patients during acute attacks than those with MS. Even in patients who do not have NMO antibodies on current testing, GFAP may be detected in the CSF to assist in diagnosis [44].

Clinical Features Typical ON ON affects otherwise healthy young individuals, usually aged 20–30 years. Women are affected 2–3 times more commonly than men. ON causes a decrease in vision over a 7- to 10-day period and is commonly associated with pain on eye movement [45]. The majority of patients with a painful ON have involvement of the orbital segment of the optic nerve. The absence of pain suggests the ON is limited to the canalicular or intracranial portion of the optic nerve [46]. The features listed in Table 12.2 reflect the “typical” ON presentation. These were the clinical characteristics seen in the 455 patients enrolled in the ONTT between 1988 and 1991 [47]. The clinical data of 110 patients with acute unilateral ON were analyzed and showed that those with MRI lesions consistent with demyelination had a significantly higher incidence of ON in spring months compared with patients with normal MRI scans. The increased incidence of ON in spring months was seen in these patients whether or not they were diagnosed with clinically definite MS on follow-up [48].

Atypical ON Atypical features include severe headache, uveitis, retinal inflammation, failure of vision to improve after 30 days, age over 50 years, and evidence of other systemic conditions. Patients presenting with atypical features should be examined further. Full history and examination should guide further investigations, such as syphilis serology and antinuclear antibodies [47]. The presence of associated polyneuropathies should lead to consideration of Guillain–Barré or Miller–Fisher syndrome [13]. Patients should also be asked about alcohol consumption, smoking, and use of other drugs such as ethambutol, isoniazid, and amiodarone, all of which are known to cause optic neuropathy [45]. The common causes of atypical ON and the mimics of ON are presented in Table 12.3. In children ON may be para-infectious, associated with measles, mumps, chickenpox, or glandular fever or following immunization [52]. In these cases patients usually present 1–3 weeks following the infection with severe, often bilateral, visual loss and papillitis [9]. There are several case reports of ON associated with a pansinusitis [53].

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Table 12.2  Clinical Profile of the ONTT Patients [47] Characteristic

Patients

Female

77%

Caucasian

85%

Age (years)

326.7

Ocular pain

92%

Pain worse on eye movement

87%

Optic disk   Normal (retrobulbar)   Swollen   Mild diffuse swelling   Mild focal swelling

65% 35% 51% 29%

No retinal or optic disk hemorrhage

84.5%

Normal vitreous

94%

Visual acuity   20/20 or better (6/6)   20/25–20/40 (6/7.5–6/12)   20/50–20/190 (6/15–6/57)   20/200–20/800 (6/60–6/240)   Count fingers or hand motions   Light perception   No perception of light

11% 25% 29% 20% 10%   3%   3%

Visual field defect of affected eye   Diffuse   Altitudinal, arcuate, nasal step   Retrochiasmal   Central, cecocentral   Chiasmal   Other

48% 20%   9%   8%   5% 24%

Abnormal MRI (one or more significant white matter lesions)

49%

In the elderly population other causes of visual loss must be considered before a diagnosis of ON is made. Non-arteritic ischemic optic neuropathy (NAION) may present after the age of 45 with a usually sudden but occasionally progressive, painless visual loss and typically an altitudinal visual field defect. Giant cell arteritis usually presents in those aged 60–80 and is typically associated with scalp tenderness, headache, jaw claudication, polymyalgia rheumatica, and malaise [45]. In a review of 67 cases of neurosarcoid, optic neuropathy was the most common manifestation, typically presenting with optic disk edema and severe visual loss. Of the 14 patients with sarcoid-related optic neuropathy, 8 had optic disk edema, 5 had

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Table 12.3  Summary of Differential Diagnoses in Atypical ON [49] Types of Pathology that can Mimic Optic Neuritis

Specific Examples

Retinal Disease

Idiopathic big blind spot syndrome Acute zonal occult outer retinopathy Central serous retinopathy

Compressive optic neuropathy

Tumor Idiopathic intracranial hypertension

Toxic optic neuropathy [45]

Alcohol/tobacco amblyopia (vitamin B deficiency) Amiodarone Ethambutol

Genetic disease [50]

Leber hereditary optic neuropathy Autosomal dominant ON (Kjer syndrome) Wolfram syndrome (DIDMOAD  diabetes insipidus/mellitus, optic atrophy, deafness)

Infections

Syphilitic ON (primary or secondary) Human immunodeficiency virus (HIV) Cat-scratch (Bartonella henselae) Tuberculosis Post-viral Aspergillus

Inflammatory

Sarcoidosis Systemic lupus erythematosus (SLE) Reiter syndrome

Vasculitis

Giant cell arteritis (temporal arteritis) Polyarteritis nodosa Wegener granulomatosis Churg–Strauss syndrome

Paraneoplastic optic neuropathy [51]

Typically lung cancer, CRMP-5 IgG positive

optic disk pallor, and 1 had an optic disk granuloma. No light perception vision was relatively common and should be considered a red flag for the diagnosis [54]. Neurosyphilis is still a significant medical problem even in developed countries, and syphilitic ocular manifestations are often not diagnosed due to the lack of typical characteristics. ON associated with syphilis can occur in secondary as well as tertiary stages; ocular involvement in syphilitic patients is suggestive of involvement of the CNS and should be considered equivalent to neurosyphilis [55].

Neuromyelitis Optica The association of acute or subacute loss of vision in one eye or both eyes caused by optic neuropathy with a simultaneous or separate attack of transverse or ascending myelopathy is referred to as neuromyelitis optica (or Devic’s disease). Though

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Table 12.4  Revised Diagnostic Criteria for NMO [57] Optic neuritis Acute myelitis At least two of three supportive criteria   Contiguous spinal cord lesion extending over at least three spinal segments   MRI at onset not meeting the diagnostic criteria for MS   NMO-IgG seropositive status

one eye is usually affected first, the second may be affected in hours or days. There are case reports of several weeks separating episodes of visual loss. It may be longer between spinal and visual symptoms; up to 10 years has now been reported [56]. The diagnostic criteria for NMO published in 2006 are in Table 12.4 [57]. The patient demographics are usually that of a slightly older group than MS (30 years versus 20 years old). There is an increased female predominance of more than 3:1 reported from samples of diverse racial and regional populations worldwide. The gender distribution appears to be similar in both childhood-onset and adult-onset cases. Female gender is associated with a relapsing course and familial disease, but the influence of gender on disease severity and treatment response is not clear [58]. It is more common in those of African and Asian ethnic backgrounds. Patients will often develop a mild febrile illness several days or weeks before the onset of neurologic deficit. Textbooks report that the vision loss is typically rapid and severe and that pain is felt in only a minority of cases [59]. However, when studied in direct comparison with typical ON, no difference was found in terms of pain and severity of vision loss between the two groups [60]. Visual field loss is typically central, when vision is sufficient to allow for fields to be plotted. The majority of patients have mild disk swelling, though appearances can range from normal to severe swelling with distention of retinal veins. The disease can be monophasic or relapsing, with a high relapse rate in the first 2 years of disease associated with a poor prognosis [61].

Assessment of the Optic Nerve Bedside Tests Reduced visual acuity in the affected eye can be mild to severe (see Table 12.1). In a young person, normal visual acuity is often 6/4.5; thus, vision of 6/6 in the affected eye can be abnormal for that patient. The test for a relative afferent papillary defect (RAPD) is an important bedside test used to determine if decreased vision in a patient is due to an optic nerve problem. Studies suggest that RAPD is mainly related to the integrity of central vision being correlated with a reduction in conventional visual evoked potential (VEP) amplitudes and intraocular mean deviation on Humphrey subjective perimetry in patients with ON [62,63].

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Loss of color vision can be inherited or acquired. Though there are many causes of acquired loss of color vision, ON causes color vision abnormalities in 88% of affected patients [47]. The ONTT patients showed mixed red-green (RG) and blueyellow (BY) color defects. BY defects tend to be more common in the acute phase of the disease, with slightly more RG defects at 6 months. It was thus concluded that the type of color defect cannot be used in the differential diagnosis of ON [64]. Red desaturation can also be measured using a red test object. Brightness perception can be tested using a penlight. Poor brightness perception is reported in 89% of patients with ON and can be used to follow the course of disease [65,66]. Contrast sensitivity is the ability of the eye to distinguish subtle degrees of contrast. Retinal, optic nerve disease, and clouding of the ocular media can impair this ability. In patients with previous ON but normal visual acuity, contrast sensitivity abnormality rates have been reported to be 99% [67]. Contrast sensitivity is an important measure of vision quality in patients after ON [68].

Visual Field and Perimetric Analysis Visual field testing can be divided into kinetic (i.e., Goldman perimetry) or static (i.e., Humphrey visual fields) testing, which can be manual or automated. Automated perimetry, though lacking in defect shape detail due to the six-degree stimulus area, proves to be more sensitive than kinetic testing in ON. However, the Humphrey visual field (HVF) analyzer sensitivity relies on a cooperative and competent patient, as well as a good technician to monitor the patient; despite this there is still high test–retest variability [69]. Patients with ON involved in the ONTT underwent HVF testing. Initial perimetry revealed that 48% had a diffuse field defect (Figure 12.1), 20% had altitudinal or arcuate defects, and 8% had a centrocecal defect [70]. These results were initially surprising as they went against the previously held belief that ON caused a centrocecal scotoma. However, it is now believed that the central scotoma found by Goldman perimetry represents diffuse suppression of sensitivity within the central 30 degrees of vision [71]. Although patients with ON can present with central visual field loss and sparing of the periphery, it is rare for the peripheral field to be abnormal in the presence of a normal central field [13]. In most cases of ON the recovery can therefore be tracked effectively using automated perimetry of the central visual field.

Visual Evoked Potentials VEPs represent a specific change in ongoing electroencephalographic (EEG) recording due to stimulation of the visual pathway to either a pattern or flash stimulus. Recordings of evoked potentials can be made with the use of electrodes applied to the scalp, with the main signal detected over the occipital cortex. The advent of VEPs in the 1970s first allowed clinicians to assess neural conduction in the optic nerve. Halliday demonstrated that diagnostic confirmation of ON can be made using full-field conventional VEP delays [72–74].

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Figure 12.1  Humphrey Visual Field analysis; gray-scale map of a patient with ON showing diffuse loss of sensitivity with a dense central scotoma.

ON can cause a loss of VEP signal amplitude as well as a delay in the signal. A loss of signal amplitude is also seen in compressive lesions of the optic nerve and latency delay is found in Parkinson’s disease and migraineurs. Overall the VEP has been shown to be very sensitive in these conditions; however, it is a nonspecific diagnostic tool [75].

Optical Coherence Tomography OCT is an attractive imaging technique because it permits the imaging of tissue microstructure in situ, yielding micron-scale image resolution without the need for excision of a specimen and tissue processing. OCT enables repeated imaging studies to be performed on the same patient to track changes. OCT is analogous to ultrasound B-mode imaging except that it uses low-coherence light rather than sound and performs cross-sectional imaging by measuring the backscattered intensity of light from structures in tissue [76]. OCT can measure in vivo axonal loss due to ON. As the retinal nerve fiber layer (RNFL) contains ganglion cell axons that are unmyelinated (in the vast majority of cases), the thickness measurements represent only axonal density, not myelin sheath changes. Evidence of RNFL thinning can be observed with OCT within 3–6 months following the onset of ON. Patients with marked loss of RNFL had a poorer visual outcome, with limited visual improvement [77,78].

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Some studies have shown a difference in the RNFL measurements between typical ON and NMO. NMO is associated with a thinner overall average RNFL compared to MS, with particular involvement of the superior and inferior quadrants. This suggests that NMO is associated with more widespread axonal injury in the affected optic nerves. OCT can help distinguish the etiology of these two causes of ON and may be useful as a surrogate marker of axonal involvement in demyelinating disease [79].

Magnetic Resonance Imaging The quality of MRI of the optic nerve has lagged behind imaging of other parts of the CNS, partly due to technical challenges related to the small size and mobility of the optic nerves. In one study MRI was able to detect abnormalities in the ON of only 80% of acute cases [80]. Artifacts caused by surrounding CSF, orbital fat, and air–bone interfaces further complicate image acquisition. Progress has been made, however, with regard to detecting optic nerve atrophy following ON, the use of fatand CSF-suppressed high-resolution imaging, and the emergence of SPIR–FLAIR for increasing sensitivity to inflammatory demyelination [81]. Magnetization transfer ratio (MTR) imaging is a quantitative MRI technique that provides information about tissue integrity in vivo, including myelin and axonal content. MTR has been shown to be reduced in ON compared to both clinically unaffected nerves from patients and control nerves. The MTR correlates with both VEP latency (myelin integrity) and OCT measures of axonal loss and thus does not distinguish the relative contributions of demyelination and axonal loss in ON [82].

Prognosis Typical ON The course of visual recovery in typical ON is often rapid regardless of treatment, with improvement noticeable within the first 2 weeks in most patients and much of the recovery occurring by the end of 1 month [13]. Over 75% of patients recover to normal vision within 6 months [83]. If recovery of vision is incomplete at 6 months, some further improvement may continue for up to 1 year. Ten years after ON in the ONTT, 70% of patients had 20/20 acuity in both eyes and 86% had 20/20 vision in one eye. Relapses of ON within this time did not appear to cause a major loss of visual function [84]. However, patients may complain of residual deficits in color vision, stereopsis, and light brightness perception, despite the apparent normalization of distance vision [85]. The only predictor of poor visual outcome found in the ONTT was poor visual acuity at the time of entry to the study. However, of the patients with visual acuity of light perception (LP) and non-perception of light (NPL), 67% recovered to a final visual acuity of 20/40 or better. Older age at onset was statistically associated with a slightly worse visual outcome [84].

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Recent studies have shown that early neuroplasticity in higher visual areas, as detected by fMRI activation of the lateral occipital complex at the onset of ON, appears to be an important determinant of recovery from ON. This was independent of tissue damage in the anterior or posterior visual pathway, including neuroaxonal loss (as measured by MRI, VEP, and OCT) and demyelination (as measured by VEP) [86].

Association with MS The incidence of ON in the United States is 3 per 100,000 people [87]. Despite this relatively low incidence and the fact that vision recovers in the majority of patients, ON is still of great significance due to its association with MS [88,89]. The risk for developing MS following ON is quite variable within the literature, reflecting differences in the populations studied; however, the majority of studies indicate a 25–35% risk [90]. In fact the most common cause of visual loss in MS patients is ON, with 90% of MS sufferers experiencing some form of ON during the course of their disease [91]. Following the release of the 10-year data from ONTT, it is now known that a single MRI white matter lesion in the brain of at least 3 mm increases the risk of future MS. In ON patients with a normal brain scan, the absence of pain on eye movement, no perception of light vision, severe optic disk swelling, hemorrhages, and exudates is associated with a low risk of future MS [92]. MRI studies show that periventricular and gadolinium-enhancing lesions on baseline scans following ON are independent predictors of clinically definite MS. When reviewing baseline and follow-up scan on the same patients, baseline periventricular and new T2 lesions at follow-up remained significant (hazard ratios 2.4 and 4.9, respectively). The authors concluded that new T2 lesions on an early follow-up scan were the strongest independent predictor of clinically definite MS [93].

Atypical ON Atypical ON is more often associated with poor visual recovery without treatment. One study looked at isolated ON in patients in whom all known causes of an optic neuropathy were excluded (i.e., sarcoid, HIV, syphilis, connective tissue disease). Of the 18 eyes (10 patients), after at least 3 months follow-up, only 33% of eyes reco­ vered visual acuity of 6/12 or better, with only 16% of eyes recovering color vision of 10/13 or better on Ishihara plate testing. This was despite initial treatment with corticosteroids in all cases [94].

Association with SLE According to case studies, patients with optic neuropathy due to SLE who are treated with steroids alone generally have a poor visual prognosis, with final vision worse than 6/60 [95]. Other observational studies report variable visual prognosis [96]. However, recent studies using alternative immunosuppression have shown excellent visual recovery in up to 50% [97].

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Association with Sarcoid In patients with sarcoid visual recovery is often poor, and patients require ongoing immunosuppression to maintain vision. Recorded visual acuity at last follow-up visit of 14 sarcoid optic neuropathy patients was improved in 5, was worse in 5, and was stable (i.e., within one Snellen acuity line of baseline) in 4.

Neuromyelitis Optica Some recovery of vision usually occurs within 1–2 weeks after symptom onset. Prognosis is generally poorer than those with typical ON, with some having severe residual visual loss. The same pattern is seen with the paraplegia: most patients recover to some extent and some are left with persistent and complete paralysis. In one study of 13 patients followed for over 8 years, only 23% were able to walk more than 100 m unaided [98,99]. Initial mortality rates were reported at 50%, but with modern treatment this is now 10%. Recurrences can occur in either eye or spinal cord [100]. Patients who have positive NMO-IgG antibodies detected are shown to have a higher Expanded Disability Status Scale (EDSS) score than those who meet the diagnostic criteria for NMO without detectable antibodies [101].

Management Typical ON Investigations A clinical diagnosis can be made in patients with typical features of ON with a high degree of certainty. In these cases ancillary testing is not required. A decision will need to be made as to whether an MRI is warranted in order to stratify future risk for MS. If the patient fails to recover vision as expected, then further investigation is warranted.

Treatment Treatment with corticosteroids in the setting of ON reportedly results in a more rapid recovery of visual function; however, it has also been reported that there was no difference in visual outcome by 6–12 months [102]. The ONTT set out to provide definitive data to evaluate the efficacy of corticosteroid treatment for acute ON. The trial enrolled 457 patients with a RAPD as well as a visual field defect in the affected eye. Other causes for ON were ruled out prior to entry in the study. The patients were randomly assigned to one of three treatments: (1) intravenous (IV) methylprednisolone (250 mg 4 times daily for 3 days) with an 11-day oral steroid taper; (2) oral prednisone (1 mg/kg once daily) for 14 days; (3) oral placebo for 14 days. The study confirmed that IV corticosteroids with an oral taper resulted in an acceleration of visual recovery, but there were no long-term benefits to visual

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outcome. Interestingly, it was also found that the first dosing regimen reduced the rates of patient conversion to MS over the first 2 years after ON. However, this effect was not sustained into the third year after diagnosis [90]. A recent Cochrane review on the management of typical ON concluded that there was no evidence that steroid treatment (IV or oral) was of benefit in terms of visual recovery with respect to acuity, field, or contrast sensitivity [103]. The ONTT initially showed an increased relapse rate in patients treated with oral steroids alone, and this has led to reluctance, particularly among ophthalmologists, to use any treatment other than IV methylprednisolone. However, a trial of 60 patients has shown that 500 mg oral methylprednisolone for 5 days with a 10-day oral taper will similarly speed up visual recovery but not alter long-term visual prognosis. In particular, there was no difference in the frequency of subsequent attacks in the treatment versus placebo groups [104]. Pathologic and MRI studies now suggest that axonal damage is an early event in the evolution of MS, and that this is primarily dependent on inflammatory processes [15]. Treatments for MS available include interferon-beta, which has a partly antiinflammatory action. The Controlled High-Risk Subjects Avonex Multiple Sclerosis Prevention Study (CHAMPS) was a randomized, double-blind, placebo-controlled trial in patients who had a first acute demyelinating event (ON, incomplete transverse myelitis, brain stem, or cerebellar syndrome) and evidence of previous subclinical demyelination on MRI. Eligible patients had two or more clinically silent brain lesions of 3 mm or more in diameter on MRI, one of which was ovoid or periventricular. Patients were randomized to receive either 30 μg of intramuscular interferon-beta-1a (Avonex) or placebo weekly. Results indicated that treatment with interferon significantly reduced the risk of developing MS, according to the Poser criteria, by 44% compared with placebo [105]. A more recent analysis of the CHAMPS data found that using more stringent MRI criteria for eligibility, as set out by Barkhof (nine or more white matter lesions with at least one gadolinium-enhancing lesion), treatment with interferon conferred a 66% reduction in risk for MS over 3 years [106]. The more recent Betaferon in Newly Emerging MS for Initial Treatment (BENEFIT) study used interferon-beta-1b in a double-blind, placebo-controlled randomized trial in patients with a first episode suggestive of MS. Patients were followed over time, with conversion to MS defined according to the McDonald criteria. Results showed that the risk of developing MS within a year was reduced by 46% when compared to the placebo group [107]. A review of all available preparations of interferon and the available studies on each concluded that treatment with all interferon-beta products is effective compared to placebo, with a possible advantage to a thrice-weekly dosing regimen rather than weekly [108]. Interferon-beta-1a has been approved for use in Europe and America in patients who have experienced a first clinical episode and have MRI features consistent with MS (ON, incomplete transverse myelitis, brain stem, or cerebellar syndrome), where alternative diagnoses have been excluded and the patient is deemed to be at high risk for developing MS. However, given that there is now a treatment to reduce the risk of

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MS in patients after ON, the need for objective tests that may reflect a higher risk for progression to MS becomes apparent. Interferon therapy is not without side effects, the most common being flu-like symptoms and depression. Thus, preventive therapy must be guided by this knowledge of which patients are inherently at a higher risk of MS. In patients with known MS, treatment with interferon-beta significantly reduces the annual relapse rate of ON [109].

Atypical ON Investigations Thought should be given to performing a lumbar puncture and additional laboratory tests, including syphilis serology, antinuclear antibodies, and a chest X-ray. Even if a patient cannot fulfill the diagnostic criteria for NMO, testing for NMOIgG in patients with recurrent or severe ON who lack convincing evidence of MS may identify patients who would benefit from immunosuppression rather than MS-directed immunomodulatory therapies [110].

Treatment Any underlying causes should be treated. Prompt diagnosis and early treatment minimizes long-term visual dysfunction [111]. In SLE-related cases IV cyclophosphamide (0.5–1 g/m2) monthly for 6 months seems to be an effective treatment for ON refractory to corticosteroids [97]. Methotrexate is effective as an adjunct to or instead of steroids in patients with sarcoid-related ON [112].

Neuromyelitis Optica Investigations NMO-IgG/APQ4 was found to have a sensitivity of 54% with a specificity of 90% when tested in patients with NMO versus those with MS [113]. Other markers of autoimmunity are often elevated in NMO patients, in the absence of clinical manifestations of their associated conditions. Examples include SLE, Sjögren syndrome, pernicious anemia, and ulcerative colitis [100]. Lumbar puncture and CSF analysis reveal abnormalities in most NMO patients. Pleocytosis of greater than 50 white cells/mm2 rarely occurs in MS, and oligoclonal bands are rarely found [114]. Elevated levels of GFAP are found in the CSF of NMO patients compared to those with MS [115]. As with all types of ON, the optic nerves will show evidence of enhancement on MRI. More specifically, the spinal cord lesions are gadolinium-enhancing and extend through several vertebral segments. In the acute phase there will also be marked swelling of the cord. At onset there are usually no other MRI brain abnormalities. Follow-up studies have shown the presence of small nonspecific lesions

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that do not fulfill the Barkhof criteria for diagnosis of MS. In particular, these lesions may be around the hypothalamus, thalamus, fourth ventricle, and cerebral peduncle [116].

Treatment IV corticosteroids are commonly used as first-line therapy due to the severity of attacks as they may increase the speed of visual recovery. The most common protocol calls for daily 1-g methylprednisolone infusions for 5 consecutive days. If the treatment plan will include an immunosuppressive regimen with a delayed onset of action, one should consider following the IV course with daily oral prednisone at a dosage of ~1 mg/kg/day. If clinical symptoms and signs progress during steroid therapy, then evidence supports rescue therapy with plasmapheresis. A total of seven exchanges of ~55 mL/kg each are administered every other day for 14 days [117]. This finding was further supported by a retrospective review in which 6 of 10 NMO patients experienced moderate or marked clinical improvement of myelitis-related symptoms and signs within days after starting plasmapheresis. Earlier initiation of plasmapheresis therapy after attack onset (less than 20 days) predicts a greater likelihood of clinical response compared with delayed therapy. There is no role for plasmapheresis in slowly progressive disease states [118]. It has been shown that early high-dose methylprednisolone is effective at preserving retinal ganglion cell axons and thus improving visual potential [119]. Long-term immunosuppression is directed toward attack prevention with the aim of protecting neurologic function and should be instituted as early in the disease course as possible. This may mean starting treatment when a relapsing course has declared itself (i.e., two or more attacks). However, the detection of NMO antibodies in new-onset patients with longitudinally extensive myelitis appears to portend a high risk of relapse within 1 year [120]. The current standard preventive approach includes azathioprine (2–3 mg/kg daily) in combination with oral prednisone (1 mg/ kg/day). In one report, most patients used maintenance doses of 75–100 mg azathioprine (a low dose) and 10 mg prednisone daily and were attack-free during the 18-month period after therapy initiation [121]. The aim of therapy is to achieve clinical remission using azathioprine and then to slowly taper the daily prednisone dosage over a few months once it is evident that azathioprine is having an immunologic effect (reduced peripheral white blood cell count and elevation in mean cell volume). Some patients may suffer relapses once the prednisone dose is reduced to less than 10 mg/day, and others may show relapses even after adequate treatment. Rituximab (anti-CD-20) therapy was recently reported to inhibit relapses in three patients treated for over 2 years with an interval of about 9 months. One year after therapy, relapses had disappeared in all cases with no adverse effects. However, during long-term rituximab treatment, attention needs to be given to infections such as progressive multifocal leukoencephalopathy [122]. Mycophenolate mofetil has also been reported to reduce the relapse rate, but in this trial with a larger population, side effects of treatment were seen in 25% of patients. A median dose of 2000 mg/day was given for a median duration of treatment of 27 months [123].

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Interferon-beta is classically used in MS to reduce the relapse rate, but it does not appear to be of benefit in NMO. This is probably due to the different immunopathogenesis between the two diseases [124]. Supportive treatment is required for severe myelitis.

Conclusion ON is a heterogeneous diagnosis, particularly with geographic and ethnic differences. Careful consideration of such factors must be taken into account when making a diagnosis, instituting a treatment regimen, and discussing prognosis. Patients who do not have a “typical” demyelinating ON may have a poor outcome if inappropriate MS treatment regimens are applied. Further work needs to be done to unravel the uncertainties regarding whether opticospinal MS is a separate entity from NMO. There is also much scope for future research in finding more effective MS treatments.

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13 Ischemic Demyelination Tiffany Chang, Roger E. Kelley Department of Neurology, Tulane University School of Medicine, New Orleans, LA, USA

Introduction The finding of white matter signal changes on magnetic resonance imaging (MRI) brain scan is being increasingly recognized as of potential clinical significance, but the pathogenesis, clinical correlation, and prognostic implications remain ill defined. They are recognized with increasing frequency in patients with risk factors for small vessel occlusive disease, including age, hypertension, and diabetes mellitus. It is not uncommon to see small areas of increased signal intensity in the subcortical white matter of patients with migraine [1] (Figure 13.1). Therefore, the collection of risk factors most commonly associated with small vessel white matter disease includes any disease process that affects the integrity of these small vessels. Although hypertensive microangiopathy is the most commonly cited mechanism, the pathogenesis appears to be related to genetic factors, endothelial dysfunction, and/or low levels

Figure 13.1  A woman in her 30s with recurrent vascular-type headache, compatible with common migraine, who demonstrates areas of increased signal intensity on FLAIR sequence of MRI. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00013-7 © 2011 Elsevier Inc. All rights reserved.

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Table 13.1  Clinical Conditions Associated with Ischemic Demyelination 1. 2. 3. 4. 5.

Small vessel cerebrovascular disease, including Binswanger’s disease CADASIL Migraine Neurodegenerative disease, including Alzheimer’s disease Nonspecific aging changes in the elderly not associated with hypertension or other potential cause of microangiopathy 6. Possible relationship with cerebral amyloid angiopathy

of free radical scavengers [2]. The potential clinical conditions associated with ischemic demyelination are outlined in Table 13.1. Most clinicians view such white matter changes as reflective of a cerebrovascular process. There appears to be an ongoing predisposition over time based upon serial studies. However, it is not uncommon at all to see a clinical and imaging picture that can mimic multiple sclerosis [2]. The most common clinical correlate appears to be lacunar stroke. This fits in effectively with the effects of lipohyalinosis on small penetrating arteries in patients with long-standing hypertension from a clinicopathologic standpoint. Therefore, one can assume that the finding of small vessel disease in stroke patients would help to support aggressive attention to any factors that might, theoretically, contribute to the ongoing pathogenesis. One entity that is being increasingly recognized, despite its relative rarity, is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). This disorder represents the potential for unique insight into small vessel cerebrovascular disease on a clearly genetic basis.

Manifestations of Hypertensive Microangiopathy There is clearly a relationship between white matter hyperintensities on MRI and hypertension. In a longitudinal study of hypertension and the evolution of white matter hyperintensities, there was a definite relationship between severe white matter hyperintensities and the duration of the hypertension [3]. Breteler et al.[4] reported a relationship not only with hypertension, but also with plasma cholesterol, prior myocardial infarction or stroke, factor VIIc activity, and fibrinogen level in subjects 65–74 years. In addition, the white matter changes correlated with cognitive impairment. The most commonly recognized clinical manifestation of small vessel occlusive disease is lacunar stroke. The most common lacunar syndromes include pure motor stroke, pure sensory stroke, sensorimotor stroke, clumsy hand dysarthria, and hemiparesis–hemiataxia. Especially with MRI brain scan, there is often a small (i.e., ,1.5  1.5 cm) area of increased signal intensity in the subcortical region visualized by either T2-weighted or fluid attenuation inversion recovery (FLAIR) imaging (Figure 13.2).

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Figure 13.2  T2-weighted MRI showing bilateral thalamic lacunar-type infarcts (arrows) in a 50-year-old man with long-standing hypertension.

Lacunar stroke is usually associated with good functional recovery after the first event. However, the risk of recurrent stroke can be on the order of 12% per year. This can translate into cumulative neurologic deficit over time, including multi-infarct dementia, pseudobulbar palsy, and bilateral long-tract signs. It is quite possible that most patients who are found incidentally to have white matter lesions have no specific clinical correlate. However, a number of studies have suggested that there is a relationship between cumulative white matter signal abnormalities, as manifested by increased signal intensity on MRI brain scan or white matter hypointensity on computed tomography (CT) brain scan, and cognitive impairment [5]. Of particular note, white matter changes have been observed in patients with pathologically confirmed Alzheimer’s disease [6], and periventricular white matter changes on MRI brain scan predict poor neuropsychological performance in Alzheimer’s disease [7]. This finding underscores the potential to see coexistent neuropathology in the elderly where small vessel cerebrovascular disease can contribute to the severity and progression of the primary dementing process.

CADASIL The characteristics of CADASIL are summarized in Table 13.2. This is a genetically transmitted disorder associated with a mutation in the NOTCH3 gene. The NOTCH3 expression is restricted to smooth muscle cells. The mutation associated with CADASIL results in accumulation of the ectodomain of NOTCH3, specifically the 210-kDa fragment, within the affected cerebral vessel.

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Table 13.2  Characteristic Features of CADASIL 1. Autosomal dominant inheritance pattern 2. Associated with white matter angiopathy with characteristic distribution of lesions seen on MRI 3. Associated with stroke-like episodes leading to progressive neurologic deficit 4. Associated with migraine-type headaches, which can be the presenting manifestation 5. Associated with dementia as the disease progresses 6. Can be confirmed by either skin biopsy, which demonstrates GOM, or genetic testing which shows a mutation in the NOTCH3 gene

Figure 13.3  Prominent white matter changes in a 36-year-old woman with transient ischemic attacks who was found to have CADASIL, as observed on T2-weighted (A) and FLAIR (B) sequences of MRI.

Pathologically, one typically sees small isolated areas of ischemic change within the periventricular and subcortical white matter. In addition, one can see lacunar-type infarction of the brain stem, thalami, and basal ganglia. The cortex and cerebellum are typically spared. The involvement of the small vessels is characterized by deposition of osmiophilic, granular, electron-dense material (GOM) within the media with secondary vessel thickening. Radiographically, there are punctuated hypointensities on the T1-weighted MRI corresponding to hyperintensities on the T2-weighted and FLAIR MRI (Figure 13.3). It is expected that the MRI brain scan will routinely be abnormal by the age of 35 years, if not sooner. The MRI brain scan can be abnormal by the age of 20 years, and a normal MRI brain scan is not compatible with symptomatic CADASIL.

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One expects to obtain a positive history of similar manifestations in the family of an affected patient in light of the autosomal dominant inheritance pattern of this disorder. However, the patient has been reported with clinical and imaging characteristics of CADASIL, but with de novo mutation of the NOTCH3 gene, specifically of Arg182Cys [8]. This has led to the speculation that CADASIL is more frequent than previously thought. The typical pattern of CADASIL is the development of migraine, which occurs in at least 40–60% of patients by the third and fourth decades. Migraine with aura and complicated migraines are seen more frequently than in the general population. Manifestations of stroke are present in the majority of patients in the fourth and fifth decades, primarily presenting with subcortical syndromes. Cognitive decline, especially executive function impairment, may begin at an early age. Dementia generally develops in the sixth and seventh decades [9], and most patients die from the disease by the seventh decade. Patients can demonstrate a fluctuating pattern, and this pattern, along with MRI findings, can mimic multiple sclerosis. Furthermore, just like with multiple sclerosis, the phenotypic expression can be relatively mild, with little in the way of neurologic deficit. In a study of the natural history of CADASIL [10] based upon pooled data in 105 symptomatic patients, the mean age of onset was 36.7  12.9 years. Stroke or transient ischemic attack was seen as the initial presentation in 43%, with a mean age of 41.2  9.2 years. Migraine was diagnosed in 40%, with a mean age of 28.3  11.7 years. Initial symptoms also included depression in 8.6%, cognitive impairment in 6%, and epilepsy in 3%. Over time, 68% of patients experienced a stroke or transient ischemic attack, and the events were recurrent in roughly 50%. Dementia was diagnosed in 42%, and a total of 6% developed epilepsy over time. During follow-up, 21% of patients died, with a mean age of 54.8  10.6 years. Of the patients who died, all but 13.6% had suffered a stroke or transient ischemic attack and 86% were demented. This study suggested a significantly shortened survival for patients with more severe CADASIL, especially those with stroke and dementia. Of note, hypertension was diagnosed in only 8 of the 105 patients (7.6%). Therefore, coexistent risk factors for small vessel disease do not appear to be an important contributor in most patients with CADASIL. Desmond et al. [11] studied the phenotypic expression in a North American family with an identified family member with CADASIL. They found an array of manifestations in multiple family members, including depression, learning disorders, migraine, and ischemic stroke. The four family members with diffuse white matter disease and lacunar-type infarctions on MRI brain scan, characteristic of CADASIL, were not hypertensive, and a number of other family members had migraine, recurrent stroke, dementia, and/or depression. Severe mood disturbance is observed in up to 20% of patients. The diagnosis was previously based on the clinical manifestations along with a positive family history. A skin biopsy demonstrating GOM can be confirmatory and has a reported sensitivity of 45% and a specificity of 100%. The MRI brain scan has features that help in distinguishing CADASIL from other disorders with white

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matter signal abnormalities. Moderate or severe involvement of the anterior temporal pole on MRI was associated with a sensitivity of 89% and a specificity of 86%. Involvement of the external capsule was associated with a sensitivity of 93% but with a specificity of only 45% [9]. There is increasing white matter involvement over time, and signal abnormalities can be detected in the presymptomatic phase of the disease. Genetic analysis, which is now commercially available, is confirmatory, and there can be a variety of different point mutations. In a study of 48 families, 73% were in exon 4, 8% in exon 3, and 6% in exons 5 and 6 [9]. Recently, a similar familial syndrome termed pontine autosomal dominant microangiopathy and leukoencephalopathy (PADMAL) has been described [12]. It is not associated with the NOTCH3 mutation and has characteristic MRI findings of infarcts in the pons and subcortical and periventricular white matter, with relative sparing of the temporal lobes. The genetics of this disease has not yet been identified. There is no specific treatment for CADASIL. Antiplatelet therapy with aspirin, clopidogrel, or the combination of aspirin and dipyridamole in extended-release form is often the first choice from an antithrombotic standpoint. Certain patients may ultimately be placed on the anticoagulant warfarin because of recurrent events despite treatment with antiplatelet agents. Some have advocated cholinesterase inhibitors in an effort to augment the cholinergic pathway in patients susceptible to a dementing illness. A randomized controlled trial assessed cognitive outcomes in CADASIL patients treated with donepezil compared with placebo. The study failed to find a significant difference between treatment groups in the primary endpoint, the cognitive subscale of the Alzheimer’s disease assessment scale. However, the donepezil group did show significant improvement on certain executive function tests [13]. Memantine may be considered from a neuroprotective standpoint; it promotes N-methyl-d-aspartate (NMDA) receptor blockage. Other approaches include optimal control of migraine, which can be associated with small vessel ischemic insults and is commonly part of the CADASIL clinical spectrum. One could make the case that perhaps a vasodilator, such as a beta-blocker or a calcium channel blocker, would be the most attractive migraine prophylactic agent. However, it is probably advisable to avoid hypotension in such a clinical setting as this could, theoretically, contribute to the pathogenesis of dementia [14]. Triptan medications are generally avoided. Furthermore, one could perhaps make a stronger case for an angiotensin-converting enzyme (ACE) inhibitor in light of reports on their efficacy in the prevention of recurrent ischemic stroke. In the same vein, one could make the case for statin therapy even if there is no clear-cut hyperlipidemia in light of reports that there may be other protective vascular mechanisms involved. In addition, control of elevated homocysteine levels with vitamin supplementation may have some theoretical benefit. In the North Manhattan Study [15], total homocysteine level was associated with white matter hyperintensity volume. Practical measures such as smoking cessation, a diet and exercise program, optimal diabetic control, and relaxation techniques are to be promoted as a general health approach and are especially pertinent in higher-risk patients such as those with CADASIL. Potential but unproven therapeutic approaches to CADASIL are summarized in Table 13.3.

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Table 13.3  Potential Therapeutic Approaches to CADASIL 1. 2. 3. 4. 5. 6.

Antiplatelet therapy Anticoagulant therapy Migraine therapy Cholinesterase inhibitor therapy NMDA receptor blockade with an agent such as memantine Optimal control of potential risk factors such as hypertension, diabetes mellitus, hyperlipidemia, and hyperhomocysteinemia

Binswanger’s Disease There has been considerable controversy over the term “Binswanger’s disease,” as it is not a particularly well-defined disorder [16]. It has been termed “subcortical arteriosclerotic encephalopathy,” and such a designation tends to emphasize the heterogeneity. It is quite possible that the majority of cases ascribed to Binswanger’s disease actually represent more severe cases of hypertensive microangiopathy or undiagnosed CADASIL. However, one does not always see significant white matter abnormalities in chronic hypertensive patients, and the degree of white matter signal abnormalities does not necessarily correlate with the duration and degree of hypertension. Other pathologic factors might well be at play in certain patients with neurologic deficits and prominent periventricular and centrum semiovale signal abnormalities by MRI brain scan or prominent hypodensity changes in the same regions by CT scan, which has been termed “leukoaraiosis.” Furthermore, it is not uncommon to see little in terms of neurologic deficit clinically in a number of these patients while the MRI brain scan reveals dramatic white matter abnormalities (Figure 13.4). This often leads patients with subtle findings on neurologic examination to be informed by the clinician reviewing the scan that they have had “multiple strokes.” Patients with prominent white matter abnormalities on MRI or CT brain scan often have cognitive difficulty beyond that compatible with age. They also demonstrate more in terms of gait instability than expected for the age-matched populations. Furthermore, it is not uncommon to find subtle long-tract signs with some loss of dexterity, prominent reflexes, and one or both upgoing toes. In addition, one may see so-called cortical release signs such as a snout, suck, grasp, or palmomental reflex. However, the age group most commonly affected by such white matter findings is also susceptible to other relatively frequent afflictions, such as primary neurodegenerative disease and cervical spondylosis with myelopathy. It is not uncommon to see a patient with progressive cognitive deficit and/or gait instability with a combination of cortical atrophy, periventricular and centrum semiovale white matter changes, and prominent ventricles on MRI brain scan (Figure 13.5). The challenge for the clinician, in such a circumstance, is to determine how much the ventricular enlargement (i.e., a possible component of normalpressure hydrocephalus) might be contributing to the neurologic picture. The management of a Binswanger’s-type clinical picture probably calls for aggressive management of risk factors that may be contributing to the pathogenesis.

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Figure 13.4  A 78-year-old man with concerns about forgetfulness. FLAIR MRI reveals prominent white matter changes despite normal cognitive testing and a normal neurologic evaluation.

Figure 13.5  A 76-year-old woman with cognitive impairment and progressive gait instability. FLAIR MRI demonstrates prominent ventricles with periventricular white matter hyperintensities.

This would include aggressive blood pressure management over time with strict adherence to more recent guidelines for blood pressure management. Specifically, a systolic pressure of roughly 120 mmHg and a diastolic of roughly 80 mmHg would be reasonable target goals. ACE inhibitors might well be the antihypertensive of

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Table 13.4  Management Approaches for the Prevention of Ischemic Demyelination 1. Maintenance of a systolic blood pressure in the 100- to 120-mmHg range and diastolic blood pressure in the 60- to 80-mmHg range 2. Use of an ACE inhibitor 3. Use of statin therapy to promote ideal control of low-density lipoprotein (LDL) and other possible neuroprotective effects 4. Optimal blood sugar control in diabetics 5. Lowering of the homocysteine level with vitamin therapy 6. Use of antiplatelet therapy, when appropriate, such as aspirin, clopidogrel, or the combination of low-dose aspirin and extended-release dipyridamole 7. Potential benefits of certain fruits, grains, and vegetables in the diet 8. Potential benefits of antioxidant vitamin supplementation 9. Effective diet and exercise program with promotion of relaxation techniques

choice, and one could certainly make a case for statin therapy as well. Potential management approaches are summarized in Table 13.4.

White Matter Disease and Cerebral Hemorrhage Underlying small vessel vasculopathy, especially in the setting of hypertension, can be associated with not only infarction, but also intracerebral hemorrhage. Smith et al. [17] reported that lobar hemorrhage was commonly associated with white matter damage and cognitive impairment. In this subset of patients, the authors speculated that possible cerebral amyloid angiopathy played a role in the clinical picture. This might help to explain the observation that leukoaraiosis is associated with an enhanced risk of intracerebral hemorrhage in patients receiving warfarin [18].

Migraine It is now well recognized that one can see white matter signal abnormality in association with migraine in relatively young patients with no other risk factors for ischemic demyelination (see Figure 13.1). This implies the potential for vascular insult, and it is now well recognized that migraine is a not uncommon cause of stroke in the young [19] (Figure 13.6). The finding of such white matter signal abnormalities should draw attention to the potential for cumulative ischemic injury over time as well as possibly identifying patients at enhanced risk for ischemic stroke. The patchy lesions that can be observed in classic migraine, for example, have been termed “microinfarction” [20]. The detection of such MRI findings might well point the clinician toward avoidance of hormonal manipulation in such individuals, especially those with stroke-like symptoms in association with their migraine, those in their 30s and beyond, those who smoke, as well as those with a history or family history of

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Figure 13.6  A 26-year-old woman with migraine, who developed vertical diplopia and left-sided numbness associated with severe throbbing headache. Right thalamic infarct is demonstrated on T2-weighted axial (A and B) and coronal (C) MRI. Cardiac and hematologic evaluations were normal. Carotid duplex ultrasound and intracranial magnetic resonance angiogram (D) were also normal.

either a well-documented prothrombotic state or a positive family history of earlyonset vascular disease. From a management standpoint, the finding of white matter signal intensity changes by MRI brain scan might well direct the clinician toward either beta-blockers or calcium channel blockers (i.e., vasodilators) for migraine prophylaxis. In addition, there might well be heightened concern in such patients about the use of vasoconstricting agents such as triptans or ergotamines. Furthermore, one must carefully address any additional contributing factors such as hyperlipidemia or a significantly elevated homocysteine level. Such management approaches are summarized in Table 13.5.

Pathogenic Models of Ischemic Demyelination As mentioned previously, the underlying vasculopathy associated with CADASIL involves degradation of vascular smooth muscle cells, deposition of GOM within the basement membrane of these vascular smooth muscle cells, and accumulation of NOTCH3 protein within the cell membrane. Cerebral blood flow studies with positron emission tomography (PET) imaging and MRI have demonstrated an apparent

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Table 13.5  Potential Approaches to the Migraine Patient with the Finding of Ischemic Demyelination 1. 2. 3. 4. 5. 6.

Aggressive prophylaxis against headache Enhanced use of vasodilator such as a beta-blocker or calcium channel blocker Consideration of an antiplatelet agent such as aspirin Avoidance of hormonal manipulation unless necessary Avoidance of vasoconstricting agents such as triptans or ergotamines Enhanced attention to smoking cessation, hypertension control, diabetes control, and lipid control when indicated

component of hypoperfusion contributing to the white matter lesions [21–23]. This could be related to small vessel diameter compromise or possibly to loss of the integrity of the vascular smooth muscle cells. In an experimental model of CADASIL [24], impaired cerebrovascular reactivity was observed. The finding of compromised cerebral autoregulation prompted the authors to speculate that increased resistance or decreased relaxation of involved vessels might be part of an early pathogenic process. In human studies of white matter disease in susceptible individuals, there is evidence of selective hypoperfusion of either subcortical white matter or so-called borderzone territories [25]. However, the susceptibility might be selective, as Gold et al. [26] reported that lacunes affecting the thalamus and basal ganglia correlated with dementia while lacunes involving white matter did not. Lacunar disease in these locations has also been associated with an increased risk of poststroke depression [27]. In terms of the pathogenesis of lacunar stroke, Doubal et al. [28] used fractional analysis of retinal vessels to determine that decreased complexity of vessel branching was associated with lacunar stroke. This was viewed as supportive of a distinct vasculopathy as such a unique vascular pattern identification persisted when the authors corrected for coexistent factors, including hypertension, diabetes mellitus, degree of white matter hyperintensity, and stroke severity. In the general population, the finding of white matter lesions and silent brain infarcts is associated with an increased risk of stroke [29]. Markus et al. [30] looked at markers of endothelial and hemostatic activation in patients enrolled in the Austrian Stroke Prevention Study. They found a relationship between cerebral white matter hyperintensities and endothelial cell activation, but not with coagulation activation. In a recent transcranial Doppler study [31], impaired cerebral perfusion was found to be possibly a significant factor in dementia. It has been recognized for a number of years that there is the potential contribution of a primary neurodegenerative component and vascular compromise component in a relatively large proportion of patients with dementia of the Alzheimer’s type or in the combined entity of Alzheimer’s disease with vascular dementia. Increasing recognition of the potential for vascular compromise to have an impact on the cumulative tissue pathology in the virtual epidemic of Alzheimer’s disease might lead to different approaches to prevention, such as with statin drugs [32].

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The role of inflammation in cerebral microvascular disease and cognition is also an area of investigation. Using diffusion tensor imaging (DTI), Wersching et al. [33] recently demonstrated an association between high-sensitivity C-reactive protein (hs-CRP) levels and areas of disrupted white matter microstructure not visible on conventional MRI scans. Elevated hs-CRP levels were also associated with cognitive decline in patients with no evidence of stroke. The roles of hs-CRP and other markers of inflammation have yet to be defined clinically in the setting of chronic microvascular disease.

Ischemic Demyelination and Ischemic Stroke Risk Patients with ischemic white matter disease are at enhanced risk for stroke [34]. In addition, white matter hyperintensities on MRI in elderly normal volunteers correlate with subtle cognitive deficit with reduced speed of complex mental processing [35]. Lacunar-type stroke has been reported to have a recurrence rate of up to 12% per year, and one can hypothesize that asymptomatic small vessel disease is presumably associated with at least some heightened risk when compared with an age-matched population without evidence of ischemic demyelination on brain scan. Multiple studies indicate that it is the confluence of the ischemic white matter lesions on MRI brain scan that is associated with the greatest risk for clinically evident ischemic stroke [2]. This confluence of larger white matter abnormalities correlates with the changes described as leukoaraiosis on CT brain scan. The Austrian Stroke Prevention Study addressed the evolution of white matter signal abnormalities on serial MRI brain scan over time [36]. The preliminary study looked at 273 middle-aged and elderly subjects in a community who were free of neuropsychiatric disease on a clinical basis. Over 3 years of follow-up, white matter lesions progressed in 17.9% of subjects, but this change was pronounced in only 8.1%. The 6-year follow-up data [37] on 296 subjects revealed detectable progression in no more than 17.2%. This would appear to indicate a relatively benign course for such patients found incidentally to have white matter lesions. However, roughly two-thirds of a subset of subjects with early confluent white matter lesions, and all of the subjects with clearly defined confluent lesions, had a pronounced increase in lesion volume. In support of this finding, a study of elderly subjects with gait abnormalities revealed an absolute white matter lesion volume increase of 1.1 cm3 over 4 years [38]. In a follow-up report to the Austrian Stroke Prevention Study [39] after 6 years, the median increase in white matter lesion load was 0.2 cm3; the maximum increase observed was 31.4 cm3. Of particular interest, the white matter lesion load correlated with both loss of brain volume and cognitive impairment. However, the correlation between cognitive function and white matter lesion load was no longer significant when the model incorporated tissue loss, suggesting that the resultant tissue loss or cortical atrophy associated with progressive white matter changes was the most important determinant of cognitive decline. This might help to explain the somewhat contradictory reports of association versus no association between white matter lesions and cognitive decline.

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There also appears to be an interaction between ischemic demyelination and stroke outcomes. White matter ischemic disease has been correlated with increased risk of stroke recurrence [40], hemorrhagic transformation after thrombolysis [41], and development of dementia following stroke [42]. The burden of white matter disease may also correlate with functional outcome after stroke. A recent study by Arsava et al. [43] found that patients with a larger volume of leukoaraiosis at the time of stroke had a significantly higher modified Rankin Scale score at 6 months. In the future, these associations may help to guide medical decision making and stratify risk for interventions in the setting of acute ischemic stroke.

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14 Inflammatory Mechanisms in

Ischemic Cerebrovascular Disease Mutsumi Nagai, D. Neil Granger Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Inflammatory Cell Recruitment in Ischemic Stroke Leukocyte Recruitment in Post-Ischemic Cerebral Microvessels Kinetics of Accumulation of Different Leukocyte Populations Overview A significant reduction in cerebral blood flow (CBF) resulting from thrombosis, embolism, or relative hypoperfusion generally produces a characteristic infarct that can be divided into two regions that relate to the potential for cell survival. The center of the infarct has the least perfusion and is called the “ischemic core,” which is associated with rapid cell death caused by proteolysis, lipolysis, energy depletion, and a severe ionic imbalance [1]. A functionally disabled but viable region of brain tissue called the “penumbra” surrounds the ischemic core, where blood flow is limited and the cells enjoy a partially preserved metabolism [2]. Unless rescued, cells in the penumbra region will suffer from membrane damage mediated by a complex cascade of events, and this will progress and deteriorate over a period of several days. After an ischemic insult, the combination of bioenergetic failure, excitotoxicity, and oxidative stress leads to microvascular dysfunction and injury. Anatomically, brain vessels are surrounded by pericytes, which are attached by neurons or astrocytes with their end feet [3]. The functionally failed neurons may transmit a danger signal (directly or via interstitial astrocytes) to brain vessels. Alternatively, injured neurons or astrocytes can secrete certain cytokines, and these neuroglial–vascular interactions leads to vessel wall injury. Concurrently with the development of vascular injury, an inflammatory response is initiated shortly after the ischemic insult, and it can last for several days [4]. The inflammatory response appears to accelerate the progression of tissue injury even if tissue perfusion is restored hours after the ischemic attack. Direct therapy for cerebral infarction caused by arterial thrombosis is thrombolysis using tissue plasminogen activator (t-PA), which has a narrow window (<3 h) for effective treatment [5]. Such an intervention has the potential to rescue the penumbra region from the catastrophic ischemic cascade that is linked to local inflammation [6] (Figure 14.1). Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00014-9 © 2011 Elsevier Inc. All rights reserved.

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Figure 14.1  (A) Time-dependent alterations of neurons and glial cells as well as macroscopic brain tissue following brain ischemia. In the ischemic core lesion, neurons and glial cells rapidly (seconds to minutes) die due to disruption of cell membranes caused by energy failure. In the penumbra, these cells can survive under conditions of minimal blood flow. However, the surviving cells are activated and secrete cytokines and other agents that initiate inflammation. Over a period of days, the inflammatory response intensifies, which renders cells within the penumbra vulnerable to necrosis and apoptosis. (B) Time-dependent appearance of different cell populations in the ischemic lesion. Neutrophils appear first (within 4 h after ischemia), followed by monocytes (within 24 h and remain after several weeks). Microglial cells accumulate between 24 and 72 h after the ischemic insult.

Cell Infiltration in the Infarct Area Under physiologic conditions, a relatively small number of leukocytes (compared to other tissue) reside in brain parenchyma, which is referred to as an immune privileged site. However, after ischemia, many kinds of cells (e.g., astrocytes, microglial cells, and endothelial cells [ECs]) are activated in brain tissue, with leukocytes and platelets infiltrating the tissue to mediate an inflammatory response. The population of the blood cells that infiltrate the brain is altered over the time course after the ischemic insult. Observations from a rodent middle cerebral artery (MCA) occlusion (MCAO) model indicate that resident microglial cells migrate into the peri-infarct area within 24 and 72 h of the ischemic insult, whereas monocytes/macrophages, which transform

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into phagocytes and salvage injured cells, appear in the core lesion at 72 h after the ischemic insult [7,8]. The polymorphonuclear leukocytes (PMNs) appear in the ischemic brain within several minutes to a few hours after the insult. Infiltrating neutrophils and macrophages have also been described in human cerebral ischemic lesions [9]. A radiologic study employing radiolabeled PMNs has documented the early PMN recruitment that occurs in post-ischemic human brain [10]. Leukocyte recruitment is observed for days to weeks after an ischemic insult [11], during which there is a switch in the infiltrating population from PMNs to mononuclear leukocytes (MNs) (Figure 14.2).

Erythrocyte

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Figure 14.2  Time-dependent changes in cerebral ECs, parenchymal cells, and recruited blood cells in post-ischemic brain. (A) Cell–cell interactions in and surrounding cerebral microvessels under normal physiologic conditions. Neurons and glial cells are attached via their end feet either directly or indirectly (via pericytes) to the blood vessel wall. Extraparenchymal vessels (e.g., pial vessels) lack these connections to the vessel wall. The brain vessels comprise three layers: the intima, media, and adventitia. The intima is covered with ECs, while the adventitia is covered with pericytes.

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Figure 14.2  (B) Early (within a few hours after ischemic insult) changes in and around a cerebral venule lying in an ischemic lesion. Astrocytes and neurons activated by the ischemic stress transmit danger signals to the vessel wall and secrete inflammatory cytokines, which in turn activate ECs to express adhesion molecules.

Astrocytes and Microglial Cells  Both of these resident cells play a role in ischemiainduced brain injury. They are activated to produce reactive oxygen species (ROS) upon initiation of the ischemic stress. Astrocytes produce and secrete various proinflammatory cytokines, chemokines, as well as the inducible isoform of nitric oxide synthase (iNOS), all of which promote inflammation. Astrocytes also express the major histocompatibility complex (MHC) and co-stimulatory molecules, which support an anti-inflammatory response [12]. Microglial cells are the resident macrophages of the brain and play a crucial role as resident immunocompetent and phagocytic cells. There is substantial evidence that microglial cells activated in response to ischemia have the potential of releasing several pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, as well as other potential inflammatory/cytotoxic molecules, including nitric oxide (NO), ROS, and prostaglandins [13]. However, there are some reports suggesting that microglial cells do not exacerbate brain injury by accelerating inflammatory response even though they appeared in the ischemic lesion and

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Figure 14.2  (C) Leukocyte recruitment paradigm after brain ischemia. Leukocytes (using PSGL-1) engage with EC E-selectin and/or P-selectin, which allow the low-affinity adhesive interaction called rolling. Subsequently, β2-integrin (LFA-1 or Mac-1) expressed on the leukocyte surface engages with ICAM-1 on ECs to mediate firm adhesion. Concomitantly, platelets adhere to both ECs and adherent leukocytes via different ligand–receptor interactions.

transformed into phagocytes. They seem to exert beneficial effects by supporting neurogenesis, as well as cell survival, migration, and differentiation [14]. Microglial cells may exert neuroprotection by producing neurotrophic molecules such as brainderived neurotrophic factor, insulin-like growth factor (IGF)-I, and several other growth factors. Hence, microglial cells seem to control inflammatory response, while acting to scavenge and repair injured brain tissue after ischemia. Neutrophils  Neutrophils adhere on the vessel wall and infiltrate into the ischemic lesion before all other subtypes of leukocyte [8,9,15,16]. Although there are many reports suggesting that infiltrating leukocytes into the lesion are associated with deterioration after ischemic stroke, there are other reports that do not support this contention [17,18]. Lymphocytes  There is a growing body of evidence that supports a role for T cells in the tissue injury following an ischemic stroke [15,19]. In a mouse model of cerebral ischemia–reperfusion (I/R), T cells appear to be major modulators of the adhesion of leukocytes and platelets in cerebral venules, and infarct size. Mice deficient in either CD4

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Transmigration

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Figure 14.2  (D) Mechanisms of leukocyte transmigration, BBB dysfunction, and amplification of the inflammatory response. Neutrophils, monocytes, and lymphocytes migrate into the brain parenchyma in response to chemoattractants such as MCP-1, MIP-1, or RANTES derived from astrocytes, neurons, infiltrating leukocytes, or ECs. Proteinases such as MMP-9 derived from ECs, neurons, and/or astrocytes digest the endothelial basal lamina to cause BBB failure. The infiltrating neutrophils secrete more MMP-9 and pro-inflammatory cytokines, which worsens the BBB dysfunction and amplifies the inflammatory response. Some PLAs detach from the cerebral endothelium and enter the systemic circulation.

and/or CD8 T cells exhibited a reduced infarct size, a lower number of adherent leukocytes and platelets in the cerebral venules, and improved neurologic outcome 24 h after reperfusion [15]. B-cell–deficient mice did not exhibit protection against the inflammation, tissue injury, and neurologic deficits induced by ischemic stroke. In vitro experiments have revealed that the adhesion of lymphocytes derived from stroke patients to cerebral endothelium was significantly higher than that noted for lymphocytes isolated from healthy subjects. The pathophysiologic relevance of this observation is unclear since it is not known whether T cells must directly interact with cerebral ECs to exert their deleterious actions during ischemic stroke. Recently, the importance of regulatory T cells (Tregs) in ischemic stroke has been elucidated. The neuroprotective potential of Tregs was demonstrated by depletion of Tregs in mice, which delayed brain damage and neuronal function. The neuroprotective role of Tregs was mediated by IL-10 [20]. Another recent study has also

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implicated IL-17–producing T cells in ischemic stroke. Although reconstitution of immunodeficient Rag-2/ mice with wild-type T cells leads to an exacerbation of ischemic brain injury, the adoptive transfer of T cells from IL-17 knockout mice into Rag-2/ mice leads to a significantly reduced infarct size. They also demonstrated that γδT cells were a major source of IL-17 and that IL-23 secreted from hematopoietic macrophages was the main stimulus for IL-17 secretion. Using either an antibody to T cell receptor (TCR) γδ or its knockout mice, they showed decreased IL-17 levels and reductions in the inflammatory response and magnitude of injury. A clinically important observation in this study was the capacity of the antibody to provide neuroprotection even though it was administered 24 h after the ischemic insult [21]. Macrophages and Monocytes  Mononuclear cells accumulate in the ischemic region following the appearance of neutrophils, and they play an important role following their transformation into macrophages. Intravital microscopic studies suggest that the leukocyte population that accumulates after 24 h of reperfusion is mainly mononuclear cells rather than neutrophils, since mice rendered neutropenic with antineutrophil serum exhibit an attenuated recruitment of adherent leukocytes in cerebral venules at 4 h after I/R, but it has little effect on leukocyte adhesion at 24 h after reperfusion [15,16].

Role of Leukocyte and EC Adhesion Molecules Intravital microscopy has been used to directly monitor and quantify the recruitment of leukocytes or platelets in post-ischemic cerebral venules. After brain ischemia, circulating leukocytes begin to roll and firmly adhere to ECs in venules (not arterioles) and then transmigrate through the vessel wall into brain parenchyma [16,22]. The process of leukocyte infiltration involves several steps (see Figure 14.2C), including activation, rolling, firm adhesion, and transendothelial migration. ECs are activated by ischemic stress, which results in the cell surface expression of adhesion molecules. Likewise, activated leukocytes or platelets coursing through the cerebral microcirculation also express adhesion glycoproteins (GPs) that allow blood cells to form adhesive bonds with EC adhesion molecules. Oxidative stress, cytokines, and chemokines are some of the long list of stimuli that can promote the expression of adhesion molecules on ECs and blood cells following ischemic stroke.

Rolling and Firm Adhesion of Leukocytes

Rolling  In non-inflamed cerebral venules, relatively few rolling leukocytes are observed. This may reflect a low expression of EC adhesion molecules, a high electrostatic charge on the EC surface, or high venular shear rates in the cerebral microvasculature [23]. ECs activated by ischemic stress express E-selectin and/or P-selectin. P-selectin is detected in 15 min and E-selectin in 2 h following brain ischemia [24]. P-selectin expression appears to be induced by a complement-dependent pathway, because its expression can be modulated by targeting the complement receptor-2 [24]. Circulating leukocytes and platelet constitutively express P-selectin glycoprotein ligand-1 (PSGL-1), and its motif sialyl-Lewisx can engage with either E-selectin or

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P-selectin. However, the engagement is too weak to arrest the leukocytes on ECs; consequently, in the presence of shear, the leukocytes roll on the EC surface [25]. Firm Adhesion  The firm adhesion of leukocytes with ECs is mediated by the engagement of β2-integrins on leukocytes and its ligand, intercellular adhesion molecule-1 (ICAM-1), on ECs [23]. The β2-integrins are heterodimeric proteins that consist of α (e.g., CD11a, CD11b) and β (CD18) subunits. Both CD11a/CD18 (lymphocyte function-associated antigen-1 [LFA-1]) and CD11b/CD18 (macrophage-1 antigen [Mac-1]) appear to mediate the leukocyte adherence noted in post-ischemic cerebral venules at 24 h of reperfusion since LFA-1 knockout or Mac-1 knockout mice exhibit much less leukocyte adhesion at that time compared to wild-type controls subjected to MCAO [26]. The upregulation of EC adhesion molecules after MCAO is modulated by several signaling pathways, including CD40/CD40L and Notch-1. CD40 is expressed on lymphocytes, monocytes, platelets, dendritic cells, ECs, and neurons, whereas CD40L is detected on activated platelets. Mutant mice that are deficient in either CD40 or CD40L exhibit lower leukocyte and platelet recruitment in postischemic cerebral venules, which is associated with attenuated blood–brain barrier (BBB) damage and a smaller infarct volume [27]. Notch-1 also seems to contribute to adhesion molecule expression and leukocyte recruitment. Inhibition of the Notchactivating enzyme γ-secretase attenuates I/R-induced brain injury. It also reduces the accumulation of leukocytes and platelets, which was associated with smaller number of CD11b-positive cells in the cerebral cortex and attenuated ICAM-1 expression on the cerebral endothelium after I/R injury [28]. This finding suggests that Notch signaling may contribute to the upregulation of ICAM-1 and other EC adhesion molecules in the cerebral vasculature injured by I/R. Many studies have focused on testing the efficacy of agents that interfere with leukocyte–EC adhesion on the severity of brain injury after an ischemic insult. L-selectin blockade alone appears to exert little beneficial effect on brain damage and neutrophil infiltration in a rabbit model of ischemic stroke [29]. However, when used in combination with t-PA, significant protection against brain injury is observed [30]. P-selectin blockade has also proven effective in different experimental settings. P-selectin knockout mice exhibit a reduction in infarct volume, better functional outcome, and a better return of CBF after I/R [31]. Similarly, an anti–P-selectin antibody was shown to reduce infarct size in a model of cerebral I/R [32]. In a different study employing a permanent ischemia model, P-selectin immunoblockade reduced infarct size and brain edema. In these studies, anti–P-selectin antibodies were administered 30 min before ischemic induction, which does not adequately reflect potential clinical use as a therapeutic agent. E-selectin immunoblockade appears to exert a therapeutic effect when administered following the induction of cerebral ischemia. Administration of an anti–E-selectin antibody reduces infarct size even when administered 90 min after ischemic insult [33]. E-selectin blockade with an analogue of sialyl-Lewisx (CY-1503) also reduces infarct size and neutrophil recruitment. Administration of a humanized monoclonal anti–E-selectin and P-selectin antibody (HuEP5C7) has been reported to reduce infarct volume and improve neurologic outcome when administered during the period of cerebral vessel occlusion, but did not exert a beneficial effect when administered after

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stroke onset [34]. Selectin blockade with a synthetic oligopeptide also has been shown to reduce brain injury in a transient but not in a permanent model of cerebral ischemia, suggesting that reperfusion is necessary for effective anti-selectin therapy [35]. A novel strategy for induction of mucosal immune tolerance to E-selectin has shown promise in preventing the genesis of ischemic and hemorrhagic strokes in spontaneously hypertensive stroke-prone rats [36], and to confer neuroprotection in a model of permanent ischemia [37]. There is considerable evidence for an increased expression of ICAM-1 in the post-ischemic brain [9]. ICAM-1–deficient mice exhibit smaller infarct sizes and less neutrophil recruitment following ischemic stroke compared to wild-type mice [31,38]. Similar protection is observed in wild-type mice receiving an ICAM-1– blocking antibody, either with or without concomitant treatment with t-PA [39]. On the basis of these promising preclinical studies with anti–ICAM-1 therapies, human trials were undertaken; however, the clinical studies failed to show an improved outcome in stroke patients [40]. LFA-1 is expressed on all leukocytes while Mac-1 is found on neutrophils, monocytes, and natural killer (NK) cells. Both β2-integrins are constitutively expressed, and increased expression and/or activation occurs following ischemic stroke. Mac-1 and CD18 knockout mice exhibit a reduced infarct volume and a lower mortality after cerebral I/R [41]. Immunoblockade of CD11b, CD18, or Mac-1 also protects against brain ischemic injury [42]. Moreover, immunoblockade of CD18 ameliorates the brain edema and infarction size while blunting the leukocyte recruitment [43]. In vitro experiments also suggest that lymphocyte adhesion on cerebral endothelium subjected to ischemia/hypoxia is dependent on LFA-1 and ICAM-1[44]. This could explain why LFA-1 blockade and Mac-1 blockade are more effective at blunting leukocyte recruitment at later times after I/R injury [26]. Collectively, the available data indicate that anti–β2-integrin and anti–P-selectin therapies are effective in models of ischemic stroke, especially if there is adequate reperfusion [45]. This possibility is supported by the observation that a combination of anti–β2-integrin (CD11/CD18) and t-PA confers a better therapeutic outcome in experimental animals than either agent alone [46]. A clinical study of acute stroke therapy (within 6 h of stroke insult) employed UK-279,276, which binds to Mac-1 antigen (CD11b/CD18). A quarter of the patients were treated with the anti–Mac-1 drug along with t-PA. While there was no evidence for effective treatment with UK-279,276, a post hoc analysis showed a slight improvement in patients who received the combination therapy [47]. Transendothelial Migration  The final step in the recruitment of leukocytes is transendothelial migration, which allows leukocytes to enter the brain parenchyma. Specialized proteins expressed in the junctions between adjacent ECs that are associated with the cytoskeleton facilitate this process, along with proteases secreted by the transmigrating leukocytes. Signaling pathways within the ECs are activated in response to leukocyte entry into the interendothelial junctions. These pathways affect the stability of the junctional proteins and/or their interactions with each other and the cytoskeleton. Another important contributor to the transendothelial migration process is chemokines, which promote the directed movement of adherent leukocytes toward the junction and to sites within the perivascular space [48].

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Contribution of Cytokines and Chemokines A variety of cytokines and chemokines are upregulated in the ischemic brain. Resident cells (glial cells and neurons) as well as recruited leukocytes (MNs, T lymphocytes, NK cells, and PMNs) secrete a number of cytokines and chemokines [49]. TNF-α, IL-1β, IL-6, IL-20, IL-10, and transforming growth factor (TGF)-β have been extensively studied in stroke, and the results are generally consistent with IL-1β and TNF-α promoting ischemic injury and inflammation, while IL-6, IL-10, and TGF-β appear to confer neuroprotection [50].

Interleukin-1β This is a major pro-inflammatory cytokine that has been widely studied. The major sources of the cytokine are ECs, microglia, macrophages, and damaged neuron or astrocytes. These cells express IL-1β mRNA as early as 15 min after an ischemic insult and produce the protein within a few hours and up to 4 days after the ischemic insult [51]. Administration of IL-1β exacerbates brain edema, infarction volume, and neutrophil infiltration after cerebral ischemia [52]. Infarct size is reduced in IL-1– deficient mice [53]. Both an anti–IL-1β antibody and an IL-1 receptor antagonist have been shown to ameliorate ischemic brain damage [52,54]. Recently, it was proposed that the enhanced IL-1β production in ischemic brain is associated with activation of Toll-like receptor (TLR)-4 and/or matrix metalloproteinase (MMP) activity [55]. While there is evidence that elevated IL-1β levels after stroke are correlated with IL-6 levels in plasma [56], studies on IL-6–deficient mice or intraventricular administration of IL-6 do not support such a linkage [57,58].

Tumor necrosis factor-α This cytokine is produced by neurons in the early stage (first hour) of an ischemic insult, with microglial cells and macrophages assuming a larger role in the later stages of the injury response [59]. TNF-α immunoblockade has been shown to reduce ischemic brain injury. However, it has been reported that blockade of the α (p55) subunit of the TNF-α receptor exacerbates the brain damage after ischemia [60].

Interleukin-10 A neuroprotective effect is observed with IL-10 [61], and it has been proposed as an indicator of clinical neurologic outcome after stroke. A reduced concentration of IL-10 within 1 h of an ischemic insult appears to indicate an exacerbation of neurologic symptoms [62]. The recent demonstration that Tregs exert a protective effect against the inflammation and tissue injury in ischemic stroke has been attributed to the ability of these cells to secrete IL-10 [20].

Chemokines MCP-1 is a potent chemoattractant that promotes the migration of monocytes and neutrophils into tissues. Injured neurons are a major source of MCP-1 in the first

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12 h after an ischemic insult, while astrocytes or microglia account for the MCP-1 in the later stages (e.g., 2 days) of an ischemic stroke [63]. While MCP-1 exacerbates brain injury due to its pro-inflammatory effects, it also facilitates the salvage function of inflammatory cells by directing them to damaged neurons. The results of a recent study revealed a role for MCP-1 in attracting neuroblasts into an ischemic locus in the brain, which was demonstrated using knockout mice for MCP-1 and its receptor (CCR2) [63].

Stromal Cell-Derived Factor-1α This is the trafficking factor for hematopoietic stem cells that influences their movement from bone marrow to peripheral blood and subsequent migration into ischemic brain tissue [64]. Recent work indicates that stromal cell-derived factor-1α treatment may rescue brain tissue after ischemia by reducing infarct volume and improving neural plasticity [65].

RANTES Regulated on activation, normal T-cell-expressed and secreted (RANTES) is a member of the CC-chemokine family. RANTES knockout mice and bone marrow chimeras produced from these mice have revealed that RANTES is an important mediator of BBB dysfunction, tissue inflammation, and platelet–vessel wall interactions in brain after I/R. Platelets were proposed as a potential source of the RANTES released into post-ischemic brain [22].

Role of Reactive Oxygen and Nitrogen Species Following an ischemic insult, brain cells produce increased amounts of reactive oxygen and nitrogen species, which can exacerbate the deleterious effects of ischemia via multiple injury mechanisms. Reactive oxygen and nitrogen molecules injure brain tissue via processes such as mitochondrial inhibition, Ca2 overload, and inflammation [66]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase appears to be a major source of the superoxide (O2) that is generated following brain ischemia [67]. The O2 formed by NADPH oxidase undergoes spontaneous dismutation to form hydrogen peroxide (H2O2), which is a potential source of hydroxyl radicals (OH). NO is produced from l-arginine by three types of NOS. In the early stage of brain ischemia, NOS types I and III are the predominant isoforms that are activated in neurons and vascular endothelium. At the later stages, NOS type II (iNOS) is activated in a variety of cells, including glial cells and infiltrating neutrophils [68]. Immediately following the ischemic insult, NO derived from endothelial NOS elicits vasodilatation, while the NO produced in the later stage by neuronal NOS or iNOS promotes tissue injury [69]. However, the results of a recent report suggest that iNOS is not always a deleterious mediator, since iNOS-deficient mice did not exhibit an exaggerated infarct volume and CBF response to brain I/R injury [70]. In addition to causing cell damage and activating different signaling pathways, oxygen-free radicals can

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activate MMP, especially MMP-9, which mediates extracellular damage and leads to BBB dysfunction.

Trigger for Post-Ischemic Inflammation Recent studies have revealed that TLRs expressed on various immune cells play an important role in the initiation of inflammatory responses in different brain cells after I/R. When any invariant- or damage-associated molecular pattern (e.g., ischemic stress-induced heat-shock protein) engages with TLR, the inflammatory signal is transmitted downstream of the NFκB or mitogen-activated protein kinase (MARK) pathways in brain cells. Then, these cells begin to produce and secrete pro-inflammatory cytokines, such as IL-1 and TNF-α and chemokines, and elicit the expression of adhesion molecules [71]. Consequently, TLR-2 and TLR-4 knockout mice are protected from ischemic brain injury [72,73]. TLRs also have a beneficial role after ischemic brain injury, when they are involved in mediating a preconditioning response prior to brain ischemia. Promotion of inflammation by lipopolysaccharide (LPS), a TLR-4 ligand, administered within 24 h of an ischemic insult worsens the injury response; however, if it is administrated more than 24 h before the ischemic insult, a beneficial effect is noted [74]. The neuroprotective preconditioning response is also manifested through TLR-2– or TLR-9– mediated pathways [75,76]. These novel findings offer hope for a potentially new therapeutic strategy for prevention of stroke-induced brain injury in high-risk patients.

Platelet Recruitment: Magnitude and Mechanisms Brain ischemia triggers marked alterations in the cerebral microvascular endothelium. Activated ECs express various adhesion molecules, which promote rolling and firm adhesion of platelets as well as leukocytes. The recruited platelets, either directly attached to ECs or bound to adherent leukocytes, promote a pro-thrombotic state that further exaggerates the perfusion deficit and state of “no flow” that accompanies cerebral ischemia [77]. Thus, the recruitment of leukocytes is accompanied by platelet recruitment after cerebral I/R.

Kinetics of Accumulation The leukocyte adhesion that occurs after cerebral I/R injury is accompanied by the recruitment of platelets. Platelet recruitment generally lags behind leukocyte recruitment. In addition, a large population of recruited platelets can be attached to the leukocytes that have already adhered on the cerebral endothelium. At 4 h of reperfusion after cerebral ischemia, neutrophil-dependent platelet is dominant, but at 24 h of reperfusion, neutrophil-independent mechanisms may have a dominant influence on platelet accumulation in cerebral venules [15]. The neutrophil-independent platelet adhesion may reflect binding to other leukocyte populations or the direct adhesion of platelets to cerebral microvascular ECs. The time-dependent changes in platelet and leukocyte adhesion in post-ischemic cerebral venules and the dependency of platelet recruitment on neutrophils have been recently addressed in detail [78].

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Molecular Determinants (Adhesion Molecules) and Dependency on Leukocyte Recruitment Major receptor–ligand interactions that contribute to platelet recruitment in microvessels include P-selectin–PSGL-1, GPIIb/IIIa–fibrinogen–ICAM-1, and von Willebrand factor (vWF)–GPIbα (Figure 14.3) [79]. The interactions between platelets, leukocytes, and cerebral vascular ECs after ischemia have been recently characterized. The contribution of P-selectin and ICAM-1 to platelet recruitment in ischemic cerebral venules was investigated using platelets harvested from P-selectin/ and ICAM-1/ mice [25]. The findings of this study were consistent with two possible mechanisms to explain platelet recruitment in cerebral venules within the infarcted region [80]. One involves the binding of GPIIb/IIIa on platelets with fibrinogen, which is deposited on endothelial ICAM-1, while the second mechanism proposes that adherent leukocytes provide a PSGL-1–expressing platform onto which platelet P-selectin can bind. The results of a different study suggest that P-selectin is more important than GPIIb/IIIa in mediating platelet recruitment in post-ischemic venules, and that mice rendered neutropenic exhibit a profound attenuation of platelet adhesion [16]. The

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Figure 14.3  Different patterns of platelet adhesion in post-ischemic cerebral microvessels. Some platelets adhere directly to ECs while other platelets attach to already adherent leukocytes.

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dependency of platelet recruitment in the microcirculation on leukocytes appears to depend on the inflammatory insult (e.g., ischemia versus TNF-α) and location of the venule (e.g., brain versus mesentery) [67,81–83]. A dependency of platelet recruitment on leukocyte adhesion is also supported by the observation that platelet accumulation in cerebral venules is delayed relative to leukocyte adhesion after I/R [79].

Platelet–Leukocyte Aggregates The attachment of platelets to leukocytes enhances the activation state of both cell types. Activated platelets promote leukocytes to produce larger quantities of superoxide and platelet-activating factor when attached to the leukocyte [84]. Moreover, platelet–leukocyte aggregates (PLAs) formed in an inflammatory locus can play a role in disseminating the inflammatory response to distant sites. PLAs formed on the vessel wall can be detached from vascular endothelium by shear forces created by the movement of blood. The appearance of a higher number of PLAs in the peripheral blood in stroke patients may reflect such a mechanism or may result from the binding of platelets to leukocytes in the circulation [85].

Consequences of Leukocyte and Platelet Recruitment Infarct Size As mentioned above, there is considerable evidence that leukocyte recruitment is tightly correlated with infarct volume after brain ischemia [78,86]. However, not all leukocyte subpopulations are associated with accelerated brain injury after stroke. Neutrophils have been shown to hasten brain injury after stroke, but there also are some reports suggesting that neutrophils do not affect infarct size [17], suggesting that the neutrophil may be a bystander rather than an effecter of tissue injury after cerebral ischemia. The contribution of lymphocytes to the brain injury response appears to be population-specific. Th1, Th17, and cytotoxic T lymphocytes (CTLs) contribute to the pro-inflammatory response, whereas Th2 and Tregs tend to downregulate the inflammatory response to cerebral ischemia. Depletion of either CD4 or CD8 T cells confers protection against brain infarction after I/R [15,78]. Platelets may also exacerbate ischemic brain injury through their interactions with adherent and/or circulating leukocytes. These heterotypic and homotypic interactions of blood cells may also contribute to the apparent “no-flow” state that often accompanies reperfusion of ischemic tissue [77]. There is no direct evidence that platelet depletion ameliorates brain infarct volume after ischemia, likely due to the consequences of platelet depletion on blood clotting and bleeding [22]. However, indirect evidence for a contribution of platelets is suggested by studies showing reductions in infarct volume following P-selectin blockade.

BBB Dysfunction The BBB is one of the most important physiologic characteristics of the cerebral vasculature. It is defined by a low permeability for small hydrophilic molecules, low

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rates of fluid-phase endocytosis, and high resistance to exchange. The BBB comprises a network of tight junctions, complex structures between adjacent ECs. In addition, there are several perivascular cells (pericytes, astroglial cells) that interact with ECs to contribute to the BBB phenotype [86]. After an ischemic insult, the BBB fails, allowing for the infiltration of leukocytes that secrete pro-inflammatory cytokines and chemokines, worsening the BBB dysfunction. The BBB disruption can lead to albumin extravasation, interstitial edema, and the movement of erythrocytes into the extravascular space. The time course of ischemia-induced BBB dysfunction is divided into early (2–24 h) and late (24–72 h) phases, which relate to distinct mechanisms of injury [87,88]. In the early phase (as early as 2 h following ischemic insult), dissolution of the basal lamina is elicited by several factors and BBB permeability rises. In neurons, glial cells, and ECs, ischemia induces oxidative stress, which triggers the production and activation of MMPs (especially for MMP-9) and other proteinases that digest endothelial basal lamina [89]. In the early phase, resident inflammatory cells are a major contributor to BBB dysfunction. However, in the late phase, infiltrating neutrophils gain importance as a contributor. Infiltrating neutrophils secrete MMP-9 as well as pro-inflammatory cytokines, which further digest the basal lamina, leading to severe BBB dysfunction. The discontinuous basal lamina ensures the easy transmigration of neutrophils through the BBB [90], which accelerates the inflammatory process and induces potentially life-threatening conditions (i.e., brain edema and hemorrhage).

Inflammatory Mechanisms in Cerebrovascular Thrombosis Arterial Thrombosis: Atherothrombosis Atherosclerosis is a major risk factor that predisposes the brain to thrombosis and cerebral ischemia. It is now widely recognized that atherosclerosis is an inflammatory disease, and that monocytes/macrophages as well as T cells contribute to plaque development. Large-scale clinical trials have revealed that the reductions in total cholesterol or low-density lipoprotein (LDL) levels in blood reduce the incidence of atherosclerosis and strokes [91].

Mechanisms for Thrombus Formation Hypercholesterolemia and other cardiovascular risk factors, including high blood pressure, diabetes mellitus, and smoking, promote the deposition of LDL beneath the ECs of large arteries, particularly those vessels exposed to high shear rates. This process involves the passive diffusion of apolipoprotein B (on LDL) into the subendothelial matrix [92]. The LDL is retained in the extracellular matrix and transformed into oxidized LDL (oxLDL) by ROS [93]. OxLDL and oxidized phospholipids derived from oxLDL activate ECs and induce an increased expression of adhesion molecules (e.g., ICAM-1, VCAM-1, or ELAM-1). Activated ECs allow leukocytes or platelets to roll and adhere, with the subsequent transendothelial migration into

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the intimal layer. Pro-inflammatory cytokines produced by leukocytes and platelets diminish endothelial barrier function, allowing easy passage of lipoproteins and the lipid components thereof into the subendothelial space. Monocytes, which become loaded with oxLDL and other lipids, transform into foam cells that accumulate to form the early plaque lesion. The atherosclerotic plaque is composed of a core lesion, proliferating smooth muscle cells (SMCs), extracellular matrix, and a fibrous cap that covers the lesion. The further deposition of extracellular components, blood cell recruitment, and neovascularization induced by cytokines and growth factors derived from infiltrating cells promote growth of the core, which leads to a thicker intimal layer that bulges into and narrows the vessel lumen. Within the core lesion, apoptosis of macrophages and other cells creates a necrotic core, and neovascularization can lead to hemorrhage of the fragile vessels. Immune cells secrete matrixdegrading proteases that cause thinning of the fibrous cap until it eventually ruptures. Exposure of the matrix beneath the ruptured plaque as well as tissue factor (TF) can initiate the coagulation cascade, producing a thrombus that can reduce blood flow at the site of development or that can dislodge to occlude a smaller downstream artery [94–96]. While monocytes/macrophages have received much attention as mediators of atherogenesis, recent studies suggest that mast cells, neutrophils, and lymphocytes also make a significant contribution to this process (Figure 14.4).

Role of Blood Cells Neutrophils Some recent studies demonstrated the importance of neutrophils in atherogenesis [97]. A histopathologic study of the human circle of Willis showed that neutrophils infiltrate into a lesion during the late stage of atherogenesis and contributes to plaque rupture by secreting elastase [98]. Another histologic study revealed neutrophil infiltration at the lesion area that is attached to the plaque and the adventitia [99]. It was reported that CXCR4 blockade exacerbates atherosclerosis in apolipoprotein E-deficient (ApoE/) and LDL receptor-deficient (LDLR/) mice, and this was associated with neutrophil accumulation in the plaque, increased inflammation, and enhanced apoptosis (Figure 14.5).

Mast Cells Mast cells appear to render atherosclerotic plaques more vulnerable to instability and rupture [100]. Mast cell–derived cytokines, TNF-α and IL-6, play a role in the formation of aneurysms and in atherogenesis, as evidenced from studies using mice that are genetically deficient in these cytokines [101].

Monocytes/Macrophages This leukocyte population seems to play a major role in all stages in atherogenesis. Transmigrated monocytes are transformed into activated macrophages by monocyte-colony stimulating factor (M-CSF) secreted by endothelial and vascular SMCs. Activated macrophages take up oxLDL through pattern-recognition receptors such as scavenger receptor type A and CD36 [102], then transform into foam

Lumen

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High blood pressure Diabetes mellitus Smoking Shear stress LDL depositionn

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ROS

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Monocyte Macrophage Foam cell

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SMC proliferation M-CSF Monocyte

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Figure 14.4  (A) Events associated with the early stage of atherothrombosis development. Circulating LDL crosses the EC layer and is retained in the extracellular matrix of the intima. The LDL is oxidized by ROS to form oxLDL, which activates ECs and promotes the expression of EC adhesion molecules that bind and recruit circulating leukocytes (monocytes). (B) Events associated with the intermediate stage of atherothrombosis. Transmigrated and accumulating monocytes transform into macrophages (foam cells) in response to M-CSF. Pro-inflammatory cytokines derived from leukocytes and ECs impair endothelial barrier function, which facilitates the passage of LDL. The accumulated foam cells, LDL, and proliferating SMC make up early plaque.

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Dead cell

Lymphocyte

Tissue factor

Angiogenesis

vWF

Plaque core

Fibrin

GPIbα

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Mature plaque

Ruptured plaque

(C)

Figure 14.4  (C) Events associated with plaque maturation and rupture. The core of mature plaque contains dead cells, newly formed blood vessels, and leukocytes. The core lesion is also covered with extracellular matrix and a fibrous cap. The immune cells secrete matrixdegrading protease, which thins the fibrous cap and renders it vulnerable to rupture. The ruptured atherosclerotic plaque exposes the extracellular matrix with its associated TF, vWF, and collagen, which activates the coagulation cascade and promotes platelet binding and aggregation.

cells. Recent studies have revealed two types of macrophages (M1 and M2) based on their contribution to inflammation. M1 has pro-atherogenic potential through the secretion of pro-inflammatory cytokines (e.g., IL-1 and TNF-α) and production of ROS, whereas M2 exhibits anti-inflammatory potential by secreting IL-10 or TGF-β [103]. Activated LDL-bearing macrophages secrete various cytokines, enzymes (e.g., cysteine-protease, serine-protease, MMP), and growth factors (e.g., platelet-derived growth factor [PDGF], vascular endothelial growth factor [VEGF], and IGF), which promote the inflammatory response with the help of T-cell–derived cytokines. This coordinated inflammatory response leads to SMC proliferation and migration into the intima. In the initial stage of monocyte adhesion and transmigration into the intima, several adhesion molecules, such as P-selectin, VCAM-1, and ICAM-1, are expressed on the ECs and facilitate monocyte recruitment. Although inhibition of VCAM-1 in animal models dramatically reduces plaque size, VCAM-1 blockade has no beneficial effect in myocardial infarction or ischemic stroke [104]. Other clinical trials of adhesion molecule blockade are ongoing. The function of monocytes/macrophages has been reviewed in detail by others [94,95]. Briefly, foam cells produce cyclo-oxygenase-1 and -2 (COX-1 and -2), which promote thromboxane A2 (TXA2) generation. TXA2 induces vasoconstriction and

VCAM-1

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Express pro-inflammatory receptor (CCR4, CXCR4, CX3CR1)

Platelet Treg

Endothelial cell

IL-1β RANTES PF-4 MIP-1α TGF-β MMP

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Figure 14.5  Cell–cell interactions among leukocytes, platelets, ECs, and SMCs during the development of atherothrombosis.

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platelet aggregation by binding thromboxane prostanoid (TP). Animal studies using a TP receptor blocker showed a beneficial effect on atherosclerosis development [105]. However, COX-2 seems to have a complex role in atherogenesis because its inhibition is associated with both beneficial and adverse pro-thrombotic effects [106].

T Cells The role of lymphocytes in the development of atherosclerotic lesions has been discussed in detail by others [96]. Briefly, emigrated T cells are activated by an interaction with antigen-presenting cells such as macrophages, dendritic cells, or ECs, and then differentiate into either T-helper (Th)1 or Th2 cells [107]. Th1 cells produce various pro-inflammatory cytokines (e.g., IFN-γ, TNF-α) and chemokines that promote plaque development and instability by stimulating the secretion of proteases such as MMP that hydrolyze the extracellular matrix [108]. Th2 cells seem to inhibit atherogenesis inasmuch as blockade of the Th2 response reveals atherogenesis [109]. Tregs, which are known to play a role in self-tolerance and autoimmunity, are detected in atherosclerotic lesions of apoE/ mice [110]. Secretion of TGF-β may account for the protective effect of Tregs [111].

Platelets A detailed discussion of the role of platelets in atherogenesis is available elsewhere [112]. Platelets appear to modulate the inflammatory response during all stages of atherogenesis. Adhesion onto injured ECs leads to platelet activation. Activated platelets produce several cytokines (e.g., CD40L, IL-1β), chemokines (e.g., RANTES, platelet factor [PF]-4, MIP-1α), and growth factors (e.g., platelet-derived growth factor (PDGF), TGF-β) and express pro-inflammatory receptors (e.g., CCR4, CXCR4, CX3CR1) and ligands (e.g., CCL5, CXCL4) [113]. This allows platelets to participate in the inflammatory response by attracting immune cells, stimulating SMC proliferation, and secreting proteases such as MMP. Following plaque rupture, circulating platelets quickly adhere onto the injured vessel wall via exposed collagen and through platelet GP receptor interactions with vWF and other GP receptors, initiating the coagulation cascade [112]. TF, which is abundant in the atherosclerotic plaque [114], also contributes to the local coagulation and thrombus development that can result in a sudden stroke.

Smooth Muscle Cells SMCs exhibit the capacity to adapt to their environment. Consequently, in response to injury, SMCs assume a secretory phenotype and release growth factors and cytokines, such as VEGF, TNF-α, and IL-1, and produce collagen and other components of the extracellular matrix. With these phenotypic changes, SMCs participate in the early phases of atherogenesis [115].

Vaccination as a Therapeutic Strategy for Stroke Vaccination has been proposed as a novel therapeutic approach for atherosclerosis [116]. Based on the concept that oxLDL is the major antigen that initiates the

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immune response that leads to plaque formation [96,117], oxLDL immunization therapy has been used to prevent lesion development in animal models of atherosclerosis; however, this approach to develop adaptive autoimmunity proved to be unsatisfactory. Subsequent studies have employed natural antibodies specific for phosphatidyl choline or apolipoprotein B peptide and showed reduced atherosclerosis [118]. The induction of mucosal tolerance for heat-shock protein 65, a potent atherogenic accelerator, or oxLDL also has been shown to significantly attenuate the development of atherosclerosis [119].

Venous Thrombosis The pathophysiology of venous thrombosis in the cerebral circulation differs from that of the arterial system. The difference includes risk factors, mechanisms of development, and properties of the thrombosis. Venous thrombosis also associates with systemic or local inflammation, as seen with arterial thrombosis. While the pathology of cerebral venous sinus thrombosis (CVST) has largely been attributed to congestion of venous circulation, recent work has implicated an inflammatory response in CVST. The pathophysiology of venous thrombosis is consistent with Virchow’s triad—that is, it involves changes in blood composition and alterations in the vessel wall and blood flow [120].

Mechanisms of Thrombus Development The first of three main causes of CVST is a change in blood composition, which involves either congenital or acquired factors [121]. The former includes factor V Leiden mutation, prothrombin-gene mutation 20210GA, and protein C or S deficiency; the latter factors include polycythemia, thrombocythemia, pregnancy puerperium, and oral contraceptives. The alteration in the vessel wall that contributes to CVST development is EC activation. EC activation can result from systemic vasculitis or an inflammatory response (due to infection) that spreads from the head and neck, such as sinusitis or otitis. The blood flow factor is generally manifested as blood flow stasis, which has been ascribed an important role in the induction of cerebral venous thrombosis. The mechanisms underlying the development of deep vein thrombosis (DVT) are more clearly delineated than those for CVST. DVT development is influenced by the presence of valves, which causes blood to swirl in the valve pocket, leading to stasis, hypoxia, and EC activation [122]. Unlike the deep veins, the venous system in the brain has no valves, but it has a well-developed collateral network such as the anastomotic vein of Trolard or the vein of Labbé, which connect the cavernous or sphenoparietal sinus to the superior sagittal or transverse sinus through the cerebral veins. Blood flow in the superior sagittal sinus (SSS), where CVST is most frequently evidenced [123], can easily change direction with any minor obstruction from backward to forward flow and drain into the other sinus via an anastomotic vein. As shown in Figure 14.6, the bidirectional venous draining system in the brain promotes blood stasis between a set of two entry points, leading to hypoxia in SSS

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Rostral

Inferior cerebral vein

Cortical vein

Minor blood flow Prone to stasis

Main blood flow Transverse sinus

Sinus confluence

Caudal

Figure 14.6  Anatomic features of the cerebral venous sinus in mice. In addition to the major draining system, the transverse sinus, the SSS can drain into the inferior cerebral vein, which drains blood anteriorly into the SSS. This anatomic feature may contribute to blood flow stasis in the SSS.

ECs. This possibility has been demonstrated in mice but not humans. Mice also have an anastomotic inferior cerebral vein and are predisposed to blood stasis in the SSS. Blood flow between a set of two entry points in the SSS in mice was very slow and readily changed direction, which has been confirmed by the authors (unpublished observation).

Mechanism of Thrombus Development in the Venous System Activated ECs express P-selectin, which allows for the binding of leukocytes or TF-expressing microparticles derived from monocytes and platelets. The accumulating TF can initiate venous thrombosis [124]. The critical role of P-selectin has been demonstrated in an animal model of venous thrombosis wherein an inhibitor of P-selectin prevented venous thrombus formation [125]. Another study has revealed that the presence of TF-bearing microparticles is significantly correlated with thrombus weight in an inferior vena cava model of murine thrombosis [126]. There is also evidence in the extracranial vasculature (e.g., vena cava, jugular vein, saphenous vein) that vessel wall-associated TF plays an equally important role in venous thrombosis. It was shown that mice deficient in blood cell-associated TF (but not on the vessel wall) do not exhibit a significantly altered thrombus formation [127]. Recently, we provided evidence that TF immunoblockade protects against photoactivation-induced thrombosis in cerebral venules. In addition, we reported that vessel wall-associated

Inflammatory Mechanisms in Ischemic Cerebrovascular Disease

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Tissue factor (TF)

P-selectin

PSGL-1

Microparticle (MP)

Fibrin

Stasis, pressure

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Inflammation (APC pathway) P-selectin vWF expression

Figure 14.7  Proposed mechanism of thrombus development in the cerebral venous system. Stasis and/or increased intravascular pressure activate ECs, which initiate thrombus development. Circulating microparticles express both TF and PSGL-1, with the latter allowing the microparticles to bind to endothelial P-selectin. The accumulation of TF-rich microparticles activates the coagulation cascade. Activated ECs also express vWF, which can promote the accumulation of platelets. The static or reduced blood flow results in the formation of a clot that is composed of a mixture of fibrin, erythrocytes, leukocytes, and platelets.

TF contributes to thrombus formation in cerebral venules, while blood cell- and/or microparticle-associated TF was more important in promoting thrombosis in cerebral arterioles [128]. Of interest was the observation that TF immunoneutralization had no effect on FeCl3-induced thrombus development in the SSS, which raises a question about a role for TF in the development of CVST (Figure 14.7). Upon activation of TF, the coagulation cascade proceeds to produce thrombin and eventually fibrin. Whether sufficient thrombin and fibrin is produced to yield a thrombus is also dependent on the efficiency of the anticoagulant pathways, including heparin-antithrombin and the protein C pathway. In a murine model of CVST, it was demonstrated that both heparin-antithrombin and the protein C pathway play an important role in slowing the development of FeCl3-induced thrombus development in the SSS [128]. Activated ECs express vWF as well as P-selectin, both of which are stored in granules called Weibel–Palade bodies. These EC adhesion molecules bind to counter-receptors expressed on leukocytes and erythrocytes as well as platelets [129]. The blood cells are mixed with fibrin and form “red clots” in veins, while the “white

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clots” in arteries are mainly composed of platelets. Venous thrombi occasionally include a platelet aggregate (lines of Zahn) that typically appears on the downstream aspect of the clot. The clot is primarily attached to endothelium via fibrin in veins, but through platelets in arteries [130].

Role of Inflammation An association between inflammation and the development of CVST is predicted from a large-scale statistical analysis of clinical data performed in 2004 [123]. For example, 12% of CVST patients have a facial or intracranial infection. Based on the existence of venous connections between the cavernous sinus and veins draining the paranasal sinus, as well as a connection between the venous drainage of the ear and the lateral petrosal sinus, it is not surprising that the inflammatory responses in sinusitis and otitis spread to the cerebral venous sinuses. While 12% of CVST cases include a preceding inflammatory response, it remains unclear how an infection at these sites can propagate the inflammation to the SSS via the cerebral venous system. Although the link between inflammation and thrombosis has been extensively addressed for DVT [130], less attention has been devoted to this process in the cerebral venous system. Nonetheless, the fact that venous endothelium is more sensitive to the initiation of an inflammatory response than arterial ECs tends to support the possibility that inflammation can contribute to the genesis of CVST [131].

Consequences of Cerebral Venous Thrombosis Once a thrombus obstructs venous flow, the subsequent pathophysiology in brain tissue comprises two components: brain edema due to venous congestion and intracranial hypertension due to obstruction of cerebrospinal fluid (CSF) circulation [132]. The brain edema associated with CVST is detected earlier than following an arterial cerebral infarction (edema usually detected within least 6 h of the infarction), which was evidenced by magnetic resonance imaging studies on the time course of edemagenesis after CVST [133]. We recently reported findings from a murine model of CVST that manifests an inflammatory response evidenced by increased inflammatory cytokine levels and leukocyte recruitment at 3 h after venous thrombosis as well as vasogenic edema detected 48 h after CVST induction [134]. In this experimental model of CVST, CBF, measured by laser Doppler flowmetry, is minimally (9.5% reduction) altered, compared to the 90% reduction in blood flow that accompanies the MCAO model of ischemic stroke. Collectively, these findings (early onset of brain edema and inflammation with little change in tissue perfusion) suggest that the underlying mechanism of the CVST injury response differs from that observed following an arterial cerebral infarction. The conventional view of how CVST leads to brain edema is venous congestion → cerebral perfusion insufficiency → hypoxia in brain cells → cytotoxic (intracellular) edema → inflammation → BBB disruption → vasogenic edema. However, our recent findings in experimental CVST [134] support a different mechanism: venous congestion → venous hypertension → EC injury due to stasis and increased venous pressure → inflammation → BBB disruption → vasogenic edema. Additional evidence for a contribution of

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inflammation in the edema development following CVST is provided by an evaluation of the participation of activated protein C (APC) pathway in CVST-induced brain injury. APC is a potent anti-inflammatory as well as an antithrombotic factor. Immunoblockade of the APC pathway promotes CVST-induced edema, while genetic overexpression of the endothelial protein C receptor in mice blunts the brain edema response [134].

Conclusions There is a large and growing body of evidence that links inflammation to cerebrovascular diseases. An inflammatory response appears to underlie the genesis of vesseloccluding thrombi that occur in both the arterial and venous systems of the brain. Inflammation has also been linked to the BBB dysfunction and tissue injury that accompanies cerebrovascular diseases. A critical element of the inflammatory and injury responses in these conditions is EC activation, which corresponds to the initiation of molecular events that promote the recruitment and activation of inflammatory cells and platelets and eventually leads to endothelial barrier failure. The responses initiated by ECs are amplified and propagated by perivascular cells (microglia, astrocytes, mast cells) and the activation products (e.g., cytokines, ROS) that they contribute to the inflammatory milieu. Other cells (Tregs) work to counteract the deleterious effects of inflammatory cells by releasing their own anti-inflammatory mediators (e.g., IL-10). The intensity and duration of the inflammatory response elicited within the vasculature are therefore determined by the balance between these pro- and anti-inflammatory signals. An improved understanding of the inflammatory response and its contribution to brain injury offers hope for the development of novel therapeutic agents that are directed toward reducing the morbidity and mortality of cerebrovascular diseases.

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15 Protection Against

Neuroinflammation by Promoting Co-activation of G Protein– Growth Factor Signaling and Metabolic Flexibility in the Brain Donard S. Dwyer1,2, Parrin Patterson1 1 

Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, USA 2  Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, USA

The Challenge of Neuroinflammation Neuroinflammation occurs in response to a variety of injuries and insults. For example, stroke/ischemia, traumatic brain injury (TBI), viral and other infections, as well as idiopathic processes (e.g., neurodegeneration in Alzheimer’s disease) can produce inflammation of the brain (Feuerstein et al., 1994; [1–3,122]). The inflammation may be transient and reversible, as in the case of mild encephalitis, or chronic and severe, as in multiple sclerosis (MS) or HIV-AIDS with neurologic symptoms. While the brain is sometimes viewed as an immunologically privileged site (partly due to the presence of the blood–brain barrier [BBB]), in fact immune surveillance of this tissue is ongoing, and the BBB is readily breached in the case of infections or major insults.

Traumatic Brain Injury In TBI, inflammation is a frequent outcome, especially in the case of injuries with brain penetration. There are acute and chronic phases of inflammation, with initial infiltration by polymorphonuclear leukocytes and macrophages and later involvement of other cell types [4]. Demyelination is a common feature of TBI [5,6]. Neurodegeneration is evident immediately in the area proximal to the injury; however, there may also be delayed loss of neurons and oligodendrocytes in the posttraumatic phase [5]. Pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α], IL-1, and IL-6) are released by activated immune cells or by resident glial cells and play a prominent role in the inflammatory process [4]. Chemokines are also Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00015-0 © 2011 Elsevier Inc. All rights reserved.

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elevated, including CCL2 and RANTES, and levels may correlate with the severity of the condition [6]. The role of cytokines in TBI appears to be complex because both positive and negative regulation of recovery has been described [4,7]. Morganti-Kossmann et al. [7] suggested that cytokines are a “double-edged sword.” Thus, TNF-α hampers the acute response to injury, but may be needed for the regenerative phase of recovery. By contrast, IL-6 is protective in the early stages of injury recovery, whereas overexpression in astrocytes causes neuronal loss and neurologic symptoms [8]. Therefore, any strategy aimed at protecting against neuroinflammation will need to take into account the potential duality of host mechanisms that respond to injury. Neuroprotective approaches may have to be tailored for specific stages of recovery or to provide trophic support across the entire spectrum of pathologic processes.

Ischemic Injury/Stroke Cerebral ischemia initiates inflammatory processes in the brain that include lymphocytic infiltration and cytokine production [9,10]. Neuronal loss is prominent at focal areas of injury, whereas the neuron survival rate is much higher at penumbral regions. White matter is also adversely affected, and oligodendrocytes appear particularly vulnerable to disruption of the blood supply [11,12]. Outright loss of oligodendrocytes is not required to cause extensive neurologic impairment; disruption of metabolic activity is sufficient to disturb myelin function and oligodendrocyte–axon interactions [11]. As in TBI, there is an elevation of pro-inflammatory cytokines after ischemic injury [6,9]. IL-1 plays a critical role in acute neuronal degeneration after a stroke [10], whereas levels of IL-6 correlate with infarct volume and stroke severity [6]. Conversely, administration of insulin-like growth factor-1 (IGF-1)—a global growth factor—reduced demyelination and loss of oligodendrocytes in sheep with experimental induction of ischemic brain injury [13]. This treatment approach may not be practical in human stroke because of limited distribution of IGF-1 in the brain parenchyma following systemic administration. Nevertheless, these findings suggest that the IGF-1 receptor and downstream signaling molecules may be attractive therapeutic targets. Ideally, effective treatment of stroke would prevent the death of injured cells in penumbral regions, promote restoration of function and metabolic activity in both neurons and oligodendrocytes, and facilitate compensatory changes in collateral pathways. Thus far, trials of neuroprotective drugs in stroke have been disappointing [14]. There are many reasons why greater success has not been achieved, including design flaws in the clinical trials, timing of the intervention relative to the initial insult, restricted targets of the drugs, and the diversity of stress/damage (e.g., excitotoxicity versus oxidative) caused by the ischemic episode. Clearly, protective and regenerative processes occur normally during recovery from stroke; the key is to know how to harness these processes and enhance the response with an exogenous therapeutic agent.

Central Nervous System Infections Perhaps the most obvious cause of neuroinflammation is infection of the nervous system. Meningitis, encephalitis, choroiditis, and cerebrovasculitis represent the

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major manifestations of central nervous system (CNS) infection, which may be caused by bacteria, viruses, fungi, or other agents [3]. In addition to causing neuroinflammation, viruses may produce other changes in the brain, including demyelination and neuronal loss [15,16]. Acute viral encephalitis is mainly caused by mumps, herpes simplex virus (HSV), varicella zoster, cytomegalovirus, and enteroviruses [3]. Initial symptoms typically consist of fever, headache, lethargy, and myalgia. There may be changes in consciousness and mental status, and seizures can occur. In addition to acute infection, some viruses (e.g., HSV and HIV-1) may persist in the CNS, producing chronic syndromes. There are several stages to the chronic infectious process: (1) initial infiltration of immune cells at perivascular sites in the CNS, followed by (2) mobilization of activated lymphocytes and macrophages in the parenchyma, and (3) ongoing maintenance of the inflammatory state by resident cells (e.g., microglia) with continuous production of cytokines/chemokines [17]. It is the latter stages of chronic infection that pose the greatest risk for oligodendrocyte damage (demyelination) and neuronal loss (neurodegeneration). In fact, neurologic symptoms may emerge at a point when there is little recoverable virus in the CNS or evidence of an active immune response [16]. MS may fit this pattern because viral infections have been difficult to establish as causal factors, despite epidemiologic evidence and results of animal studies that implicate viruses in the development of the autoimmune response [18,19]. In this chapter, we will focus on MS as the prototype neuroinflammatory condition for treatment with neuroprotective strategies. As with other neuroinflammatory conditions, CNS infections elicit the production of cytokines (TNF-1, IL-1, IL-6, etc.), chemokines (CXCL10, CCL2, CCL3, etc.), and matrix metalloproteinases (MMPs) [16]. These substances are generally associated with injury to the CNS, but they may also be beneficial, depending on the circumstances, as observed earlier for TBI. In addition, various CNS infections produce changes in metabolic parameters, in particular glucose levels in the cerebrospinal fluid (CSF) [3]. Bacterial and fungal meningitis are frequently accompanied by a decrease in CSF glucose levels, as is inflammation of the ventricles [3]. Viral infections have also been reported to reduce glucose metabolism in the brain [20,21], and the reductions correlate with decline in cognitive function in AIDS dementia [22]. The decrease in energy supply and metabolism as the result of infection may suggest a novel therapeutic approach aimed at reversing this situation.

MS: Neuroinflammation, Ionic Imbalance, and Energy Depletion MS is a chronic inflammatory disorder of the brain characterized by functional disruption and/or loss of pathways controlling sensation, movement, and cognition [23]. It afflicts over 2 million people worldwide and is the most common cause of neurologic impairment in young adults [23,24]. Although its cause is not known, MS is widely believed to result from an immune response in the brain that targets myelin components and is possibly triggered by a viral infection or defective regulation of

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immunity [18,19,25]. The reader is referred to other reviews for more in-depth analysis of MS and its origins [26–28]. Here, we will focus on the major factors that contribute to the pathogenesis of this disease. Regardless of the cause of inflammation in MS, treatment of this disorder will require mitigation of the adverse consequences of the inflammatory process, namely demyelination, disruption of axonal conduction, metabolic defects, and, ultimately, neurodegeneration. Based on the current literature (e.g., see [29–33], a hypothetical scheme for the initiation of MS is presented here to highlight the pathologic processes that need to be targeted with new therapeutic approaches (Figure 15.1). An initial inflammatory event in the brain stimulates the production of cytokines, chemokines, MMPs, and lytic proteins, which directly and indirectly (via actions on local blood flow) impair oligodendrocyte function. These factors also reduce the metabolic efficiency of neurons, leading to disruption of metabolite shuttles between cells, including the export of N-acetyl aspartate (NAA) to oligodendrocytes for myelin production. In parallel, there is loss of trophic support, possibly through antagonism

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Figure 15.1  Novel therapeutic approach for neuroinflammation. Inflammation in the brain may result from different insults that produce a common outcome—local production of cytokines, chemokines, and other inflammatory mediators. These factors compromise energy metabolism, antagonize growth factor signaling, and induce a catabolic state in the brain (red blocked line). These adverse effects could potentially be counteracted (green arrows) with growth factors (e.g., NGF), co-activators (e.g., adenosine analogs), and/or metabolic regulators (e.g., thiamine). The goal would be to restore optimal function of neurons and oligodendrocytes as described in the text. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this book.)

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(by cytokines such as TNF-α) of signaling pathways normally activated by growth factors (e.g., IGF-1 or neurotrophins). It is well established that TNF-α and IL-1 cause insulin resistance [34,35] and oppose trophic actions mediated by certain growth factors [36,37]. The decrease in NAA production contributes to a loss of myelin integrity, which is then exacerbated by the ongoing inflammatory response. Upon demyelination, neurons redistribute voltage-gated Na channels along their axon to maintain conductance down the fiber. Consequently, ionic concentrations in demyelinated axons are disturbed, and there is a compensatory increase in Na–KATPase activity, which eventually takes a toll on cell energetics. As local ATP levels fall, removal of Na is coupled to increased exchange with extracellular Ca. At the same time, more glutamine is used as a supplemental energy source, and glutamate may become limiting for GABA synthesis. This leads to a relative excess of excitatory neurotransmission and further buildup of intracellular Ca levels. Because glucose metabolism is already reduced by cytokine/chemokine activity (and lack of trophic support), neurons have difficulty maintaining cellular levels of ATP, and mitochondrial function is further compromised. The combination of inefficient glucose metabolism (oxidative stress), increase in cytosolic Ca, and defective growth factor signaling initiates catabolic processes in vulnerable cells. Together with the decline in oligodendrocyte function, there is loss of neurotransmission in affected pathways that gives rise to the symptoms of MS. Several aspects of the above scheme merit additional discussion in the context of therapeutic approaches for MS. Pro-inflammatory cytokines, chemokines, and their receptors are clearly attractive targets for drug development but are beyond the scope of this chapter. In addition, excitotoxicity by excessive glutamate may contribute to neuronal loss in MS, but perhaps more fundamental is the failure of the nervous system to counter-regulate this response. This failure appears to be linked to the metabolic disturbances that accompany chronic inflammation in the CNS. It has long been known that patients with MS have defective regulation of glucose metabolism, specifically abnormal glucose tolerance tests and relative insulin resistance [38–40]. There is also a decrease in glucose metabolism in affected brain areas detected by positron emission tomography (PET) analysis [41,42], and reduced levels of lactate in the CSF of MS patients have been reported [43]. Mitochondrial dysfunction in MS is well established [31] and has been proposed as the cause of neuronal degeneration [32,44]. Levels of glutamate and glutamine (an alternative energy source) may either be elevated or reduced in MS patients [43,45], which might reflect variable disturbances in energy metabolism as a function of the intensity of active disease processes. Finally, NAA levels are uniformly decreased in MS brain tissue [45,46], which indicates a reduction in neuronal metabolic activity and diminished availability of NAA for oligodendrocytes. A common thread that runs through the pathogenetic mechanisms of MS is a failure of trophic support mediated by growth factor signaling, and an associated impairment in energy production [44,47–49]. Consequently, in the next two sections, we describe biologic aspects of energy metabolism and trophic function that will need to be addressed for effective treatment of MS and other neuroinflammatory conditions.

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Metabolic Flexibility: Regulation of Energy Substrate Usage in the Brain Glucose is the main energy source for the adult brain [50]. However, various metabolites or derivatives of glucose (e.g., pyruvate, lactate, and glutamine) may serve as the ultimate fuel at any given moment. In addition to its primary role as a source of energy in glycolytic and oxidative pathways, glucose is used to generate reducing equivalents (NADPH) for the synthesis of lipids, sterols, and nucleotides via the pentose phosphate pathway; as a structural component of glycoproteins; and for the synthesis of neurotransmitters (e.g., glutamate and GABA) [50]. Use of glucose and its metabolites in these various pathways requires different complements of enzymes, cofactors, and regulatory molecules. When the cell is geared up for high levels of activity in one pathway, it cannot simultaneously sustain high activity in another. The system (enzymes and regulatory molecules) must be adjusted to maximize output from one pathway, often at the expense of another (e.g., anaplerotic versus cataplerotic cycles). Likewise, the system must be adjusted to use different sources of energy (e.g., glucose versus glutamine) to meet the cell’s energy requirements in a dynamic environment. We have previously proposed the term “metabolic flexibility” to describe the cellular adaptation to shifting supplies of fuel and the cell’s energy currency (ATP/ADP/AMP) [51]. Inflammatory processes, mediated largely by secreted cytokines and chemokines, appear to alter insulin/IGF-1 sensitivity, reduce glucose utilization, and tilt cells toward a catabolic state [52,53]. Consequently, one goal of treatment would be to reverse this trend and optimize energy utilization. If the objective includes restoration of NAA levels to support oligodendrocyte function, there must be adequate availability of glucose and glutamine to permit siphoning off of acetyl-CoA and aspartate for the synthesis of NAA. Thus, the anabolic process of myelin production and maintenance can proceed effectively only during periods of relative energy excess due to either an increase in the energy supply or the efficiency of metabolism. High-level neural activity in widely distributed pathways can dramatically increase the demand for energy in the brain. This demand is met, in part, through the use of supplemental fuel sources such as amino acids, and secondarily by shifting the ratios of phosphorylated adenine nucleotides in the cell. Thus, the level of ATP is determined not only by its rate of synthesis in glycolytic and oxidative pathways, but also by the purine nucleotide cycle coupled to the activity of adenylate kinase [54,55]. In the purine nucleotide cycle, adenosine monophosphate (AMP) is converted to inosine monophosphate (IMP) via the actions of AMP deaminase, and IMP is then reconverted to AMP via adenylosuccinate as an intermediate [54]. Aspartate provides an amino group in the process and is released as fumarate, which can feed into the tricarboxylic acid (TCA) cycle. A major outcome of the purine nucleotide cycle is a reduction in the level of AMP in the cell, especially during times of energy insufficiency and oxidative stress. The decrease in AMP then shifts the activity of adenylate kinase toward the production of ATP from two ADP molecules [56]. In addition, the cycle provides energy substrate (fumarate) for oxidation or anaplerotic processes [57]. When aspartate is converted to fumarate, less will be available for other purposes, such as the synthesis of NAA.

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Various signals regulate the tenor of energy metabolism in cells, and a better understanding of how they accomplish this feat may provide a basis for improving metabolic function in MS. Growth factors increase glucose uptake and utilization [58,59] via activation of Akt [60]. Akt regulates a switch between oxidative phosphorylation and aerobic glycolysis that favors the use of glutamine as energy substrate [61]. By contrast, cAMP enhances both the glycolytic and oxidative pathways of glucose metabolism [62], whereas activation of the purine nucleotide cycle inhibits the glycolytic pathway [63]. Small molecules have been reported to shift the metabolic bias of neuronal cells and to protect against glucose or glutamine deprivation [51]. Dibutyryl cAMP and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) both protect neurons against glucose deprivation, apparently by activating AMP kinase and stimulating the purine nucleotide cycle. On the other hand, dibutyryl cAMP and adenosine kinase inhibitors promote neuronal survival in glutamine-free medium containing low levels of serum growth factors [51]. In this case, cAMP appears to activate coupling between glycolytic and oxidative metabolism of glucose in the TCA cycle. There may also be down-modulation of the purine nucleotide cycle. These studies provide initial proof of concept for the idea that small molecules can be used to promote metabolic flexibility in neurons. This topic will be discussed further in the section on the pharmacology of neuroprotection.

Co-activation of Trophic Factor Receptors in the Brain Trophic factors in the brain stimulate cell proliferation and differentiation and promote growth in differentiated cells, including neurite output and myelin production. These processes are energy-intensive, and consequently trophic factors also upregulate glucose metabolism [58,59]. In addition, myelin provides survival and differentiation signals to neurons, whereas demyelination triggers neurodegeneration. Various groups have reported disturbances in trophic function in MS patients and in animal models of demyelinating disease [47–49]. There has been some success treating experimental allergic encephalomyelitis (EAE) with growth factors belonging to the IL-6 superfamily [48]. Moreover, it has been suggested that brain-derived neurotrophic factor (BDNF) produced by immune cells in MS lesions is neuroprotective in vivo [64]. However, clinical trials of neural growth factors for other neurologic diseases have mostly been disappointing, probably as a result of short protein half-lives, failure to adequately cross the BBB, and/or poor distribution within the brain parenchyma. Consequently, a small-molecule approach to stimulating growth factor receptors in the brain would be highly desirable as a therapeutic strategy. Recent advances related to transactivation of growth factor receptors may offer such a possibility. Growth factor receptors for IGF-1, BDNF, nerve growth factor (NGF), and other neurotrophins phosphorylate downstream substrate on tyrosine residues and are thus referred to as receptor tyrosine kinases (RTKs). There is crosstalk between RTKs and G proteins derived from G protein-coupled receptors (GPCRs) such that

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G proteins can transactivate RTKs and vice versa [65–67]. We prefer to use the term “co-activation” to describe this crosstalk because it is more reflective of the purpose of the response as discussed here. To establish and maintain functional neural–glial interactions, brain cells receive various signals and inputs, which include cell–cell contacts, trophic factors, neurotransmitters, and nutrient availability. Some signals regulate proliferation and survival, whereas others serve to confirm that the correct connections have been made—for example, there is compatibility between neurotransmitter release from apposing nerve terminals and the expression of neurotransmitter receptors on the receptive cell. Consequently, cells must integrate a complex array of signals to determine their survival, location, and functional activity in the brain. The strength of the individual signals is important; however, coincidence detection (co-activation) may be key to the response. Thus, co-activation ensures that cells receive coincident signals (as a type of failsafe mechanism?) before embarking on potentially irreversible (differentiation) and energetically costly processes [68]. RTK signaling may determine growth/differentiation outcomes, whereas GPCR signaling would confirm that additional environmental and/or location conditions are right, especially in the context of nutrient availability to support growth, differentiation, etc. Together, these systems can amplify overlapping signals (e.g., Akt) or enlist nonoverlapping pathways to bring about major morphologic and functional changes in the cell. Co-activation is mediated by direct interactions between components of the RTK and GPCR signaling pathways. The most striking example is co-immunoprecipitation of the IGF-1 receptor and Gα subunits [69], but other interactions involve β-arrestin, G protein-coupled receptor kinases (GRKs), and adaptor proteins such as Grb2 [70–72]. Interestingly, knockout of the G protein, Gαi2, causes defective signaling via the insulin receptor and severe insulin resistance in mice [73]. Thus, the goal of coactivation would be to compensate for the relative lack of RTK signaling with a drug that targets the G protein component of receptor crosstalk. This strategy might complement efforts aimed at the discovery and development of small molecules that directly replace neurotrophins by acting as agonists at specific growth factor receptors.

Neuroprotection: Pharmacologic Approaches Several broad strategies were outlined earlier for potentially improving metabolic function and trophic activity in MS and other neuroinflammatory diseases. In this section, we will discuss pharmacologic aspects of the three main targets, namely growth factor receptors, crosstalk between RTKs and GPCRs, and regulation of metabolic flexibility. These targets are shown schematically in Figure 15.1, along with examples of drugs or small molecules that affect each process.

Small-Molecule Agonists of Growth Factor Receptors Recombinant growth factors may yet succeed as therapeutic agents in MS, despite initial cause for doubt. As an alternative to administering polypeptide growth

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factors, various groups are seeking to develop small-molecule agonists of growth factor receptors. A variety of methods can potentially be used to achieve this goal. A standard industry-based approach would be to devise a binding assay that measures ligand–growth factor receptor interactions, and then perform high-throughput screening (HTS) of compound collections to identify “hits” (i.e., molecules that activate the target receptor). Alternatively, chemists can use the growth factor as a starting point to design small-molecule peptide mimetics that can be synthesized by conventional methods. The goal is to recapitulate the activity of the whole polypeptide chain in a low-molecular-weight mimic. Structure-based drug design (SBDD) can be used if the three-dimensional (3D) structure of the target receptor is known, and there is a strategy to identify potential leads that bind to the target. Fragment-based drug design relies on detection of structural perturbations (usually by nuclear magnetic resonance [NMR]) upon the binding of small test compounds to the receptor. By chemically linking active fragments, larger compounds are generated with higher affinity for the receptor and better coverage of the binding site. Receptor-fragment co-crystal structures also provide a valuable starting point for SBDD. Alternatively, the 3D structure of the receptor can be used as a target in docking runs—sophisticated computational methods that allow “in silico” screening of small-molecule structures for their propensity to interact with a virtual receptor. Fortunately, the 3D structures of a growing list of neurotrophic factor receptors have been solved and are available for SBDD. The list includes the IGF-1 receptor [74], the p75NTR subunit complexed with NGF [75], and the ligand binding domains of TrkA, TrkB, and TrkC [76]. The IGF-1 receptor is an attractive target for developing neuroprotective agents because it positively affects the growth of those brain cells most affected by neuroinflammation. Moreover, IGF-1 has been shown to protect oligodendrocytes against demyelination in response to ischemic injury in sheep [13]. The structure of an IGF-1 monomer consisting of leucine-rich repeat-1 (L1), cysteine-rich (CR), and L2 domains (L1-CR-L2) has been solved to 2.6 Å resolution. However, this fragment does not bind ligand, probably because the fibronectin type III-1 (FnIII-1) domain is missing [74,77]. The homodimer structure of the insulin receptor ectodomain, which does bind insulin with high affinity, reveals that dimerization and intramolecular interactions create the high-affinity ligand-binding site at an interface involving the L1 and FnIII-1 domains [78]. Thus, a combination of X-ray crystallography and molecular modeling provides atomic coordinates for the IGF-1 receptor for docking or other structure-based approaches. Many docking algorithms are commercially available, and in the public domain, DOCK Blaster is a powerful and free alternative [79]. Thus far, IGF-1 has mainly been considered a target for antiproliferative therapy in cancer because its major downstream effector, Akt, has been implicated in the regulation of tumor growth. However, a number of growth factors that activate Akt are used therapeutically (e.g., insulin and follicle-stimulating hormone) without a significant increase in cancer. Furthermore, it should be possible to avoid chronic overstimulation of Akt by adjusting the dosage or the timing of delivery of the smallmolecule agonists of growth factor receptors. A preferable alternative might be the development of small-molecule co-activators of growth factor signaling (see the next section).

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In the case of the NGF receptor, TrkA, SBDD has been successfully applied to the discovery of initial lead compounds [80]. The crystal structure of NGF complexed with domain 5 of TrkA (TrKAd5) was used as a source for automated docking. Approximately 2 106 compounds were screened “in silico,” and follow-up analysis identified a total of 455 candidates, which were further evaluated in binding assays. This approach yielded 15 compounds that bound to TrkA with IC50s for inhibition of NGF binding in the range of 50–1000 μM [80]. The next step will be to use this strategy to discover small-molecule ligands that activate TrkA similar to NGF.

Small Molecules for Co-Activation Co-activation with small molecules has the distinct advantage that it avoids chronic stimulation of signaling pathways (such as Akt) because the full response is observed only in the co-presence of growth factor. Thus, the in vivo effects of a compound would be coordinated with natural fluctuations in trophic factors, but would serve to amplify the signal. In addition, the concentration of the small molecule required for co-activation may be much less than would be needed to stimulate significant activation of signaling on its own. Two small molecules, lysophosphatidic acid and adenosine, have already been reported to co-activate signaling via growth factor receptors [81,82], so there is clear precedent for the approach. How might low-molecular-weight compounds produce co-activation? In the 1990s, several groups demonstrated that small molecules could directly activate Gαi/o proteins by binding to a Gα protein interface with the GPCR and Gβγ [83–87]. A peptide component of wasp venom, mastoparan, was critical to defining the site of action of direct G protein activators that bypass the GPCR [88]. Small-molecule activators include histamine, various lipoamines (e.g., compound 48/80 and spermine), tetracaine, diphenhydramine, and promethazine [87,89]. The amino terminus of Gαo contributes to the binding site for mastoparan [88], and this site is linked to the GTP/GDP binding pocket. In addition, this region of the heterotrimer interface is a recognition site for G protein regulators such as RGS proteins and calmodulin [90]. Thus, small molecules may mimic naturally occurring ligands, mastoparan and spermine, and bind to Gα or Gβγ to activate G protein signaling. While GPCR- and RGS protein-derived peptides have been synthesized and shown to regulate G protein signaling, it would be preferable to obtain small molecules to accomplish this task [89]. A few marketed drugs have been reported to directly activate G proteins, including propranolol and amiodarone [85,86]; however, they must be used at supra-physiologic concentrations to produce this effect [87]. Several groups have succeeded in synthesizing compounds that specifically target Gα or Gβγ subunits to modulate their activity. Based on the structure of active lipoamines, Manetti et al. [90] designed nonpeptide molecules that directly activate Gαi proteins as judged by increased binding of GTPγS to Gαi. While typical hits from this series showed activity in the range of 10–100 μM, the most potent compound was active at ~1 μM. Manetti et al. [90] described two main requirements for biologic activity: (1) a long hydrophobic chain (~10–15 methylene groups) and (2) at least two nitrogen atoms that would be positively charged at physiologic pH. Some of the compounds

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showed four- to fivefold greater activity against Gαi3 than Gαi1 or Gαo, whereas others from their series were two- to threefold more active against Gαo than Gαi. Previous work has shown that compounds that activate Gαi/o do not affect Gαs proteins. Moreover, benzalkonium chloride is an agonist of Gαo but an antagonist of Gαi, whereas suramin, a G protein antagonist, shows 10-fold greater selectivity for Gαs than Gαi/o [83,89]. Together, these studies demonstrate that small molecules can be used to selectively bind to and activate various Gα subunits. A complementary approach was taken by Bonacci et al. [91], who sought to design compounds to specifically interact with Gβγ subunits of the receptor heterotrimer. Previous work by this group identified a protein–protein interaction hot spot on the βγ subunits involved in the recognition of effector molecules [92]. Candidate ligands for Gβγ were identified by virtual screening and follow-up testing in binding assays. Several compounds were discovered that bound to Gβγ, some with affinities in the low micromolar range. Although most of the compounds were antagonists of Gβγ function, one potentiated the activation of phosphatidylinositol-3 kinase (PI3K), which suggests there is selectivity in the modulation of downstream effector molecules, despite similar binding affinities for Gβγ. It is not known whether any of these compounds produce co-activation in cells. However, the success of Manetti et al. [90] and Bonacci et al. [91] confirms the biologic significance of the G protein interface and its viability as a small-molecule target. The phenomenon of co-activation of growth factor signals in neuronal cell lines is illustrated by the data in Figure 15.2. For these studies, SH-SY5Y human neuroblastoma cells were incubated in serum-free medium to eliminate exogenous growth factors, which produces a gradual decline in cell viability. Insulin or drug (olanzapine and aripiprazole) was then added alone or in combination, and enhancement of cell viability was measured after several days of culture. Whereas drug alone causes a slight, but nonsignificant, increase in cell viability, insulin greatly enhances cell proliferation and survival (Figure 15.2A). This effect is further augmented by addition of drug. Insulin promotes growth by activating the PI3K-Akt signaling pathway. Olanzapine has also been shown to activate Akt [93]. To evaluate the role of PI3K in co-activation, selective inhibitors of this enzyme were added to the cell cultures. It is clear from Figure 15.2B that PI3K is required for the response because LY294002 and wortmannin both abolished the combined effects of insulin and drug on the growth of SH-SY5Y cells. While these studies confirm a role for PI3K-Akt in co-activation, other downstream signaling molecules (e.g., extracellular regulated kinase (ERK)) may also contribute to this response. It was previously shown that second-generation antipsychotic drugs can potentiate the actions of NGF on PC12 cells [51,94]. While causing little neuronal differentiation on their own, the drugs greatly amplified the effects of suboptimal concentrations of NGF. This response required Gαi/o for full expression, as judged by the fact that pertussis toxin blocked the effects of the drugs on neurite outgrowth. To explain these results, we suggested that the drugs stimulated crosstalk between Gαi/o proteins and neurotrophin receptors [94]. Recent findings in Campylobacter elegans confirm that antipsychotic drugs, including olanzapine, produce co-activation, and strictly require the presence of a growth factor receptor (DAF-2, the C. elegans insulin/IGF-1 receptor) to activate Akt signaling [95]. A Gβ subunit (GPB-1) is also essential for this response.

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Figure 15.2  Co-activation of signaling by insulin plus olanzapine or aripiprazole. (A) SH-SY5Y human neuroblastoma cells were cultured in triplicate in 96-well plates in serum-free medium. Insulin (62.5 nM) and/or drug (at the concentrations indicated) were added and cell viability was measured after 3 days with a formazan-based dye indicator as described previously [93]. The data are derived from two experiments and are expressed as the percentage of control wells (serum-free medium plus solvent). Statistical significance was determined with Student’s t-test, and significant differences from the insulin-alone group are indicated with asterisks, *P<0.05. (B) Cells were cultured as in (A) except that PI3K inhibitors, LY294002 (LY; 10 μM) or wortmannin (WM; 10 μM), were added prior to insulin and drugs. Significant enhancement of cell viability by drug plus insulin, as compared to insulin alone, is indicated by asterisks (*P<0.05; **P<0.01), whereas significant inhibition of co-activation is indicated by pound symbols, ##P<0.01.

Furthermore, olanzapine served as a template structure for searching chemical libraries and identifying small molecules that produce similar neuroprotection and enhancement of neurite outgrowth [51]. In this context, it is noteworthy that xaliproden, a neuroprotective drug previously in clinical development, also enhances the actions of NGF

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and enables functional signaling by neurotrophin-3 [96], although co-activation has not been formally demonstrated.

Neuroprotection Through Regulation of Metabolic Flexibility NGF, IGF-1, and other growth factors increase glucose uptake and metabolism, in part, to provide energy for sustaining proliferation and building materials for biosynthesis. Akt is downstream of most growth factors and not only regulates glucose transport but also causes a metabolic shift toward glycolysis and away from glucose oxidation [61]. This shift toward glycolysis is probably required in rapidly proliferating cells because ATP production via oxidative phosphorylation may be limited while there is ongoing duplication of the mitochondria. Glucose itself may be limiting in the brain during high levels of electrical activity, or when concurrent biosynthetic demands are extensive. For example, if glucose is simultaneously needed for (1) ATP production, (2) conversion to glutamate, GABA, and acetylcholine for neurotransmission, (3) the synthesis of phospholipids, (4) reducing equivalents (NADPH) for lipid synthesis and to combat oxidative stress, and (5) NAA for myelin production, it may be difficult to match supply with demand. Therefore, it is essential that the brain budgets its available energy substrates quickly and flexibly in response to constantly shifting conditions. Neuroinflammatory disorders such as MS are associated with disturbances in glucose and energy metabolism in the brain [41,42] that may ultimately cause demyelination and neurodegeneration. In the remainder of this section, we will discuss approaches for addressing this defect by promoting efficient energy production with metabolic regulators. Thiamine provides a useful starting point for this discussion. Thiamine is an essential co-factor for three key enzymes involved in glucose metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase from the pentose phosphate pathway [97]. Chronic deficiency of thiamine is associated with a spectrum of neuropsychiatric symptoms that manifest in beriberi and Wernicke-Korsakoff syndrome [98]. Symptoms of fatigue, muscle pain, atrophy, paresthesia, weight loss, anorexia, edema, and cardiac failure are common in beriberi, whereas progressive polyneuritis with sensorimotor deficits and psychosis may also be observed in Wernicke-Korsakoff syndrome [98]. In the brain, thiamine deficiency is accompanied by demyelination (also in peripheral nerves), myelinolysis, and evidence for degenerative lesions and necrosis [99]. These effects are largely reversed by administration of thiamine, but this may take time, reflecting regenerative processes. Thiamine protects neuronal cultures in vitro and promotes survival of hippocampal neurons [100]. In humans, thiamine administration may facilitate recovery from TBI, which is associated with thiamine deficiency [101], while in rats, benfotiamine protects against diabetic retinopathy [102]. In MS patients, thiamine may slow disease progression [103]; however, because it was administered in combination with liver extract, additional testing is needed to establish whether this is indeed a viable adjunctive therapy. Nicotinamide is another vitamin-derived (from niacin) co-factor that plays an essential role in energy metabolism. It produces neuroprotective effects through

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activation of Akt and also reduces inflammation [104]. Niacin was recently reported to reduce neuroinflammation and stimulate oligodendrocyte proliferation and axon regeneration in a mouse model of EAE [105]. Nicotinamide in the form of nicotinamide adenine dinucleotide (NAD) serves as a co-factor in various metabolic reactions aimed at energy production. The possible role of NAD in MS has recently been discussed at length by Penberthy and Tsunoda [106], along with the potential for supplementation with nicotinic acid as a therapeutic strategy. By ensuring that NAD is not limiting for energy production, it may be possible to maximize metabolic flexibility in the brain. Riluzole is an intriguing drug that may promote metabolic flexibility in the brain. It is a neuroprotective agent that is approved for the treatment of amyotrophic lateral sclerosis (ALS). Although riluzole is noted for antiglutamatergic properties, its molecular mechanism of action has not been firmly established. Chowdhury et al. [107] showed that riluzole increased glucose metabolism in the prefrontal cortex and hippocampus of rats. It was also shown to preserve glucose metabolism and gray matter volume in the cortex of patients with Huntington’s disease [108]. In animal models of traumatic spinal cord injury, riluzole protected against oxidative stress [109] consistent with a role in optimizing energy metabolism. The findings of Kalra et al. [110] are especially relevant for this discussion and potentially for the treatment of MS. Kalra et al. [110] showed that riluzole produced a significant increase in NAA in the motor cortex of ALS patients treated for only 3 weeks with the drug. Thus, various studies demonstrate that riluzole positively affects glucose metabolism in the brain, and show initial proof of concept for promoting metabolic flexibility with low-molecular-weight compounds that cross the BBB. Additional drugs used to treat disorders of the nervous system have shown promise as prototypes for optimizing glucose metabolism in neuronal cells. As mentioned earlier, several antipsychotic drugs protect neuronal cells against various insults. Olanzapine, quetiapine, and clozapine activate Akt and enhance neurite outgrowth in combination with NGF [94]. In addition, olanzapine and clozapine increase the expression of neuronal glucose transporters (GLUT3), and olanzapine also stimulates glucose transport in PC12 cells [111,112]. Although the signaling pathways involved in these responses have not been definitively established, the fact that the antipsychotic drugs activate Akt may provide a clue about the molecular mechanism of action. Moreover, these drugs protect neuronal cells against both glutamine and glucose deprivation, suggesting that they can cause a shift in energy utilization in vitro. The concentrations of drug required to stimulate GLUT3 expression and to protect cells against energy shortage are somewhat higher than those achieved in patients, which would limit the utility of these drugs as metabolic regulators in vivo. Nevertheless, these studies show that it is possible to upregulate glucose transport and metabolism with small molecules. In addition to existing drugs that affect energy metabolism, there are several lowmolecular-weight compounds that merit further evaluation as potential modulators of metabolic flexibility. cAMP and cell-permeable analogs produce many desirable effects on neurons. We already mentioned earlier that dibutyryl cAMP promoted metabolic flexibility by enhancing oxidative metabolism of glucose during glutamine deprivation and stimulating the purine nucleotide cycle during glucose deprivation [51].

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cAMP has long been known to aid regeneration of transected/injured nerves [113–115]. It also stimulates neurite outgrowth from neuronal cell lines and enhances neurite output in response to NGF [116]. cAMP is reported to enhance regrowth after neuronal injury, to act as a guidance cue for axons, and to enhance growth-associated gene transcription [117]. The work of Filbin et al. [115] on cAMP may have particular significance for the treatment of MS and demyelinating disorders. This group showed that cAMP can overcome the negative effects of myelin on the regeneration of axons from lesioned neurons [115]. There are two stages to this response: (1) an acute phase initiated by PKA, but independent of gene transcription and (2) a second stage mediated by transcriptional regulation [115]. The transcription factor cAMP response-element binding protein (CREB) is downstream of PKA and plays a significant role in the latter response. Interestingly, the type 4 phosphodiesterase inhibitor, rolipram, increases cAMP levels and protects marmosets against demyelination in an EAE model of MS [118]. However, more selective phosphodiesterase inhibitors may be needed to avoid the serious side effects of rolipram in patients [119]. Thus, elevation of cAMP might be beneficial in MS because it would reduce neuroinflammation, enhance the efficiency of energy metabolism, stimulate neurite outgrowth/repair, and optimize myelin–axon interactions. Other adenosine nucleotides may regulate metabolic flexibility by activating 5'-AMP kinase (AMPK) or the purine nucleotide cycle (and adenylate kinase) to conserve ATP levels in neurons. We previously showed that AICAR protects PC12 cells against glucose deprivation [51]. This nucleoside activates AMPK, which regulates the metabolism of alternative fuel sources and preserves ATP levels by inhibiting anabolic processes and ion fluxes. The antidiabetic drug metformin also activates AMPK. Recently, both AICAR and metformin have been reported to provide therapeutic benefits in autoimmune models of MS [120,121], although the precise mechanism of action was not established. AICAR does not protect against glutamine deprivation, whereas two different inhibitors of adenosine kinase are protective [51]. Glutamine is a key component of the purine nucleotide cycle, which reduces the level of AMP in the cell and drives the production of ATP from ADP by adenylate kinase. When glutamine is lacking, reduction of AMP can be achieved by inhibiting adenosine kinase. This would also promote the consumption of pyruvate instead of glutamine, and enhance the coupling between glycolysis and oxidation of glucose. These various findings show that it is possible to optimize energy metabolism with small molecules. However, before drugs such as metformin, rolipram, riluzole, or nicotinamide could be recommended for primary or adjunctive therapy in MS, they would have to undergo clinical testing to determine whether they are both safe and efficacious. Nevertheless, the future of the general strategy looks promising at this point.

Additional Issues for Neuroprotective Strategies To derive therapeutic agents from the strategies outlined here, several issues will need to be addressed. The first concern is related to the selectivity of the drugs for their

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target proteins (e.g., Gαi versus Gαs) and cells (neurons and oligodendrocytes versus microglia, lymphocytes, etc.). For co-activation, preliminary data suggest that it should be possible to design compounds with the requisite selectivity in terms of their target proteins. This may be less likely for compounds that optimize glucose meta­bolism; however, their positive effects on energy production should generally be beneficial. In fact, a broad-based therapeutic approach may be required, given the diversity of cells adversely affected in neuroinflammatory conditions. A second issue concerns the timing of drug administration—that is, is the approach mainly neuroprotective versus regenerative in nature? Depending on the stage of the disease, neuroprotection may provide little clinical benefit because the damage has already been done. In more advanced stages, effective treatment may need to include drugs that stimulate repair and regeneration of injured cells. Co-activation of growth factor signaling and optimization of energy metabolism may both protect against ongoing injury and promote tissue repair. The challenge will be to temper growth promotion so that regenerative processes predominate over excessive proliferation. Another factor that may limit the utility of a drug is the potential emergence of an adaptive, counter-regulatory response to chronic treatment. Because co-activation depends on coincident signaling by growth factors released under natural conditions, this approach may not elicit strong counterregulation. However, we might expect modest downregulation of the G protein target in cells. Similarly, drugs that enhance metabolic flexibility may not provoke an opposing response because they promote homeostasis rather than shifting the cell to an extreme metabolic state. Moreover, the drugs derived from these approaches will be broadly protective by design. Consequently, they may cause fewer side effects or may limit the severity of any side effects through their protective actions. The marginal efficacy of current drugs for MS and neuroinflammation provides motivation for exploring new treatment options. A better fundamental understanding of co-activation and the factors that regulate metabolic flexibility may ultimately lead to the identification of superior drug targets in the future.

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16 Mesenchymal Stem

Cells, Inflammation, and Neurodegenerative Diseases

Behrouz Nikbin1, Yadollah Shakiba1, Mandana Mohyeddin Bonab1, Alireza Minagar2, Stephen Jaffe2 1 

Department of Immunology and the Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran 2  Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Introduction Inflammation is one of the most complicated processes in the human body, and the promoting and inhibiting mechanisms of inflammation are significant players in the pathogenesis of various pathologic conditions. Basically, inflammation is the body’s defense mechanism against endogenous and exogenous noxious stimuli and is controlled by the immune system. Based on its duration, inflammation may be divided into acute and chronic forms. Acute inflammation is usually utilized for elimination of pathologic, tissue-damaging factors and subsides after the elimination of these stimulatory factors. Generally, acute inflammation is a protective reaction against disease. By contrast, in chronic inflammation the inflammatory process persists, with periodic reactivation and eventual tissue destruction and fibrosis. Inflammation orchestrated by immune system activation plays a crucial role in tissue damage, repair, and remodeling [1]. Persistence of inflammation can induce phenotypical and epigenetic changes in immune cells and fibroblasts [2]. The fibroblasts that originate from mesenchymal stem cells (MSCs; see Table 16.1 for explanation of all abbreviations) modulate the immune system and stimulate tissue regeneration. When these cells confront persistent inflammation, they induce tissue fibrosis by secreting extraordinary amounts of extracellular matrix [3]. During tissue injury resulting from various underlying pathologies, alterations in cytokine production are observed in the inflamed microenvironment. These cytokines bind to and interact with their specific receptors on different cells and induce aggregation, stimulation, and differentiation of these cells, either locally or systemically. In addition, the produced trophic factors at the site of inflammation will facilitate migration of stem cells, particularly MSCs, and stimulate them to differentiate Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00016-2 © 2011 Elsevier Inc. All rights reserved.

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Table 16.1  Abbreviations Definition

Abbreviation

Amyloid beta Alzheimer disease Activated leukocyte cell adhesion molecule Acute lung injury Amyotrophic lateral sclerosis Antigen-presenting cell Adult stem cell Brain-derived neurotrophic factor Basic fibroblast growth factor Bone marrow Dendritic cell Concanavalin A Cyclooxygenase-2 Cytotoxic T-lymphocyte antigen-4 Chemokine ligand Chemokine receptor Experimental autoimmune encephalomyelitis Embryonic stem cell Forkhead box P3 Granulocyte colony-stimulating factor Glial cell-derived neurotrophic factor Green fluorescent protein Granulocyte–macrophage colony-stimulating factor Graft-versus-host disease Huntington’s disease Hepatocyte growth factor Human leukocyte antigen Hematopoietic stem cells Intercellular adhesion molecule-1 Indoleamine-2,3-dioxygenase Interferon-gamma Immunoglobulin G Inhibitor of IkappaB kinase 2 Interleukin Interleukin-2 receptor Induced lung injury Lymphocyte function-associated antigen Lipopolysaccharide Macrophage colony-stimulating factor Monocyte chemotactic protein-1 Major histocompatibility complex Macrophage-inflammatory protein-2 Mixed lymphocyte reaction Matrix metalloproteinase Multiple sclerosis Mesenchymal stem cell

AB AD ALCAM ALI ALS APC ASC BDNF bFGF BM DC Con-A COX-2 CTLA-4 CXCL CXCR EAE ESC FOXP3 G-CSF GDNF GFP GM-CSF GVHD HD HGF HLA HSC ICAM-1 IDO IFN-γ IgG IKK2 IL IL-2R ILI LFA-1 LPS M-CSF MCP-1 MHC MIP-2 MLR MMP MS MSC (Continued)

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Table 16.1  (Continued) Abbreviations Definition

Abbreviation

Nuclear factor-KappaB Neural stem cell Natural killer Osteopontin Peripheral blood mononuclear cell Parkinson’s disease Platelet-derived growth factor Platelet endothelial cell adhesion molecule-1 Prostaglandin E2 Phytohemagglutinin Rheumatoid arthritis Stromal cell-derived factor T helper Transforming growth factor beta Tumor necrosis factor Tissue plasminogen activator Regulatory T cell Vascular cell adhesion molecule-1 Vascular endothelial growth factor Very late antigen-4

NF-κB NSC NK OPN PBMC PD PDGF PECAM-1 PGE2 PHA RA SDF TH TGF-β TNF t-PA Treg VCAM-1 VEGF VLA-4

and secrete specific factors [4]. MSCs exist in human bone marrow in very small quantities. Beside bone marrow, MSCs can be found in other organs and tissues such as liver, kidney, adipose tissue, and cord blood. Organ-specific MSCs are probably involved in local regeneration of tissues [5]. The parental origin of MSCs can influence their phenotype and function. Because MSCs are rare cells in bone marrow and have no specific markers, they commonly are isolated by adherence to plastic and then expanded ex vivo for research purposes or clinical applications. It is not yet clear whether in vitro expanded MSCs are exactly similar to their progenitors. However, proliferation of MSCs on plastic surfaces can change their phenotype and function. Using flow cytometry, the ex vivo expanded MSCs are found to be negative for hematopoietic markers such as CD14, CD45, and CD34 and positive for other markers such as CD166, CD73, CD90, and CD105 [6,7]. However, the multipotency of these cells has been proven in vivo. After injection of MSCs into fetal sheep during the early phase of gestation, these cells successfully graft and demonstrate site-specific differentiation [8]. Jiang et al. [9] described a group of MSCs in bone marrow, muscle, and brain, designated multipotent adult progenitor cells, that can differentiate from a single cell into all types of germ layer cells. Coculture of this type of MSC with astrocytes generates neuronal-like cells, confirming the significant plasticity of MSCs [10]. MSCs also have immunomodulatory potential and secrete various growth factors. Administration of these cells ameliorates the consequences of iatrogenic damage in different animal model tissues such as kidney, brain, lung, and heart by reducing inflammation through secretion of various antiinflammatory factors [11]. In this chapter we review the immunomodulatory properties of MSCs and their treatment applications in some neurodegenerative disorders.

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MSCs and Inflammation MSCs interact with immune system cells at different levels. MSCs can modulate the immune system by expression of various receptors as well as secretion of different soluble factors. Thus, these cells may influence every single immune reaction. MSCs’ inhibitory effects can influence both naïve and memory cells [12]. These apparent suppressor functions are mediated by soluble factors, and separation of MSCs and lymphocytes by a semipermeable membrane does not influence these inhibitory properties [13,14]. Interestingly, when the supernatant from MSC cultures is added to a mixed lymphocyte reaction (MLR), the inhibition does not occur. It would seem that MSCs do not secrete these factors continually, and the secretion type depends on the MSCs’ microenvironment [15]. So far, various factors, including IL-10, prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), and indoleamine-2,3-dioxygenase (IDO), have been proposed as necessary factors for MSC immunomodulatory activity (Figure 16.1). In summary, the suppressor effect of MSCs on immune cells is a complex and multifactorial process that depends on signals from the surrounding microenvironment [16].

Indirect interaction CD80, CD86 CD40, CD1a HLA-DR

CD1b

DC reg

Proliferation Cytokine production Cytolytic activity HGF TGF-β

Active DC IL-12 IL-2 TNF-α

IL-10 TGF-β

NK cell

Proliferation cytotoxicity

PGE-2, IDO

MSC

Active T cell TNF-α IFN-γ

Soluble factors

Foxp3 CD25 CD4 T reg

IL-4 IL-10

B cell

IgG IgM IgA

Proliferation differentiation chemotaxis

Figure 16.1  The modulatory effect of MSCs on immune cells. Soluble factors like TGF-β, HGF, PGE2, IL-10, and IDO secreted from MSCs affect the major immune cells and alter their responses from activatory to regulatory functions. MSCs change naïve T cells into regulatory T cells and DC1s to DC2s, and inhibit antibody production by B cells and the cytolytic activity of NK cells.

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MSCs and Dendritic Cells Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that are involved in capturing, transferring, and presenting antigens to effector immune cells, and thus they play the main role in the initiation of immune responses. Mature DCs induce the development of effector T cells—in contrast to immature DCs, which induce peripheral tolerance by eliminating T cells and activating regulatory T cells (Tregs) [17]. The two subsets of DCs, DC1 and DC2, activate TH1 and TH2 responses, respectively. The change in DC specificity is a strong mechanism for organizing the cellular and humoral responses that conduct the final effector activities of TH1 or TH2 cells, memory and regulatory T cells. DCs are derived from CD34 bone marrow stem cells and can be generated in vitro in the presence of IL-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) [18]. By capturing, transporting, and presenting antigens to effector immune cells, DCs play a unique role in the activation of naïve T cells [19]. From processing to presenting of antigens, DCs upregulate the expression of major histocompatibility complex (MHC)-II and co-stimulatory molecules CD80 and CD86 on their surface; this process is known as DC maturation [20]. MSCs inhibit upregulation of CD80, CD86, CD40, CD14, and MHC-II molecules during DC differentiation as well as inhibiting the expression of CD86, CD83, CD40, and MHC-I during DC maturation. They also reduce DC endocytotic capacity and their production of IL-2, IL-12, and tumor necrosis factor (TNF)-α, while increasing IL-10 secretion [21–24]. By these mechanisms, MSCs arrest the maturation of DCs and shift them to the regulatory APC type. In this way, the inflammatory response changes to an anti-inflammatory response, and T-cell activities are inhibited. Other experiments demonstrate that IL-6 secreted from MSCs plays a role in maintenance of the immature DC immunophenotype [25]. Since MSCs induce tolerogenic DCs, they can be interesting tools in the treatment of graft-versus-host disease (GVHD) and autoimmune diseases. Zhang et al. [26] proposed that DCs are the first target of the MSCs’ immunosuppressant activity. MSC-treated DCs change their phenotype by overexpression of myeloid markers such as CD11b and decreased expression of functional markers Ia, CD80, CD86, and CD40, making them similar to immature DCs. In contrast to the normal immature DCs, adding bacterial lipopolysaccharide (LPS) to MSC-treated DCs does not reverse the expression of these markers. Therefore, MSCs’ activity leads to generation of DCs with a specific phenotype (MSC–DC). MSC–DCs produce large amounts of IL-10 and TGF-β and less amounts of IL-12. MSC–DCs, compared to mature DCs, have more potent phagocytotic activity, and in the presence of LPS, they diminish lymphocyte stimulation. MSC–DCs inhibit concanavalin A (Con-A)-induced lymphocyte stimulation and decrease levels of IL-2 and IFN-γ in culture supernatant. The regulatory function of MSC–DCs is dependent on cell–cell contact and is mediated through jagged-2 protein, a transmembrane protein from the notch family that plays a role in their regulation, differentiation, and growth [26]. Recently, some reports have shown that regulatory DCs can ameliorate autoimmune diseases in animal models. For instance, Usui et al. demonstrated that regulatory DCs generated from monocytes treated with GM-CSF, TGF-β, and IL-10

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suppress autoimmune uveoretinitis in mice. They reported that lymph node cells obtained from mice treated with regulatory DCs secrete lower amounts of IFN-γ but higher amounts of IL-10. Moreover, the number of antigen-specific T cells was lower in comparison to controls [27]. Another study showed that injection of regulatory DCs into animals with autoimmune gastritis downregulates the inflammatory process by increasing the number of CD4CD25FOXP3 regulatory T cells in peripheral blood [28]. Similar results were observed in animals with allograft skin transplantation and type 1 diabetes after injection of in vitro generated regulatory DCs [29]. Up to the present time, no study has evaluated the efficacy of MSCmediated tolerogenic DCs in animal models of inflammatory diseases. However, these cells would probably serve as good candidates for treatment of autoimmune diseases with or without MSC coadministration.

MSCs and T Lymphocytes Similar to thymic epithelial cells, MSCs express lymphocyte function-associated antigen 1 (LFA-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) markers when they interact with T cells [29]. A growing body of evidence indicates that MSCs inhibit alloreactive reactions. Coculture of autologous MSCs with mitogen-stimulated T cells inhibits T-cell proliferation, cytokine secretion, and cytolytic functions [12,14,30–32]. This inhibitory activity has been observed even when the MSCs and lymphocytes are not HLA matched [30]. It appears that allogeneic MSCs have even stronger immunosuppressor activity than autologous cells [33]. MSCs downregulate cyclin D2 and thereby stop the proliferation of stimulated T cells in the G0/G1 cell cycle [34]. The antiproliferative effect of MSCs is not accompanied by apoptosis [30]. This means that the elimination of the MSCs will restart T-cell proliferation. Many reports have demonstrated the inhibitory effect of MSCs on T cells via secretion of different soluble factors. This effect may be attributed to such compounds as transforming TGF-β and HGF [13,31,34,35]. Certain reports indicate that tryptophan degradation in culture medium by IDO secreted from IFN-γ–stimulated MSCs can be another mechanism of MLR inhibition [36,37]. This finding, however, has been questioned by others [24,35]. Most of these controversies appear to be due to (1) different methods of MSC separation (MSC populations isolated by different methods are not equal) or (2) different immunosuppression assay methods (lymphocyte subpopulations, stimulatory factors, and the time of assay are not the same in these different assay methods). For example, it has been shown that adding MSCs to MLR cultures increases the levels of IL-2 and IL-2R receptor (IL-2R) and IL-10. Whereas adding MSCs to the phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte cultures diminished the levels of IL-2 and IL2-R, it had no effect on the level of IL-10 [14]. It is generally thought that the inhibition of T cells by MSCs is not through apoptosis [31,35]. However, one study reported that MSCs induce apoptosis in activated T cells but not in resting T cells [37]. As mentioned above, after elimination of MSCs from culture and restimulation, T cells proliferate

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normally. But Maccario et al. [33] reported that after MSCs’ elimination, only CD4 T cells proliferate, and CD8 T cells do not. Regulatory T lymphocytes (Tregs) are a subset of T cells that express CD4, CD25, and transcription factor FOXP3 [38]. These cells suppress the function of CD4 effector T cells, CD8 cytotoxic T cells, natural killer (NK) cells, B cells, and APCs [39]. Coculture of MSCs with peripheral blood lymphocytic monocytes increases the number of CD4CD25 FOXP3 Tregs, with or without cytotoxic T-lymphocyte antigen-4 (CTLA-4) [24,33]. Increasing the number of Tregs can be considered as an alternative mechanism of T-cell suppression, because elimination of Tregs does not affect the MSCs’ inhibitory function [12,21]. In an animal model of collagen-induced arthritis, injection of MSCs reduced the incidence and severity of arthritis by increasing IL-10 production as well as the number of Tregs [40]. In conclusion, MSCs stop T-cell proliferation, reduce secretion of IFN-γ, and increase production of IL-4 and IL-10. Therefore, MSCs shift the immune response from a pro-inflammatory to an anti-inflammatory or tolerogenic state. These changes in the cytokine profile are accompanied by a shift from DC1 to DC2, which finally expands Tregs development [24,33].

MSCs and B Lymphocytes B (lymphocytes) cells constitute a potent arm of acquired immunity. Activation and differentiation of B cells lead to generation of antibody-producing plasma cells as well as memory B cells. Animal studies demonstrated that MSC-secreted factors inhibit pokeweed mitogen-induced B-cell proliferation [41]. Another study showed that MSCs inhibit proliferation, activation, and IgG production by hyperactive B cells in BXSB mice (a systemic lupus erythematosus model) [42]. In vitro studies showed that incubation of MSCs with B cells significantly decreases their proliferation and differentiation to antibody-producing cells as well as their chemotaxis [43]. Similar to T cells, B cells do not undergo apoptosis upon interaction with MSCs. However, their cell cycle is arrested at the G0/G1 phase, and therefore they cannot produce IgG, IgM, or IgA. MSCs downregulate the expression of chemokine receptor-4 (CXCR4), CXCR5, and CXCR7 and reduce B-cell chemotaxis in response to chemokine ligand-12 (CXCL-12) and CXCL-13. This eventually can stop B-cell migration to secondary lymphoid organs. All these functions are probably monitored by soluble factors, most of which remain only partially recognized [44]. Since B cells play a pivotal role in the pathogenesis of autoimmune diseases, B-cell inhibition by MSCs may be a therapeutic option.

MSCs and NK Cells NK cells play a major role in innate immunity and are the first line of defense against infections and tumor cell generation. Using the NK cells from fresh peripheral blood, one preliminary study revealed that MSCs are not subject to lysis by alloreactive NK

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cells [45]. However, another study demonstrated that cytokine-activated NK cells have strong cytolytic activity against autologous and allogeneic MSCs [46]. It seems that MSCs suppress IL-2– or IL-15–induced NK cell proliferation and IFN-γ production, but MSCs do not inhibit the lysis of K562 cells by fresh NK cells [32,33]. It has been reported that MSCs reduce the cytotoxic activity of IL-2–activated NK cells [47]. Interestingly, Sotiropoulou et al. demonstrated that MSCs reduce NK cells’ lytic activity against HLA-I–positive cells but not against HLA-I–negative cells. They also reported that inhibition of NK cells’ cytolytic activity by MSCs is probably due to downregulation of the γc chain resulting from MSC secretion of PGE2 and increased IDO activity [48]. Adding MSCs to conventional peripheral blood mononuclear cells (PBMCs) decreases the expansion of NK cells [33]. Coculturing PBMCs and MSCs without any triggers induces NK cells’ activation by upregulating CD69 expression and by the production of IFN-γ and TNF-α. This effect of MSCs on NK cells depends on cell-to-cell contact and requires LFA–ICAM-1 interaction [49]. Interestingly, although IFN-γ protects MSCs from NK cell’s cytolysis by upregulation of HLA-I molecules, masking HLA-I molecules does not affect MSC lysis [46,49].

Immunogenicity of MSCs Autologous MSCs are the most commonly used stem cells for immunoregulation and regenerative purposes, but their expansion from bone marrow is time-consuming and is not suitable for emergent medical conditions. Because of these limitations, engraftment of allogeneic MSCs is much more attractive therapeutically. The immunogenicity of MSCs remains controversial. Some evidence demonstrates that allogeneic MSCs are not immunogenic, escaping from host immune cells by different mechanisms, including low-level expression of MHC and production of costimulatory molecules as well as secretion of suppressive cytokines. In addition, it has been shown that direct coculture of MSCs with allogeneic lymphocytes does not elicit proliferative reactions [12,24,31,50]. These findings have been observed with both rat and mouse MSCs, and even xenogeneic MLRs [30,51,52]. Animal studies have shown that allogeneic MSCs can prolong skin allograft survival in immunocompetent baboons, and may prevent the rejection of allogeneic tumor cells in immunocompetent mice [51,53]. Saito et al. [54] demonstrated that MSCs can be tolerated in a xenogeneic environment and migrate to injured pericardium. Deng et al. [55] also reported that bone marrow MSCs (BM-MSCs) were tolerated for longer periods, and they suggest that these cells may cause hematopoietic chimerism. A few human studies have confirmed that allogeneic MSCs can be non-immunogenic or hypo-immunogenic. Sundin et al. [56] reported that after transplantation of allogeneic MSCs, no antibody was found against them, whereas reactive antibodies against fetal bovine serum (a part of the culture medium of MSCs) were detectable. Horwitz et al. [57] transplanted HLA-matched allogeneic bone marrow to patients with osteogenesis imperfecta and reported that these cells engrafted and improved the patient’s clinical condition. Another study showed that allogeneic MSCs injected

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into patients with metachromatic leukodystrophy and Hurler syndrome engrafted and reversed the disease pathology in some tissues, without any adverse effects such as GVHD or alloreactivity [58]. Interestingly, in a recent study, third-party donor mesenchymal stromal cells injected along with cord blood cells reduced the rate of GVHD without significant side effects [59]. However, the non-immunogenicity of MSCs has been questioned by other investigators. Nauta et al. [60] showed that injection of donor MSCs increases the rejection of transplanted allogeneic donor bone marrow cells in an animal model. They also reported that allogeneic MSCs induce memory T-cell responses in immunocompetent hosts [60]. Another study reported that allogeneic HLA-mismatched MSCs are immunogenic and rejected by immune cells of healthy animals [61]. Grinnemo et al. [52] demonstrated that injection of allogeneic MSCs into rat myocardium is associated with significant infiltration of inflammatory cells and subsequent rejection. Altogether, these data show that more controlled trials are required for better clarification as to the safety or immunogenicity of allogeneic MSCs.

MSC Migration into Inflamed Tissues and Tumors To date, many studies have demonstrated the tropism, migration, and homing of MSCs to inflamed tissues. In these studies, labeled, injected MSCs (genetically transfected with fluorescent proteins, or genetically different from the host’s cells) have been traced and separated from animal tissues. The results indicate that MSCs migrate to, infiltrate, and become part of the host tissue [62,63]. MSCs’ migration to inflamed tissue is a well-controlled process that is mediated by selectins, integrins, and chemokines. In fact, the MSC mechanism of tissue migration is similar to that of leukocytes, but different adhesion molecules are utilized. Leukocytes use L-selectin and E-selectin for rolling, whereas L-selectin has not been found on MSCs, and the role of E-selectin has not been well studied [62]. MSCs do not express surface markers such as platelet endothelial cell adhesion molecule-1 (PECAM-1), CD34, CD14, and CD45, which play important roles in leukocyte migration through endothelium [62,64]. Ruster et al. reported that P-selectin plays a role in MSC trafficking. These investigators studied the rolling process of MSCs in the ear vein of a mouse after intravenous injection. MSC rolling was significantly reduced in the P-selectin knockout mouse, and administration of P-selectin neutralizing antibody exerted the same effect [65]. Since the P-selectin ligand (P-selectin glycoprotein ligand-1) and CD34 are not present on MSCs, it seems they use a different ligand for adhesion to P-selectin on the endothelial cell [62]. The roles of chemokines and their receptors in leukocyte migration into inflamed and damaged tissues have been well documented. A large number of chemokine receptors such as CCR1, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR4, CXCR5, and CX3CR1 have been recognized on the surface of MSCs, with binding of these receptors to specific chemokines facilitating MSC migration [62,65–68]. The expression of chemokine receptors on the surface of MSCs is reduced during in vitro

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passage; this point should certainly be considered when using MSCs for tissue regeneration or treatment of inflammatory diseases [68]. Stromal cell-derived factor (SDF) or CXCL12 is a very important chemoattractant for the migration of injected MSCs into inflamed tissues. The CXCR4 molecule, a receptor for SDF, is present on MSCs [69]. Tissue SDF and its receptor on MSCs are upregulated during inflammation [70,71]. Abbot et al. [72] demonstrated that during tissue necrosis, such as with myocardial infarction (MI) animal models, the level of CXCL12 increases significantly in inflamed but not adjacent tissues. They showed that peripherally injected MSCs (48 h after MI) migrate only into necrotic tissue [72]. The role of the SDF/CXCR4 axis in MSC migration has been proven by several observations: (1) administration of CXCR4 antagonists inhibits MSC migration to the inflamed site and (2) transduction of myocardial cells by CXCL12 coding adenovirus increases MSC migration significantly. The results of this study showed that SDF/CXCR4 plays an important role in MSC migration, and this migration is enhanced by the presence of inflammation [72]. In addition to the affected tissues, the blood level of CXCL12 increases after MI and accelerates MSC migration from the bone marrow to the damaged tissue [73]. In other studies, the key role of SDF/ CXCR4 in MSC migration to ischemic and inflammatory central nervous system (CNS) sites has been demonstrated; and it has been shown that blockade of CXCR4 signaling pathways inhibits SDF-induced MSC migration [74,75]. CXCL12 is also produced by MSCs and in turn increases their growth, development, and survival [76]. Migration of MSC has been studied during cerebral ischemia. After induction of cerebral ischemia (animal studies), the level of monocyte chemoattractant protein-1 (MCP-1) is clearly elevated in the injured tissue. An extract of inflamed cerebral tissue acts as a chemotactic factor for MSCs via receptor CCR2, and antiMCP-1 antibodies stop MSC migration [77]. Pittenger et al. [78] showed for the first time the presence of integrins on MSCs, which play a role in their differentiation and migration. In this study, they proved that α1, α2, α3, α4, αv1β1, β3, and β4 molecules, along with ICAM-1, ICAM-3, VCAM1, ALCAM/CD166 (activated leukocyte cell adhesion molecule), and endoglin/ CD105 adhesion molecules, are present on MSCs [78]. Although ICAM-1 expression on endothelial cells has been implicated in MSC migration, it is not known which ligands present on MSCs interact with this molecule. To date, only one study has examined MSC rolling on endothelial cells in vitro. Ruster et al. confirmed that β1 integrin and very late antigen-4 (VLA-4)/CD-49D molecules are expressed by 50% of MSCs. They showed that MSCs bind to endothelial cells in a P-selectin–dependent manner and interact with VLA-4/VCAM-1, which process promotes firm adhesion onto the endothelial cell. Anti-integrins and anti–VCAM-1 antibodies inhibit MSC adhesion to endothelial cells. These findings suggest that VLA-4/VCAM-1 molecules are essential for MSC-endothelial adhesion. Therefore, it may be concluded that MSC adhesion to and rolling on the endothelial cell are related to the presence of P-selectin and VLA-4/VCAM-1 molecules [65] (Figure 16.2). TNF-α, the main elevated cytokine at an inflamed site, causes MSC migration from the bone marrow into the circulation and then into the affected tissue [79]. The peak of TNF-α secretion occurs during the first 24 h of inflammation. Therefore,

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MSC P-selectin ligand

VCAM-1

P-selectin Induction of MSC immigration

VLA-4

Eondothelial cell

MSC

Normal cell

Inflammed cell

Normal cell

TNF-α, IL-β, BFGF, SDF-1 local inflammatory cytokins

Figure 16.2  Migration of MSCs to inflamed sites. MSCs, via their surface receptors, respond to chemical signals from inflamed sites produced by such compounds as SDF, TNF-α, and IL-1β. Their migration mechanisms are similar to that of leukocytes, but different molecules are involved.

TNF-α acts as a chemoattractant cytokine for MSC migration in the early stages of inflammation. TNF-α secretion, in addition to that from macrophages and inflamed tissues, also occurs from cells of mesenchymal origin [80]. TNF-α, by activation of the inhibitor of IkappaB kinase 2 (IKK2), a key enzyme for NF-κB signaling, facilitates the migration and passage of MSCs through the basement membrane [81]. TNF-α, by increasing matrix metalloproteinase-9 (MMP-9) protease, destroys laminin and denatures collagen (an essential component of the basement membrane), which improves MSC migration [81,82]. Moreover, TNF-α increases VCAM-1 and CD44 expression on MSCs, and thereby plays an important role in MSC migration to inflamed tissues [81].

MSCs and Tumors Solid tumor cells infiltrate normal tissues and form a microenvironment populated by large amounts of inflammatory cells and cytokines. The secreted cytokines from inflammatory as well as tumor cells promote the engraftment of MSCs [83]. Moreover, treatment options such as tumor irradiation increase MSC migration to the

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tumor site by elevating the levels of inflammatory mediators [84]. Intravenously or intra-arterially injected MSCs migrate to tumor sites [85], and their attraction to the tumor is the basis for using them as vectors for transferring antitumor agents into the cancerous tissue. Engineered MSCs secreting IFN-β have been used for controlling tumor growth [85,86]. In tumor tissues, cytokines such as TNF-α, IL-1β, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and plateletderived growth factor (PDGF), and chemokines such as CCL2, CCR8, and SDF are responsible for MSC migration into the tumor [83]. Moreover, the exacerbation of inflammation associated with treatments (e.g., tumor irradiation) increases endothelial permeability and further elevates MSC traffic to the tumor site [79,84,86]. As noted above, MSCs are known to express some chemokine receptors, which may be responsible for their tumor-homing process. CCR2 is an inducible surface marker of MSCs; its expression is usually low but it increases in the presence of TNF-α or with tumor irradiation [62,87,88]. CCL2, a ligand for CCR2, is overexpressed in tumors and is related to the tumor’s progression. CCL2 attracts monocytes and suppressor T cells and increases tumor survival [89]. Anti-CCR2 antibodies decrease the homing of MSCs into irradiated tumors [84]. Manipulation of CCL2 and its receptor can be considered a tool for controlling MSC migration to tissues.

Migration of Differentiated and Undifferentiated Stem Cells A major question pertaining to the clinical use of stem cells is whether they should be administered as differentiated or undifferentiated. In an animal model, ex vivo expanded human neural progenitor cells were injected into rats with 6-hydroxydopamine (6-OHDA)–induced parkinsonism. After transplantation, these cells distributed throughout the brain without specific accumulation at injured sites [90]. In another similar study, pre-differentiated neural stem cells (NSCs) expressing neural markers were used [91]. There was no improvement in the animal’s behavior after transplantation of these cells into the striatum, although neural markers were still being expressed [91]. These studies indicate that the striatum may not be a suitable microenvironment for transplanted stem cells, with poor results in terms of their survival, migration, and differentiation. In another study, pre-differentiated and undifferentiated human neural cell precursors were injected into a healthy rat’s hippocampus. The investigators observed that undifferentiated cells survived longer than the pre-differentiated ones. Moreover, the pre-differentiated cells did not leave the hippocampus, whereas undifferentiated cells migrated [92]. This probably occurred because pre-differentiated cells do not express stem cell factor receptor until later in their life cycle. Therefore, they cannot respond to stem cell factor, which induces cell survival and migration and is secreted by damaged tissue [93]. Several investigators reported that MSCs can be differentiated to neural phenotypical cells using specific culture media [94,95]. It is not yet clear whether these cells would function as neurons. We believe that specific tissue microenvironments contain the necessary factors for inducing stem cell differentiation; for example, retinoic acid, a neural cell inducer, can be found in high concentrations in the striatum [96].

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Anti-Inflammatory Activities of MSCs in Clinical and Experimental Studies MSCs can be considered an alternative therapeutic option for inflammatory conditions of different organs. A discussion of MSCs as anti-inflammatory therapy in different diseases follows. In the autoimmune diseases, MSCs have been used as anti-inflammatory and regenerative agents. In animal models of experimental autoimmune encephalomyelitis (EAE), MSCs have decreased disease severity [97,98]. The probable mechanism underlying this may be induction of T-cell anergy, or reduction of inflammation along with oligodendrocyte stimulation and myelin regeneration. In a clinical trial we performed involving a cohort of MS patients, autologous MSCs were injected intrathecally in 10 patients with secondary progressive MS (none responding to conventional therapies) and followed for 1 year. One patient’s Expanded Disability Severity Scale (EDSS) score improved significantly; EDSS progression stopped in four patients, but five others showed progression. MRI lesion burden decreased in five patients and increased in five, including four patients with new enhancing lesions [99]. Statistical analysis showed no significant change at 12 months after treatment in terms of EDSS score or MRI lesion burden from the pretreatment baseline, but there was a significant treatment response with respect to relapse rate decreasing as the number of MSCs injected increased; and there was a statistical trend toward reduction in relapse rate with treatment (Michael Glabus, unpublished data). In another study, intravenous injection of BM-MSCs into rats with dextran-induced colitis improved the inflammation in comparison to the control group. This study showed that MSCs accumulated at sites of inflammatory cell infiltration. The expression of inflammatory factors, including TNF-α, IL-1β, and cyclooxygenase-2 (COX-2), was significantly reduced in inflamed colonic tissue in the MSC-treated group [100]. Gupta et al. [101] reported that MSC injection significantly limited E. coli endotoxin-induced lung injuries (ILI) in mice. They demonstrated that MSC injection downregulated the endotoxin-induced pro-inflammatory response, reducing TNF-α and macrophage-inflammatory protein-2 (MIP-2) levels, and upregulated the expression of the anti-inflammatory cytokine IL-10 [101]. These findings implied that early improvement was due to the anti-inflammatory effect of MSCs rather than engraftment and tissue regeneration. Injection of human adipose-derived MSCs (hADMSCs) into DBA/1 mice with collagen-induced arthritis stopped the disease process if injected in the early phase of the disease [40]. However, in animals with advanced arthritis, only disease severity was reduced. The investigators concluded that hADMSCs induce collagen type II (CII)-specific CD4, CD25, and FOXP3 Tregs, which can suppress CD4 autoreactive T cells, probably by enhancing IL-10 and TGF-β production [40]. In similar research, the anti-inflammatory effect of hADMSCs on collagen-specific T cells and synovial cells of rheumatoid arthritis (RA) patients was evaluated. The study found that hAD-MSCs inhibit collagen II-specific T-cell proliferation and CD4 and CD8 inflammatory cytokine secretion, and stimulate production of anti-inflammatory cytokines by monocytes and TH2 cells. The investigators cocultured monocytes with hAD-MSCs and observed that both

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cells produced large amounts of IL-10. However, when these cells were cultured alone, even in the presence of LPS, neither type secreted IL-10. On the other hand, when hAD-MSCs were cocultured with PBMCs from RA patients, the percentage of Tregs increased [102]. In a mouse model of endotoxin-ILI, BM-MSCs decreased the severity of ILI by inhibiting systemic inflammatory responses. In both in vitro and in vivo experiments, the investigators demonstrated that in animals treated with MSCs, the levels of endotoxin-induced pro-inflammatory cytokines were less than in control groups. MSCs did not affect IL-10 production but increased granulocyte colony-stimulating factor (G-CSF) secretion. Moreover, neutrophil infiltration into the lungs of the treated group was less than that in the control group. MSCs were localized in lung tissue transiently but did not express lung cell phenotypes [103]. In acute lung injuries (ALI), MSCs interact with lung cells via two mechanisms: stem cells influence lung cell function, and lung cells change MSC function. Endotoxin-treated lung cells produce chemoattractant factors that influence MSC migration, and MSCs inhibit lung cell secretion of inflammatory cytokines. Therefore, it may be concluded that MSCs change the endotoxin-specific responses from pro-inflammatory to anti-inflammatory. IL-10, HGF, and TGF-β, which are continually secreted by MSCs, are factors involved in the anti-inflammatory action of MSCs [31]. PGE2 (a product of the enzymatic metabolism of arachidonic acid), IDO, and insulin-like growth factor binding protein all play roles in the induction of immunosuppression attributed to MSCs [24,36,104]. The immunosuppressive activity of MSCs is not only due to the fact that they are stem cells, but also because they maintain their inflammatory inhibitory properties even after differentiation into other cells [105–107]. In ischemic kidneys treated with MSCs, the levels of pro-inflammatory cytokines (IL-1β, TNF-α, IFN-γ) and nitric oxide synthase expression were reduced and anti-inflammatory cytokines (IL-10, TGF-β) were upregulated [108]. MSCs increase kidney tubular cell survival, and apoptosis of those tubular cells is one of the factors responsible for end-stage kidney disease. Therefore, MSCs may be important tools for treatment in these various settings [109]. In MI, MSCs decrease the size of tissue fibrosis through secretion of MMPs [110]. Two other factors secreted by MSCs are osteopontin (OPN) and macrophage colony-stimulating factor (M-CSF). Depending on the stage of tissue damage, OPN, by acting on TH1 and TH2 cells, plays a pro-inflammatory or anti-inflammatory role. OPN regulates cytokine secretion by macrophages, mediates adhesion and migration of cells, inhibits accumulation of macrophages in vessels, and also plays a role as a survival factor [111,112].

MSCs and Neurodegenerative Diseases In recent years, a better understanding of the biology of stem cells has suggested new methods for treatment of a wide variety of neurodegenerative diseases. Currently, there is considerable interest in the therapeutic application of stem cells in diseases such as multiple sclerosis (MS), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Both adult and embryonic stem cells (ESCs) have been proposed for the treatment of CNS autoimmune and neurodegenerative diseases.

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Considering MSCs’ potential for differentiation and tissue repair, these cells are a primary focus. The capacity of MSCs to migrate to damaged tissue sites, modulate inflammation locally and systemically, and differentiate to replace lost tissue cells has attracted research attention. The observation that MSCs can differentiate into neurons both in vitro and in vivo, indicated by expression of neural markers, has created controversies since the expression of surface markers does not prove that MSC-derived neurons are functionally active [113,114]. However, in vitro MSC differentiation into neural cells supports the possibility of their future use in the treatment of neurodegenerative diseases. MSCs can be used therapeutically as differentiated or undifferentiated cells, but the direct role of MSCs in CNS tissue repair remains controversial. Certain studies suggest that BM-MSCs can directly repair tissue through two mechanisms: (1) transdifferentiation (direct differentiation of transplanted cells into neurons) [115–117] and (2) cell fusion (fusion of transplanted cells with preexisting neurons and heterokaryon formation) [118]. These observations provide evidence that supports the direct role of MSCs in neural tissue regeneration. In animal models, intravenous or intracranial injection of BM-MSCs can induce partial remyelination in proportion to the number of injected cells [119,120]. Injection of green fluorescent protein (GFP)expressing bone marrow stromal cells harvested from transgenic mice into the demyelinated spinal cord of immunosuppressed rats improved axonal conductivity [121]. Weimann et al. [117] found the Y chromosome in cerebellar Purkinje neurons of a woman transplanted with a man’s bone marrow cells. The results of this study confirmed the direct role of MSCs in neural replacement and raises the possibility of using BM-MSCs as a source for neural tissue regeneration [117]. In contrast, some evidence from EAE animal models indicates that differentiated MSCs do not migrate to CNS or repair neurons [122–124]. Currently, there is no strong evidence that integrated MSC-derived neurons are functional in neural networks. However, the neuroprotective role of MSCs has gained more support. MSCs inhibit neuronal apoptosis, which may be a potential mechanism for restricting tissue degeneration [125,126]. MSCs, via secretion of various trophic factors, cytokines, chemokines, and antioxidant molecules, increase neuronal survival [127–129]. MSCs normalize inflamed microenvironments and stimulate proliferation of in situ neurons and oligodendrocytes [124,130,131]. In conclusion, based on the currently available scientific data, it appears that the neuroprotective and tissue regeneration properties of MSCs are mostly mediated through inhibition of inflammation and stimulation of in situ progenitor cell proliferation. The use of autologous or allogeneic MSCs for the treatment of neurodegenerative diseases is a new strategy and just in its infancy. Due to the small number of studies investigating the efficacy of MSCs in the treatment of neurodegenerative diseases, we have also reviewed the treatment applications of ESCs and other adult stem cells (ASCs) in the following sections.

Stem Cells and MS MS is a chronic inflammatory, demyelinating, and neurodegerative disease of the CNS usually affecting young adults. The pathophysiology and treatment of MS

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remain problematic. However, the role of an immune mechanism affecting the certain neuronal and myelin antigens has received much attention. Formation of the demyelinating lesion results from inflammatory infiltration of activated leukocytes, especially in perivenular areas [132]. In early phases of the disease, spontaneous axonal remyelination appears to occur via the activity of endogenous progenitor oligodendrocytes, which normalizes neuronal action potentials [133]. Unfortunately, due to the chronicity of the disease, the accumulation of demyelinating damage cannot be totally compensated by this endogenous process, and clinical signs become apparent [134]. Nonspecific immunosuppressors, the conventional treatment for MS, reduce anti-myelin lymphocytic activity. These immunomodulatory treatments have a restricted effect on the disease because they do not inhibit the inflammatory process at all desired sites [135]. Moreover, these drugs have no effect on endogenous brain stem cells in terms of induction of tissue regeneration. Therefore, stem cells can be considered a major candidate for MS treatment, with their ability to migrate to inflamed sites, induce tissue regeneration, and modulate inflammation. There are usually two sources for stem cell therapy: ESCs and ASCs. ESCs can be isolated from a blastocyst’s inner mass. These cells have the potential to differentiate into any cell, including neurons. One study showed that injection of ESCs into rats with demyelinating lesions produced remyelination of brain and spinal cord axons [136]. The tumorigenicity and difficulty of preparation are restrictions for ESC therapy [137]. In contrast, ASCs are much less tumorigenic and can be used without provoking ethical debate. Among stem cells, ASCs are the most frequently used stem cells for attempting immunomodulation and tissue regeneration in both humans and animal models. In the EAE animal model, early administration of MSCs produces maximum therapeutic efficacy. When MSCs, either syngeneic (mouse) or xenogeneic (human), are injected into the EAE animal model, disease symptoms are reduced [97,138]. Intravenous injection of MSCs induces peripheral tolerance followed by decrease of autoreactive T and B cells [97,122]. Intraventricular and intraperitoneal injections of MSCs in EAE have the same immunomodulatory effect as intravenous injection [123,139]. In most studies, no evidence was observed for neuronal transdifferentiation of MSCs [122,123,140]. However, MSCs appear to reduce axonal damage and increase neuronal survival significantly [122,126,139]. These cells also mediate oligodendrocyte proliferation and induce remyelination, and they are tolerogenic [124]. Generally, the results of animal studies indicate that MSCs accelerate CNS repair in EAE. Collectively, MSCs produce neuroprotection by decreasing inflammation and immune cell activity, as well as by secreting trophic and anti-apoptotic factors [141]. To date, MSCs have been used for MS treatment by several research groups. Although allogeneic MSCs have been well tolerated in the treatment of inflammatory conditions such as GVHD, autologous MSCs have the same immunomodulatory effect without presenting ethical problems [142]. Therefore, it is preferable to use autologous MSCs for clinical trials. In preliminary clinical trials, intravenous or intrathecal injection of MSCs has been well tolerated, and their efficacy has been suggested [99,143,144]. In human trials, there are no reports showing effect differences between intravenous and intrathecal MSC injection. Although the intrathecal injection of MSCs is associated with a greater number of stem cells arriving at

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inflamed and damaged sites, reports indicate that intravenous injection of MSCs is sufficient for producing an immunomodulatory effect and neuroprotection [97,140,145]. The results of these preliminary studies demonstrate that the administration of human BM-MSCs to MS patients can be done without significant side effects [99,145].

Stem Cells and PD PD is a chronic neurodegenerative disease of the nervous system manifested prima­ rily by resting tremor, rigidity, and hypokinesia. Other clinical features include a gait disorder, depression, dementia, and autonomic disturbances. The underlying neuropathology of PD involves the progressive degeneration of dopaminergic striatal neurons. Non-dopaminergic systems such as the lower part of the brain stem and the cortex can be affected as well. Although current treatment partially reduces the clinical manifestations of PD, more effective therapies that block and restore the pathologic changes are needed [146]. Different factors, including neuroinflammation, are involved in the pathogenesis. The COX enzyme and inflammatory mediators such as nitric oxide are increased in patients with PD. Nonspecific inhibitors of COX such as aspirin or specific inhibitors such as meloxicam may provide neuroprotection in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion in mice [147]. Other anti-inflammatory agents such as tetracycline derivatives induce neuroprotection in animal models of PD. In the MPTP model, minocycline inhibits iNOS and NADPH oxidase, modulates microglial responses, and stops the destruction of dopaminergic neurons [148]. Stem cells have been evaluated for their cellular regeneration ability in animal models of PD, and observed functional improvement probably involves both immunomodulation and tissue regeneration [149]. So far, three sources of stem cells have been used for PD treatment in humans and in animal models: ESCs, NSCs, and MSCs. ESCs can be transplanted after differentiation into NSCs (i.e., neuronal progenitor cells). NSCs and MSCs are somatic stem cells and can differentiate into mature neurons. Both bone marrow and cord blood stem cells have the neuronal lineage differentiation potential [150]. Studies with PD patients demonstrate that intrastriatal transplantation of human fetal mesencephalic tissue (rich in potential dopaminergic neurons) may serve as an effective neuronal replacement strategy. During a 10-year follow-up, even though there was persistence of PD pathology, the transplanted cells were functional and appeared to improve the clinical manifestations [151,152]. These grafts normalized dopamine release from the striatum and improved hypokinesia [153]. Although transplantation of fetal mesenchymal tissue showed encouraging results in PD, difficulty in obtaining this tissue has limited its application. Kim et al. [154] studied the protective and anti-inflammatory effects of MSCs against LPS-induced inflammation in the dopaminergic system using animal models. Their results indicate that treatment of these animals with MSCs significantly reduces microglial function and LPS-induced TNF-α secretion, confirming MSCs’ ability to protect dopaminergic neurons by these anti-inflammatory mechanisms [154]. Some investigators demonstrated that iatrogenic inflammation-induced

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damage increases the viability of transplanted MSCs and attracts them to the site of injury in an animal model of PD [155]. Once stimulated, MSCs produce trophic factors [156], and it has been demonstrated that infusion of trophic factors such as glial cell-derived neurotrophic factor (GDNF) shows a positive treatment effect in some PD clinical trials [157]. In one study prior to the transplantation of NSCs, the investigators genetically manipulated these stem cells to express GDNF. Following transplantation, these cells differentiated into astrocytes and neurons in a PD animal model [158].

Stem Cells and ALS ALS is a progressive degenerative disorder of motor neurons in the cerebral cortex, brain stem, and spinal cord. Profound muscular weakness progresses rapidly and causes death in a few years. Stem cell therapy is a potential treatment for this incurable disease. Recent studies have used a new strategy of in vitro stem cell differentiation to create specific neuronal types for transplantation, and potentially, cortical neuronal network repair [159]. Miles et al. [160] reported that mouse ESC-derived motor neurons transplanted into motor neuron-injured rat spinal cord survived, and their axons grew into the ventral roots. Whether these neurons can be integrated into the local neuronal motor circuits and restore voluntary movement and reflexes remains unknown. Kerr et al. transplanted ESCs intrathecally and showed that these cells distributed into the spinal cord and migrated to injured sites. Motor function recovered partially in the test group followed for 24 weeks after transplantation, whereas control animals remained paralyzed. The researchers concluded that these transplanted cells protected host neurons from apoptosis and facilitated recovery of their cellular functions [161]. In animal models of ALS, transplantation of human NSCs into the spinal cord partially blocked disease progression and improved functional activity [162]. Recently, the results of a phase I clinical trial showed that intrathecal injection of autologous BM-MSCs into 10 patients with ALS was safe. Therefore, this procedure may be considered for investigation as a novel therapeutic option [163].

Stem Cells and Stroke Stroke remains a significant cause of neurologic disability and death worldwide. The two major forms of stroke are ischemic, the dominant form, and hemorrhagic. Ischemic stroke produces varying degrees of brain tissue infarction, and when extensive it can lead to long-term, major sensorimotor deficits. Conventional treatments, including surgical interventions, and various types of anticoagulation, including administration of tissue plasminogen activator (t-PA), can neither replace lost neurons nor improve impaired cerebral function after the acute phase [159]. Restoration of neuronal function in the chronic phase of stroke requires neurogenesis, angiogenesis, and activation of synaptic plasticity [164]. To date, there are several reports that different stem cell types and their derivatives, of both rodent and human origin, can differentiate into neurons and restore function after transplantation in ischemic

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stroke animal models [165,166]. Daadi et al. [167] reported that NSCs derived from human ESCs will migrate to ischemic brain tissue in the rat model. These cells improved limb performance in the test group 2 months after transplantation [167]. Moreover, these researchers showed that grafted human NSCs differentiated into neurons and oligodendrocytes in ischemic brain regions and were functional when studied electrophysiologically [168]. In human trials, transplantation of cultured human neuronal cells (LBS-neurons derived from the human NT2/D1 precursor cell line) into patients with ischemic stroke has been shown to be safe [169,170]. Stilley et al. [171] reported that neural cell transplantation in patients with basal ganglia stroke improves their cognitive function [171]. Bang et al. [172] were the first to use autologous MSCs for the treatment of stroke patients. The results showed that neurologic outcomes in MSCtreated patients improved during follow-up at 3, 6, and 12 months [172]. These observations provide evidence that replacement of functional neurons using stem cell grafts is possible in the stroke-damaged brain, and suggest that these cells contribute to the observed behavioral improvements.

Stem Cells and Alzheimer’s Disease Alzheimer’s disease (AD) is an irreversible, progressive brain disease characterized by degeneration and loss of neurons throughout the brain, particularly in the basal forebrain, amygdala, hippocampus, and cortical areas. Patients suffering from AD experience problems with memory, thinking, attention, expressive language, and comprehension. The pathology in AD is characterized by both deposition of large amounts of insoluble amyloid-β (AB) peptides as amyloid plaques, and neurofibrillary tangles, although the toxic agents may actually be the soluble oligomers of AB [173]. To date, no curative treatment is available for AD, and conventional drugs like acetylcholinesterase inhibitors are only symptomatically effective but do not influence neuronal regeneration [174]. Since stem cells can differentiate into neurons, have high migratory capacity after brain transplantation, and can also be genetically modified in order to carry pharmacologicals, they may be a novel tool for restoring neuronal function and/or replacing lost neurons in AD [175]. Several animal studies suggest that stem cells can be useful for improving the function of aged brains. Qu et al. [176] reported that injection of in vitro expanded human NSCs into the brains of 6-month-old and 24-month-old rats improves their cognitive function. They also noted that injected human NSCs engrafted into rat brains and differentiated into astrocytes [176]. In another study, human umbilical cord blood cells (rich in MSCs) were injected into a transgenic mouse model of AD. Animals in the test group had extended survival in comparison to the control group [177]. Coculture of NSCs with postmortem brain slices from AD patients increased neuronal cell survival. Moreover, this protective effect persisted even after separation of NSCs by a semipermeable membrane, suggesting that soluble factors secreted from NSCs were responsible [178]. Recently, an interesting study demonstrated that NSCs improve cognition in a transgenic AD mouse model through secretion of brain-derived neurotrophic factor (BDNF), a pivotal factor for neuronal survival and

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function [179]. These findings suggest that transplanted stem cells, and especially NSCs, may prove to be a novel treatment for AD. Considering the safety and regenerative potential of MSCs, it would appear that clinical trials for evaluating this therapeutic option can begin in the near future.

Stem Cells and Huntington’s Disease Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive cognitive impairment, involuntary choreiform movements, and neuropsychiatric symptoms [180]. One of the main pathologic findings in HD is selective death of GABAergic projection neurons residing in the striatum [181,182]. Despite identification of the gene mutation responsible for HD, and its resultant abnormal protein, the mechanisms underlying the pathogenesis of HD remain largely unknown. Based on the pathologic observation noted above, it may be possible for intrastriatal transplanted GABAergic neurons to replace lost neurons and restore neuronal circuitry function. Previous studies have shown positive results for transplantation of fetal striatal precursor cells in animal models of HD [183,184]. Transplanted striatal tissue from healthy animals can integrate, survive, and differentiate within the striatum of an HD transgenic mouse model in a manner comparable to that seen in control mice [185]. Lee et al. [186] demonstrated that intravenous injection of human NSCs induces functional recovery in an HD animal model. Their results demonstrated that intravenously injected human NSCs can migrate into the striatal lesion, decrease the striatal atrophy, and induce long-term functional improvement [186]. Lescaudron et al. [187] treated quinolinic acid-induced striatal damage in rats with autologous BM-MSCs. They concluded that the transplants reduced working memory deficits, probably by releasing growth factors that increased cellular survival [187]. Freeman et al. [188] transplanted human fetal striatal tissue into patients with HD. Postmortem analysis of one of the study patients revealed that the transplanted tissue survived and differentiated in affected areas despite the presence of disease pathology. Moreover, no histologic evidence of immune rejection, including microglia and macrophage infiltration, was found [188]. Another similar study also reported that transplantation of human fetal neuroblasts into the striatum of five patients with HD was associated with improvement in motor and cognitive function in three [189]. Supplying fetal striatal cells for transplantation is difficult due to scarce amounts as well as the obvious ethical problems. NSCs seem to be an ideal source for transplantation in patients with HD because of their regenerative and immunosuppresor potential. BM-MSCs from HD patients carry the mutant Huntington gene, and thus autologous transplantation would not be a suitable therapeutic option [190].

The Routes and Timing of Injection of Stem Cells MSCs may be administered systemically (i.e., intravenously) or directly to the damaged and/or inflamed site. In treating CNS diseases, MSCs can also be injected intrathecally. In an interesting study, Sokolova et al. [191] induced inflammation in one

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lobe of the prostate gland in an animal model and then injected cultured MSCs in three ways: intravenously, into tissues adjacent to the inflamed lobe, and into the intact prostate lobe [191]. They observed that MSCs injected into the adjacent tissue did not migrate into the inflamed site, while MSCs injected into the healthy lobe migrated into the inflamed lobe. Intravenously injected MSCs migrated and distributed uniformly into the inflamed lobe. Therefore, the investigators concluded that direct tissue injection is more invasive and less efficient (at least for the damaged prostate gland), and the intravenous injection method was best [191]. Other studies have demonstrated that migration and survival of different types of transplanted stem cells are related to the pathologic environment of the recipient’s tissue [192,193]. Inflammation and tissue damage will increase the survival and stimulate the migration of transplanted stem cells. The injection of BM-MSCs as well as total tissue grafting into healthy brain tissue produced undesired reactions; however, the same injection into damaged brain tissue increased migration and survival of these cells [194,195]. Jain et al. [90] reported that cultured human neural progenitor cells transplanted to the 6-OHDA–damaged striatum of the rat PD model migrated to different brain sites but not specifically to the sites of cell loss. They concluded that this negative result was due to the absence of suitable survival factors, or the presence of apoptotic signals at the affected sites [90]. Certain observations have shown that when MSCs are used as a therapeutic agent, the timing of injection can be very important, because the immunomodulatory functions of MSCs are closely related to the microenvironment at the target location [196,197]. This means that MSCs need to receive proper signals from cytokines in the microenvironment in order to become activated. For instance, IFN-γ is one of the strongest signals for induction of the suppressor function (of T-cell and NK-cell proliferation) of MSCs [47]. This action of IFN-γ may be due to induced IDO upregulation [36]. TNF-α also increases MSCs’ suppressor activity through upregulation of PGE2 and COX-2 [198]. It thus appears that T-cell– and DC-derived cytokines are able to induce the immunomodulatory action of MSCs.

Side Effects of Stem Cell Therapy Every new treatment is associated with its particular side effects. Therefore, prior to making any treatment decisions, one should weigh potential benefits against risks, especially adverse effects. Treatment applications of MSCs in human disease are not an exception. With all the reported positive effects of MSC therapy, their adverse effects must also be considered. One of the most important of these is their tumorigenicity. MSCs, by secreting pro-angiogenic factors (VEGF, IL-6) and matrixdegrading enzymes (MMPs) [53,79], and by their ability to migrate to tumor sites, can enhance tumor growth [85,199]. In vitro and in vivo studies have elucidated the role of MSCs in tumor growth [200,201]. However, other studies have shown the tumor inhibitory effect of MSCs, such as the suppression of Kaposi sarcoma growth [202]. This MSC effect on Kaposi sarcoma is probably due to the expression of E-cadherin and also direct cell contact. These contradictions could be due to the

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heterogeneity of MSCs as well as tumor tissues. However, genetically manipulated MSCs may be useful for tumor treatment. In different animal studies, engineered MSCs expressing IFN-β suppressed angiogenesis and tumor growth in different cancers such as melanoma, breast cancer, and gliomas [85,86,203].

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17 Inflammatory Mediators in Obstructive Sleep Apnea

David E. McCarty, Andrew L. Chesson Jr. Sleep Medicine Program, Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Introduction to Obstructive Sleep Apnea Obstructive sleep apnea (OSA) is a disorder characterized by instability of the upper airway, resulting in periodic alternation between functional obstructions to airflow during sleep and relative restoration of breathing, typically in the setting of an arousal or awakening [1]. Obstructions may be partial (hypopneas) or complete (apneas) and may be accompanied by one or more of the following: intermittent hypoxia, intermittent or sustained hypercarbia, sleep architecture disruption, frequent nocturnal awakenings, and/or subjective reports of nonrestorative sleep [2]. The number of apneas or hypopneas a patient experiences per hour of sleep is termed the apnea hypopnea index (AHI), which is often used as a marker for disease severity; the long-term prognosis—particularly the risk for mortality—is heavily dependent on this metric [3]. The diagnosis of OSA can be made when a patient has an AHI of at least 15 per hour of sleep (regardless of symptoms or other cardiovascular risk factors), or when a patient with symptoms of sleep impairment or cardiovascular disease is found to have an AHI of at least 5 per hour of sleep [4]. Patients often seek medical attention due to the presence of daytime neurocognitive impairment symptoms (Box 17.1) or socially disruptive snoring. Asymptomatic patients may seek medical attention due to concerns about the potential long-term health risks of untreated OSA, particularly those associated with cardiovascular disease. Box 17.1  Daytime Neurocognitive Impairment Symptoms [4]

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Fatigue or malaise Attention, concentration, or memory impairment Poor performance at school or work Daytime sleepiness Mood disturbance or irritability Motivation, energy, or initiative reduction Proneness for errors or accidents at work or while driving Tension, headaches, somatic symptoms related to disturbed sleep

Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00017-4 © 2011 Elsevier Inc. All rights reserved.

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Risk Factors and Epidemiology of OSA Typical risk factors for OSA include obesity, adenotonsillar hypertrophy, retropositioning of the mandible, obstructive nasal conditions (such as turbinate hypertrophy and nasal polyposis), large neck circumference, relative macroglossia, and midface hypoplasia [5]. Race appears to influence the presentation as well, with OSA being more common and more severe in African-Americans [6,7], Pacific Islanders, and Hispanics [8,9] compared with Caucasians. In younger adults, OSA is more common in men than women. This disparity tends to dissipate as people age—menopause increases the prevalence of OSA in women by approximately a factor of 3 [10]. OSA occurs in up to 3% of preadolescent children [11], where it is thought to have a causative role in attentional difficulties and hyperactive behavior [12]. In an unselected, community-dwelling adult population, OSA is found in up to 4% of men and 2% of women [13], and mounting evidence links the disorder to adverse cardiovascular outcomes, such as incident hypertension, ischemic heart disease, cerebrovascular accidents, heart failure, and atrial fibrillation [14]. A past history of congestive heart failure raises the prevalence to one in two [15], while a past history of stroke increases the prevalence to about two in three [16]. As obesity rates increase in developed countries, and as public and professional awareness of OSA rises, future increases in the incidence and prevalence of OSA are expected [17]. The pathophysiologic mechanism for the elevated cardiac risk in patients with OSA likely involves several pathways, including hyperarousal of the sympathetic nervous system [18,19], inflammatory mediators and reactive oxygen species (ROS) [20,21], and possibly chronic mechanical disruption to neurovascular structures of the head and neck due to vibratory trauma from snoring (Figure 17.1) [22–27]. The mechanism underlying daytime impairment symptoms in patients with OSA includes chronic partial sleep deprivation, the effects of which are in part functionally accomplished via inflammatory signaling. The fact that some patients with OSA manifest daytime impairment [28] while others don’t [29] invites speculation about whether these two groups may have fundamental differences in this inflammatory response and, by extension, differences in the overall future health risk.

Chapter Objectives This chapter will detail the current understanding of the role of inflammation in the cardiovascular, pulmonary, and neurocognitive morbidity associated with OSA. To do this, we will first review the current data supporting a link between OSA and various diseases understood to be potentiated by inflammatory processes, including cardiovascular disease, the metabolic syndrome, and asthma. The association between OSA and daytime neurocognitive impairment (particularly daytime sleepiness) will also be explored. Following this, we will review the evidence supporting the roles of oxidative stress and pro-inflammatory mediators, which are thought to be central to the pathophysiologic mechanism underlying some of these problems.

Intermittent hypoxia and oxidative stress

Head and neck vibration and trauma from air turbulence and snoring

Chronic sleep deprivation

Influences of genetic susceptibility? Apo E deficiency?

Metabolic dysregulation

Dysregulation of leptin and ghrelin

Metabolic syndrome/ insulin resistance

Sympathetic excitation

Increased risk for arrhythmias, including atrial fibrillation

Endothelial damage to vessels of the head and neck

Increased blood pressure

Systemic endothelial damage and dysfunction

Increased risk for obesity

Systemic inflammation: elevation in TNF-α, IL-6, NF-ĸB, CRP, PGD2, LTE4

Increased risk for myocardial dysfunction and CHF

Localized inflammation of upper airway

Inflammatory Mediators in Obstructive Sleep Apnea

Obstructive sleep Apnea syndrome: cyclic upper airway collapse during sleep

Neuromuscular damage to upper airway Hypercoagulability Increased bronchial hyperreactivity

Increased risk for atherogenesis

Daytime neurocognitive impairment and excessive sleepiness

Increased risk for asthma

Steroid use

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Figure 17.1  Potential mechanisms linking OSA to various adverse outcomes (see text).

Increased risk for stroke

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OSA and Cardiovascular Risk Since the first polysomnographic description of OSA in 1965 [30], the association between it and subsequent cardiovascular risk has been suspected. Attempts to prove a causal association between OSA and cardiovascular disease are complicated by the presence of multiple confounders—including obesity, diabetes, alcohol use, smoking, age, and hypertension—that often co-migrate with OSA, thus making causality difficult to determine. In the past decade, the Sleep Heart Health Study [31] and the Wisconsin Sleep Cohort [32] showed a definitive significant association between OSA and arterial hypertension, even after correcting for potential confounders of gender, age, and body habitus. Among patients with refractory hypertension— defined as uncontrolled blood pressure despite treatment with three medications at optimal doses, including a diuretic—OSA appears to be particularly common, occurring in over 80% of these patients [33]. Ambulatory blood pressure data taken from the Wisconsin Sleep Cohort showed a linear relationship between 24-h blood pressure and AHI, independent of obesity, age, and gender [34]. Further analysis of the Sleep Heart Health Study cohort also pointed to a “dose-response” effect, with higher likelihood of self-reported symptoms of congestive heart failure, stroke, and ischemic heart disease occurring with worsening degrees of OSA [35]. These data were corroborated by an 18-year follow-up report on the Wisconsin Sleep Cohort, which showed a twofold increase in rate of death among those with severe OSA (defined as AHI30), compared to those with less severe disease, even after adjustment for age, gender, and body mass index (BMI) [3]. In the same sample, cardiovascular disease was almost twice as likely to be the cause of death for patients with severe OSA compared to those without.

OSA and the Metabolic Syndrome The metabolic syndrome (previously known as “syndrome X”) refers to a collection of metabolic alterations and physical findings that confer an elevated risk for the development of type 2 diabetes mellitus and arterial atherosclerotic disease: visceral/ abdominal adiposity, hypertension, low HDL cholesterol, elevated triglycerides, and insulin resistance [36]. Other metabolic disorders that tend to co-migrate with the metabolic syndrome include nonalcoholic fatty liver disease and polycystic ovarian syndrome, though these are not required to make the diagnosis. Numerous studies point to an association between OSA and the metabolic syndrome [37,38], which has prompted the offering of the memorable term “syndrome Z” to describe patients who manifest both disorders (see Figure 17.1) [39]. As might be expected, teasing out whether the association between OSA and metabolic syndrome implies causality is difficult, given that both problems tend to co-migrate with obesity. In a Korean study, snoring—a marker for the presence of OSA—was correlated with the risk for hypertension independent of BMI [40]. In another study, a history of snoring correlated with elevation in hemoglobin A1C values in middle-aged adults in a manner

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independent of age and abdominal obesity [41]. This finding corroborated findings from the Nurses’ Health Study, which indicated a strong link between snoring and subsequent development of type 2 diabetes, independent of age and body habitus [42].

OSA and Daytime Neurocognitive Impairment The phenotype of the “classic” presentation of OSA with hypoventilation—that of the morbidly obese, plethoric, somnolent male—was perhaps most memorably portrayed by Charles Dickens in his early serial novel The Posthumous Papers of the Pickwick Club [43], later memorialized in the medical literature under the pseudoeponym “Pickwickian syndrome [44].” In the story, Dickens portrays Joe—the “fat boy”—as inattentive and constantly falling asleep at inopportune times, allowing fodder for light comedy. Over the past three decades, however, OSA has been shown to be no laughing matter, with scientific evidence for significant neurocognitive dysfunction [45], increased risk for accidents [46–49], and decreased quality of life [50,51]. Cognitive and neurobehavioral deficits across many domains—including cognitive processing, memory, vigilance, ability to divide attention, and executive functioning—have been linked to OSA [52]. Classically, the sleepiness associated with OSA has been presumed to result from chronic partial sleep deprivation, resulting in enhanced homeostatic sleep pressure. However, this explanation fails to explain why some OSA patients develop sleepiness while others do not. In fact, the daytime impairment suffered by patients with OSA is worse than would be expected to occur as a result of sleepiness alone. To illustrate this point, Greenberg et al. demonstrated measurable neuropsychological deficits among patients with OSA, showing worse performance measurements compared to patients with nonapneic primary hypersomnia syndromes matched for the degree of sleepiness. Furthermore, the degree of neurocognitive dysfunction in patients with OSA in this study correlated with the degree of nocturnal hypoxia [28]. If sleepiness associated with OSA were a factor of chronic partial sleep deprivation, then one might expect nonsleepy OSA patients to have “more normal” indices of sleep architecture compared with their sleepy counterparts. In fact, no such relationship exists. A study comparing nonsleepy and sleepy patients with OSA revealed no striking practically relevant differences in indices of OSA severity, the severity of oxygen desaturation, sleep microarchitecture, sleep staging, or sleep efficiency between the two groups [53], implying that there must exist an individual susceptibility to the development of symptoms of sleepiness as a result of the physiologic pressure of OSA. Further underscoring this notion is another study of apparently healthy adults (i.e., those with no current evidence of cardiovascular disease, no sleep-related complaints, and no daytime sleepiness) that revealed that over half of those over the age of 65 years had polysomnographic evidence of OSA (AHI>15 per hour), with one in four having an AHI above 30 [54]. The mechanism for the apparent lack of impact of OSA in this population is not known, and the implications for management of OSA patients in this age group for the purpose of cardiovascular risk stratification are the subject of considerable debate.

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Taken together, these data suggest that OSA-related daytime neurocognitive impairment results from a combination of chronic partial sleep deprivation and the interplay between nocturnal intermittent hypoxia and an individual patient’s physiologic response to it. Some evidence suggests that this interplay is at least in part mediated by inflammation (see later section on prostaglandin D2 [PGD2]), with individual susceptibility factors potentially influenced by genetic factors (see Figure 17.1). For example, apolipoprotein E (ApoE) deficiency is understood to be a major risk factor for Alzheimer’s disease [55]. One study found that exposure to nocturnal intermittent hypoxia—simulating OSA in a mouse model—induced increased evidence of inflammation and oxidative stress as well as worse behavioral deficits in ApoE-deficient mice compared with wild-type mice [56].

The Overlap Between OSA, Obesity, and Asthma Over the past two decades, Western countries have seen a dramatic increase in the prevalence of obesity, with approximately one in three adults in the United States now meeting criteria for obesity (Figure 17.2) and two in three meeting criteria for overweight status. Along with this disturbing trend, the prevalence of asthma has also grown steadily, with a 74% increase in self-reported asthma documented between 1980 and

Figure 17.2  U.S. state-by-state prevalence of obesity, defined by body mass index of 30 kg/m2, for the year 2008. Source: Center for Disease Control Behavioral Risk Factor Surveillance System, http://www. cdc.gov/brfss/.

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1996 [57]. The parallel increases in prevalence of these two disorders have prompted epidemiologic inquiry into a potential link between obesity and asthma. Though some data are conflicting, a preponderance of evidence shows a link between obesity and asthma (see Figure 17.1) [58,59]. In the United States, the prospective cohort Nurses’ Health Study revealed that weight gain preceded the development of asthma, implying the possibility of causality [60]. The largest population study to date involved over 135,000 Norwegian adults, showing a linear increase in asthma prevalence with increasing BMI [61]. In men, for every unit of BMI over 20, the asthma incidence increased by 10%. The same trend was seen in women, with a slightly smaller slope, with a 7% increase for each BMI increment over 22. Furthermore, the association remained robust even after correcting for smoking, age, education, and physical activity. Given the increasing prevalence of asthma and OSA, it is not surprising to find that these conditions often coexist within the same patient, an issue that can complicate and confound attempts at treatment [62]. The two conditions share several risk factors, including obesity, postmenopausal status, chronic rhinitis, and nasal polyposis. The coexistence of asthma with OSA is not wholly accounted for by the presence of obesity. For example, one small prospective study in Israel evaluated polysomnographic findings in a group of 22 nonobese adults with medically refractory, steroiddependent asthma. All but one of these patients had OSA, as defined by an AHI of at least 5, with the mean AHI being over 15 [63]. The authors of this study concluded that chronic steroid use, through a myopathic mechanism, may decrease the anatomic stability of the upper airway, thus predisposing to airway collapse, a mechanism cited by other authors as well (see Figure 17.1) [64]. Other data suggest the possibility of a bidirectional relationship between OSA and asthma. One of the fundamental elements of the pathophysiology of asthma— in addition to airway inflammation—is bronchial hyperreactivity. Using methacholine challenge testing in a group of adults without demonstrable evidence of asthma or allergies, Li and Li demonstrated bronchial hyperreactivity in 25% of patients with OSA, compared with 0% of patients with simple snoring (see Figure 17.1). Furthermore, bronchial hyperreactivity was shown to improve after several weeks of continuous positive airway pressure (CPAP) treatment [65]. Other groups have reported improvement in nocturnal asthma control following CPAP treatment for management of OSA [66,67], a factor that has led some groups to suggest that OSA must be considered in the differential diagnosis of medically refractory asthma [68].

OSA and Oxidative Stress Oxidative stress occurs in the setting of incomplete reduction in molecular O2, leading to an increase in the intracellular concentration of cytotoxic ROS. Cellular and subcellular damage caused by ROS can include DNA mutagenesis, cellular apoptosis, lipid peroxidation, and depletion of intracellular energy stores (adenosine triphosphate [ATP]). Oxidation of low-density lipoproteins (LDLs) is seen as a key step in the development of atherosclerosis. In normal homeostasis, oxidative stress

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serves as a signaling mechanism to regulate intracellular machinery, with a strict balance struck between ROS and endogenous antioxidants. In pathologic states involving intermittent hypoxia, such as OSA, the balance is tipped toward ROS species, which are thought to be produced through the xanthine oxidase pathway during reperfusion [69]. This contributes to chronic inflammatory damage to and ongoing dysfunction of the endothelium, as well as propagation of atherosclerotic lesions (see Figure 17.1). In the setting of more severe hypoxia, such as acute coronary syndrome, low oxygen tension is thought to be a trigger to activate polymorphonuclear neutrophils, priming them to adhere to the endothelium and release more free oxygen radicals, thus potentially contributing to reperfusion injury [70]. Several studies have shown that OSA patients have elevated markers for oxidative stress. Barcelo et al. [71] demonstrated an increase in LDL oxidation in OSA patients compared to nonapneic controls. Lavie et al. [72] found similar results, with elevated indices of oxidative stress biomarkers thiobarbituric reactive substances (TBARS) and peroxides in OSA patients with and without current cardiovascular disease, compared with nonapneic controls. Both Barcelo and Lavie found improvement in the markers of oxidative stress among OSA patients using CPAP after several months. Schultz et al. found that neutrophils taken from patients with OSA had markedly enhanced superoxide release after stimulation with a bacterial tripeptide, compared not only with nonapneic controls but also with lung cancer patients, matched with the OSA patients for comorbidities. CPAP was found to reduce neutrophilic superoxide release within days of starting therapy [21].

Systemic Inflammatory Mediators Associated with OSA TNF-α Tumor Necrosis Factor (TNF-α) is a pro-inflammatory cytokine produced by, among others, macrophages, lymphoid cells, and monocytes. Interaction with membranebound TNF receptors leads to activation of two major nuclear transcription factors, nuclear factor B (see later) and c-Jun; these transcription factors lead to numerous and diverse cellular responses, including cell proliferation, oncogenesis, cell death, and immune and inflammatory responses [73]. Uncontrolled activation of TNF-α has been associated with the pathogenesis of numerous diseases, including multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Of particular importance, TNF-α has also been implicated in the pathogenesis of atherosclerosis, primarily via downstream elaboration of vascular cell adhesion molecule-1 (VCAM-1), which promotes adherence of monocytes and T lymphocytes to nascent atheromas [74]. TNF-α may also play a role in insulin resistance, and elevated levels of this cytokine have been found in patients with metabolic syndrome [36]. Patients with OSA have higher levels of TNF-α compared to nonapneic controls, an association that remains robust even after controlling for BMI [75–77]. Furthermore, use of CPAP has been shown to reduce TNF-α levels in patients with OSA [77,78].

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The evidence regarding the role of TNF-α in the pathophysiology of daytime neurocognitive impairment symptoms associated with OSA is mixed. One study involving adults with OSA complicated by excessive daytime sleepiness showed that administration of the TNF-α antagonist etanercept not only markedly decreased daytime sleepiness symptoms but also led to a decrease in AHI [79], suggesting not only that TNF-α may mediate daytime sleepiness, but also that the relationship between OSA and inflammation may be bidirectional. Another group demonstrated no significant difference in TNF-α levels between sleepy and nonsleepy patients with OSA, suggesting that if TNF-α is a factor in producing sleepiness, it is only one piece of a larger puzzle [80].

Leukotriene E4 Leukotrienes are inflammatory mediators that are derived from the 5-lipoxygenase pathway of arachidonic acid metabolism. Leukotriene E4 (LTE4) has vasoactive properties, leading to smooth muscle cell contraction and proliferation, vascular permeability, and edema, and increasing the expression of adhesion molecules and monocyte activation [81]. These actions likely mediate the association that has been documented between LTE4 and several pro-inflammatory cardiovascular conditions, including acute coronary syndromes [82], coronary artery disease [83], and diabetes [84]. The excretion of urinary LTE4 is considered a reliable biomarker for systemic production of pro-inflammatory leukotrienes [85]. A recent investigation found that urinary LTE4 excretion was elevated in nonobese patients with OSA compared to BMI-matched nonapneic control patients [86]. Overweight and obese patients with OSA had higher LTE4 excretion compared with nonobese OSA patients, suggesting that OSA and obesity have additive effects on this inflammatory marker. Interestingly, in the same study, CPAP reduced urinary LTE4 excretion only in the nonobese OSA patients, while obese patients experienced no change.

NF-kB and Chronic Intermittent Hypoxia NF-kB is a cytosolic protein that, under unstimulated conditions, exists in an inactive form, bound to an inhibitor, IkB. After exposure to stimulating signals—including infection, ROS, and intermittent hypoxia—NF-kB splits from its inhibitor and moves to the cell nucleus, where it functions as a transcription factor for numerous constituents of the inflammatory cascade, including cytokines (such as TNF-α, IL-6, and IL-8), adhesion molecules (VCAM, intercellular adhesion molecule (ICAM), E-selectin), and coagulation factors (factor VIII, tissue factor). NF-kB is considered to function as a sort of “master switch,” involved in the transcription of numerous pro-inflammatory genes, and is implicated in the pathogenesis of numerous inflammation-dependent diseases, including myocarditis, dilated cardiomyopathy, ischemic heart disease with reperfusion injury, and atherosclerosis [87]. The hypoxia associated with OSA differs from that seen in other primary pulmonary diseases—which typically cause sustained hypoxia—in that OSA produces a state of chronic intermittent hypoxia (CIH). CIH appears to affect cellular machinery

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differently than chronic sustained hypoxia does, in a manner enhancing the proinflammatory state. In an in vitro model, Ryan et al. [88] showed that CIH, but not sustained hypoxia, produced a selective activation of NF-kB with the downstream elaboration of TNF-α. This finding was corroborated by Greenberg et al. [89] in vivo, showing that CIH in a mouse model led to increased NF-kB activation, with greatest activation in vascular smooth muscle and endothelial cells. In the same paper, they showed that OSA in humans was associated with elevated NF-kB levels in monocytes, a finding that resolved following treatment with CPAP. Whereas CIH tends to lead to inflammatory elaboration, chronic hypoxia tends to lead to an adaptive cellular response. Stabilization of hypoxia-induced factor-1 (HIF-1) occurs in a cell under chronic hypoxic conditions, leading to increased synthesis of factors that result in improved tissue oxygenation, blood flow, and glycolytic activity, such as erythropoietin, nitric oxide synthase, and vascular endothelial growth factor (VEGF) [88,90].

Interleukin-6 Interleukin-6 (IL-6), a pro-inflammatory protein produced by T cells, macrophages, and adipose tissue, is understood to play a role in atherogenesis [74,91,92] and is predictive of future cardiovascular morbidity [93] and mortality [94]. Multiple investigators have found OSA patients to have higher IL-6 levels compared with nonapneic patients [80,95,96], and treatment of moderate to severe OSA with CPAP leads to reduction in measured IL-6 [96]. However, the elevation in IL-6 may simply be a factor of OSA that co-aggregates with obesity. One large study evaluated 385 adults with polysomnography, followed by analysis of IL-6 and soluble IL-6 receptor on awakening. In this study, after adjustment for age and body habitus, IL-6 did not have a significant correlation with the presence of sleep-disordered breathing. In the same study, the authors found that soluble IL-6 receptor did correlate with the severity of sleep-disordered breathing, in a manner independent of the patient’s body habitus. Normally—that is, in the nondiseased state—the elaboration of IL-6 appears to be heavily sleep dependent, with increased signaling during sleep compared with the waking state [97,98]. One study using intranasally delivered IL-6 in humans demonstrated an increase in sleep efficiency and slow-wave activity among subjects receiving IL-6 compared with controls, a factor suggesting that this cytokine may play some role in the severity of sleepiness in OSA [99]. Another study showed IL-6 levels increased with total sleep deprivation, suggesting a potential role of IL-6 in the mechanism of homeostatic sleep pressure [100]. However, another study that compared IL-6 levels in sleepy and nonsleepy patients with OSA showed no difference between the two groups [80].

C-Reactive Protein C-reactive protein (CRP) is a nonspecific marker for inflammation that is stimulated by IL-6 and has proven to be a reliable marker for future risk of atherosclerosis and

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coronary artery disease [101–104]. It appears to be directly involved in the atherogenesis process via the induction of adhesion molecules ICAM-1, VCAM-1, and E-selectin on endothelial cells [105]. The relationship between CRP and OSA is complicated by the fact that CRP is also correlated with BMI. Several studies have shown that OSA in adults is associated with increased CRP, even after controlling for BMI [106–108], a finding that has also been noted in children [109]. Gozal et al. [110] recently found that children with OSA had higher levels of CRP compared with controls, and furthermore that CRP was highest among patients with OSA complicated by daytime impairment symptoms. However, a recent large study cast doubt on this association, finding instead that CRP was linked to BMI but not OSA. In this study, nonapneic patients were compared with BMI-matched patients with OSA, and with obese OSA patients. CRP was found to be elevated significantly only in the obese OSA group [111]. Whether or not CPAP influences CRP levels in OSA is also the subject of some controversy. Some studies have shown that CPAP reduces CRP levels in patients with OSA [96,112], but others have shown no effect [111,113]. The apparent discrepancy could be explained by a synergistic effect between obesity and OSA in the production of CRP. There are several mechanisms likely to be responsible for the elevation in CRP in patients with OSA. Intermittent hypoxia with reoxygenation—resulting in increased expression of NF-kB and IL-6 (see earlier)—appears to be the mechanism most specific for OSA, though, as mentioned earlier, CRP is also elevated in obesity due to production by adipocytes [114]. CRP is also produced as a result of nonspecific stressors, including sleep deprivation [115].

Prostaglandin D2, Cyclo-Oxygenase 2, and Sleepiness As mentioned previously, the issue of why some patients with OSA develop daytime sleepiness while others don’t is an intriguing and as yet incompletely answered question. Some evidence suggests that the process may be mediated in part by inflammation. In the early 1980s, Ueno et al. were interested in the biologic function of PGD2, which had recently been discovered to be heavily concentrated in the central nervous system tissue of rats, monkeys, and humans. In a series of trials, they found that an infusion of even small doses—as little as one femtomole (1015 mol)—of PGD2 into the third ventricle in a rat model could induce excess sleep. This finding was dose-dependent and was specific for PGD2 [116,117]. PGD2 is produced from prostaglandin H2 (PGH2), which itself is produced from arachidonic acid in steps under the control of cyclo-oxygenase 2 (COX-2), a factor that may explain why infusion of diclofenac sodium—a nonspecific inhibitor of COX-2 as well as its sister enzyme cyclo-oxygenase 1 (COX-1)—produced decreases in sleep in a similar rat model [118]. Moreover, Li et al. showed in a rat model that intermittent hypoxia was capable of inducing COX-2 and appeared to function as a mediator for neurobehavioral abnormalities resulting from this stressor [119]. Furthermore, Jelic et al. recently demonstrated a similar trend in harvested venous tissue in humans, showing that patients with OSA had a fivefold increase in COX-2 levels compared with

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nonapneic controls, a discrepancy that was found to normalize upon treatment with CPAP [120]. Lipocalin-type PGD synthase (L-PGDS) is the enzyme directly responsible for producing PGD2 in the human brain and is a protein abundantly found in the cerebrospinal fluid [121]. This enzyme, also known as β-trace, has been found to exhibit circadian variation, with a peak in the evening, consistent with the hypothesis that PGD2 operates in a homeostatic manner for sleep regulation [122]. Recently, Barcelo et al. [123] found higher levels of L-PGDS among OSA patients with excessive daytime sleepiness, compared with nonsleepy OSA patients [123]. Taken together, these data imply that PGD2, under the control of COX-2, may function as a homeostatic regulator of the sleep drive, with excessive sleepiness in OSA possibly driven by this substance as well.

Leptin, Ghrelin, and OSA Adipose tissue—in particular, visceral fat deposits—produces a collection of cytokines, some of which have pro-inflammatory effects. One of the most important of these is leptin, a hormone that helps to regulate appetite as well as energy expenditure. While leptin does not have inflammatory activity per se, it tends to correlate with multiple other indices of inflammation, including BMI, insulin levels, and TNF-α [124]. Recently Wolk et al. [125] found that human endothelial progenitor cells are equipped with leptin receptors, implying a potential mechanism for vascular pathophysiology associated with hyperleptinemic conditions. Leptin was discovered serendipitously, following a spontaneous mutation in a laboratory mouse (termed “ob/ob”), leading to a strain of massively obese offspring [126]. Later work illuminated the many physiologic functions of leptin. Physiologically, as leptin levels rise, appetite is suppressed. Ob/ob mice given supplemental leptin decreased their food intake and lost weight, demonstrating leptin’s role in appetite control. O’Donnell et al. [127] showed that leptin-deficient mice have a blunted hypercapneic ventilator response as well, suggesting that leptin may play a role in the development of hypoventilatory syndromes such as obesity hypoventilation syndrome (OHS) and OSA (Figure 17.3). Obese patients—particularly those with the metabolic syndrome—tend to have chronically elevated leptin levels, leading to speculation that the metabolic syndrome is essentially a “leptin-resistant” state [128,129]. Several studies have shown that patients with OSA have higher leptin levels compared with BMI-matched controls [129–131], but other studies have found that the elevation in leptin among obese OSA patients is largely due to the obesity [132]. Effective CPAP has been shown to decrease leptin in obese OSA patients independent of any change in BMI in a few studies [131,133], while another group showed that CPAP decreased leptin levels in nonobese OSA patients only, with the obese patients having no significant improvement [132]. Further research is needed to clarify the role played by leptin in the pathophysiology of OSA. Ghrelin, a peptide produced primarily by cells in the fundus of the stomach, is now understood to be one of the more important regulators of appetite in a manner that tends to oppose the actions of leptin [134]. In normal physiology, ghrelin rises

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ob/ob

Wild type

200

Aw ak e

Minute ventilation (ml/min)

300

ake Aw

100

EM

NR

NREM

REM

REM

0 0

3

5

Inspired CO2 (%)

8

0

3

5

8

Inspired CO2 (%)

Figure 17.3  The relationship between minute ventilation and inspired CO2 is blunted across all sleep/wake states in the ob/ob (leptin-deficient) mouse, compared with the wild type. Source: O’Donnell et al. Am J. Resp. Crit. Care Med. 152, 725–31.

prior to a meal and decreases immediately afterwards. Obesity has been associated with decreased ghrelin levels, which rise dramatically and chronically after weight loss, thus stimulating a chronic increase in appetite and frustrating long-term efforts to keep weight off [135]. Harsch et al. [136] found ghrelin levels to be elevated in patients with OSA, with levels dropping after 2 days on effective CPAP treatment, suggesting a mechanism to explain the profound difficulty that patients with untreated OSA have in losing weight (see Figure 17.1).

Summary Obstructive Sleep Apnea (OSA) is a syndrome of cyclic upper airway collapse during sleep, leading to intermittent hypoxia and sleep fragmentation. OSA leads to daytime neurocognitive impairment symptoms and is a risk factor for the development of cardiovascular disease including hypertension, stroke, heart failure, and arrhythmias. In addition, OSA may be independently linked with proinflammatory conditions including asthma and the metabolic syndrome. The mechanisms underlying the daytime impairment of OSA include increased homeostatic sleep pressure resulting from chronic partial sleep deprivation, as well as increased systemic inflammation

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induced by chronic intermittent hypoxia. The proposed mechanisms underlying the increased cardiovascular risk seen with OSA include activation of the sympathetic nervous system, physical vibratory trauma (from snoring) to structures of the head and neck, elaboration of oxidative stress and reactive oxygen species, and a systemic proinflammatory response. Systemic markers of inflammation associated with OSA include tumor necrosis factor a, leukotriene E4, C-reactive protein, interleukin 6, nuclear factor kB, and prostaglandin D2. Factors which influence an individual’s susceptibility to the development of an inflammatory response to OSA may include genetics (Apolipoprotein E deficiency) and the presence of obesity, which is, itself, a proinflammatory state. The adipokine leptin and its counterregulatory hormone, ghrelin, are improperly regulated in OSA, and likely also play a role in the development of a systemic proinflammatory state.

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[108] Saletu M, Nosiska D, Kapfhammer G, Lalouschek W, Saletu B, Benesch T, et al. Structural and serum surrogate markers of cerebrovascular disease in obstructive sleep apnea (OSA): association of mild OSA with early atherosclerosis. J Neurol 2006;253:746–52. [109] Larkin EK, Rosen CL, Kirchner HL, Storfer-Isser A, Emancipator JL, Johnson NL, et al. Variation of C-reactive protein levels in adolescents: association with sleep-disordered breathing and sleep duration. Circulation 2005;111:1978–84. [110] Gozal D, Crabtree VM, Sans Capdevila O, Witcher LA, Kheirandish-Gozal L. C-reactive protein, obstructive sleep apnea, and cognitive dysfunction in school-aged children. Am J Respir Crit Care Med 2007;176:188–93. [111] Ryan S, Nolan GM, Hannigan E, Cunningham S, Taylor C, McNicholas WT. Cardiovascular risk markers in obstructive sleep apnoea syndrome and correlation with obesity. Thorax 2007;62:509–14. [112] Patruno V, Aiolfi S, Costantino G, Murgia R, Selmi C, Malliani A, et  al. Fixed and autoadjusting continuous positive airway pressure treatments are not similar in reducing cardiovascular risk factors in patients with obstructive sleep apnea. Chest 2007;131:1393–9. [113] Barcelo A, Barbe F, Llompart E, Mayoralas LR, Ladaria A, Bosch M, et al. Effects of obesity on C-reactive protein level and metabolic disturbances in male patients with obstructive sleep apnea. Am J Med 2004;117:118–21. [114] Alam I, Lewis K, Stephens JW, Baxter JN. Obesity, metabolic syndrome and sleep apnoea: all pro-inflammatory states. Obes Rev 2007;8:119–27. [115] Meier-Ewert HK, Ridker PM, Rifai N, Regan MM, Price NJ, Dinges DF, et al. Effect of sleep loss on C-reactive protein, an inflammatory marker of cardiovascular risk. J Am Coll Cardiol 2004;43:678–83. [116] Ueno R, Ishikawa Y, Nakayama T, Hayaishi O. Prostaglandin D2 induces sleep when microinjected into the preoptic area of conscious rats. Biochem Biophys Res Commun 1982;109:576–82. [117] Ueno R, Honda K, Inoue S, Hayaishi O. Prostaglandin D2, a cerebral sleep-inducing substance in rats. Proc Natl Acad Sci USA 1983;80:1735–7. [118] Hayaishi O. Prostaglandin D2 and sleep. Ann N Y Acad Sci 1989;559:374–81. [119] Li RC, Row BW, Gozal E, Kheirandish L, Fan Q, Brittian KR, et al. Cyclooxygenase 2 and intermittent hypoxia-induced spatial deficits in the rat. Am J Respir Crit Care Med 2003;168:469–75. [120] Jelic S, Padeletti M, Kawut SM, Higgins C, Canfield SM, Onat D, et al. Inflammation, oxidative stress, and repair capacity of the vascular endothelium in obstructive sleep apnea. Circulation 2008;117:2270–8. [121] Urade Y, Hayaishi O. Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase. Biochim Biophys Acta 2000;1482:259–71. [122] Jordan W, Tumani H, Cohrs S, Eggert S, Rodenbeck A, Brunner E, et al. Prostaglandin D synthase (beta-trace) in healthy human sleep. Sleep 2004;27:867–74. [123] Barcelo A, de la Pena M, Barbe F, Pierola J, Bosch M, Agusti AG. Prostaglandin D synthase (beta trace) levels in sleep apnea patients with and without sleepiness. Sleep Med 2007;8:509–11. [124] Mantzoros CS, Moschos S, Avramopoulos I, Kaklamani V, Liolios A, Doulgerakis DE, et al. Leptin concentrations in relation to body mass index and the tumor necrosis factor-alpha system in humans. J Clin Endocrinol Metab 1997;82:3408–13. [125] Wolk R, Deb A, Caplice NM, Somers VK. Leptin receptor and functional effects of leptin in human endothelial progenitor cells. Atherosclerosis 2005;183:131–9.

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18 The Role of Neuroinflammation in Parkinson’s Disease Xi Chen, Malú G. Tansey Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA

Introduction to Parkinson’s Disease Overview Parkinson’s disease (PD) is the second most common neurodegenerative disorder, after Alzheimer’s disease (AD). It is characterized by progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) that results in a drastic depletion of DA in the striatum, to which these neurons project (nigrostriatal pathway), yet to a lesser extent other dopaminergic neurons are also affected. Another pathologic feature of PD is the presence of intraneuronal proteinaceous inclusions called Lewy bodies, which are composed of α-synuclein in association with other proteins such as ubiquitin and neurofilament proteins [1]. PD prevalence is age-associated, with an average age at onset of 55; approximately 1% of 65-year-olds are affected, and this increases to 4–5% in 85-year-olds [2]. The primary symptoms of PD are resting tremor, rigidity, slowness of movement, and postural instability. Other symptoms may include depression and other emotional changes; difficulty in swallowing, chewing, and speaking; urinary problems or constipation; skin problems; and sleep disruptions. Currently, no proven treatment can halt or even slow the progression of this disease. Although DA supplementation is currently used for PD as a means to replace the diminished tone of dopaminergic output from SNpc, it only alleviates the symptomatic motor dysfunction, and its effectiveness declines as the disease progresses [3].

Etiology PD is a neurodegenerative disorder with a complex, multifactorial etiology. In recent years, there has been increasing evidence to support a role for genetic factors in its cause. This has come from twin and family studies, the mapping and cloning of genes that are associated with the development of PD, and analysis of potential susceptibility genes. There is also evidence indicating that environmental factors may play a role in the disease process. It is likely that for most cases, there is a complex interplay between these genetic and environmental influences in the causation of PD [4,5]. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00018-6 © 2011 Elsevier Inc. All rights reserved.

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Epidemiologic studies and pathologic analyses demonstrate that sporadic PD accounts for about 95% of total PD cases [4,6,7], but familial forms of the disease linked to mutations in a restricted number of genes account for 4%, and these patients develop early-onset disease before age 50 [8,9]. For non-familial forms of PD, the prevailing view is that multiple causes such as genetic predispositions, environmental toxins, and aging are important factors in disease initiation and progression [10]. The finding that the single greatest risk is age implicates cumulative central nervous system (CNS) damage as a causative mechanism (reviewed in [7]). At the cellular level, three types of cellular dysfunction may be important in the pathogenesis of PD: reactive oxidative and nitrosative stress, mitochondrial respiratory defects, and abnormal protein aggregation [4,11]. Inhibition of complex I of the mitochondrial electron transport chain is selectively dysfunctional in DA neurons in sporadic PD [12]. DA neurons are particularly sensitive to the toxic effects of mitochondrial complex I toxins, such as rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [4,11]. Within the midbrain, the substantia nigra appears to be among the most vulnerable regions because of DA metabolism, which produces highly reactive species that oxidize lipids and other compounds, increase oxidative stress, and impair mitochondrial function [13]. Genetics has generated tremendous excitement and new energy in PD research, but only about 2–3% of the late-onset cases and ~50% of early-onset cases are due to genetic causes [5]. Currently, there are two autosomal dominantly inherited mutations that cause PD. Mutations in α-synuclein generate one form of autosomal dominant PD, and mutations in LRRK2 are the most frequent genetic cause of autosomal dominant PD [14]. α-Synuclein was the first gene linked to familial PD. The identification of mutations in α-synuclein catalyzed the identification of several other PD-linked genes and fueled additional genetic research in PD. α-synuclein is the major structural moiety of Lewy bodies and Lewy neurites [15], two key pathologic hallmarks of PD, illustrating a potential mechanistic relationship between familial and sporadic PD. α-synuclein is a protein that is expressed throughout the brain and has been implicated in learning, synaptic plasticity, vesicle dynamics, and DA synthesis [16]. Normally α-synuclein exists in an unstructured state. Three point mutations in α-synuclein (A53T, A30P, and E46K) increase its propensity for misfolding, and cause familial PD [17,18]. The toxicity of aggregated α-synuclein seems to occur through mitochondrial dysfunction and proteasomal and lysosomal impairment and disruption of ER-Golgi trafficking, which initiate feed-forward mechanisms that lead to further injury [19–22]. A number of α-synuclein transgenic mice (WT, A53T, A30P) have been reported using a variety of promoters, but none of these models accurately represent PD, in that there is no progressive loss of DA neurons. Several mutations in LRRK2 cause autosomal dominant PD. Leucine-rich repeat kinase 2 (LRRK2) is a large protein with multi-domains that is localized to membranous structures [23]. Mutations that segregate with PD are concentrated in the GTPase and kinase domains [24]. LRRK2 may play a role in neuronal outgrowth and guidance [25,26], but its normal physiologic function, phospho-substrates, binding partners, and regulators of kinase and GTPase activity have yet to be confirmed or clarified [14].

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Most of the current LRRK2 transgenic mice have abnormalities in the nigrostriatal system, such as stimulated DA neurotransmission or behavioral deficits, which are DA-responsive, but they do not exhibit more substantial pathology such as neurodegeneration of DA neurons [27,28]. LRRK2 and α-synuclein are likely to share common pathogenic mechanisms, as overexpression of LRRK2 greatly enhanced and knockout of LRRK2 reduced the neuropathologic abnormalities in A53T α-synuclein transgenic mice [29]. There are currently four autosomal recessive mutations that cause PD. Homozygous and compound heterozygous mutations in the genes that encode parkin, PTENinduced kinase 1 (PINK1), oncogene DJ, and ATP13A2 have been identified as four recessive forms of parkinsonism [14]. Mutations in the parkin gene were originally identified as the genetic cause of autosomal recessive juvenile parkinsonism in Japanese families [30,31]. More than 100 mutations in parkin have been reported, accounting for 50% of familial PD cases and at least 20% of young-onset sporadic PD cases [31]. Parkin has E3-ubiquitin ligase activity and can conjugate ubiquitin to proteins, thereby targeting them for degradation by the proteasome [32,33], strengthening the hypothesis that proteasome dysfunction and resulting protein aggregation are pivotal to PD pathogenesis [34]. Several parkin knockout mouse models have been reported, but none of them have any substantial dopaminergic or behavioral abnormalities [35–38]. Some of the parkin knockout mice have subtle abnormalities in either the DA nigrostriatal circuit or the locus coeruleus noradrenergic system [35,38]. Mutations in PINK1, the second most common autosomal recessive mutation following parkin, contribute to almost 1–7% of early-onset PD cases [17]. Structurally, PINK1 contains a conserved serine/threonine kinase domain with an N-terminal mitochondrial targeting motif [39]. PINK1 is localized to the mitochondrial intermembrane space and membranes of the mitochondria [40]. Similar to parkin knockout mice, PINK1 knockout mice did not exhibit any major abnormality [41–43]. Specifically, the number of DA neurons, the level of striatal DA, and the level of DA receptors are unchanged. Like the parkin knockout mice, PINK1 knockout mice display deficits in nigrostriatal DA neurotransmission [43]. Both parkin and PINK1 knockouts also have mild mitochondrial defects [41,44]. Mutations in DJ-1 are a rare cause of PD [45]. Overall, less than 1% of earlyonset parkinsonism is due to DJ-1 recessively inherited deletions and missense mutations [46]. DJ-1 is a member of the ThiJ/PfpI family of molecular chaperones, which are induced during oxidative stress, and the protein primarily exists as a dimer that is localized to mitochondria [47,48]. DJ1 is a redox-sensitive molecular chaperone with a variety of diverse functions [49–51]. In cellular models, it regulates redoxdependent kinase signaling pathways and acts as a regulator of antioxidant gene expression (reviewed in [50]). DJ-1 functions in vivo as an atypical peroxiredoxinlike peroxidase, where it protects against oxidative stress in mitochondria [52]. DJ-1 also functions as a redox-sensitive RNA-binding protein [53]. Consistent with its role as a chaperone, DJ-1 is thought to have a variety of other pleiotropic functions (reviewed in [49–51]). Similar to parkin and PINK1 knockout mice, DJ-1 knockout mice do not exhibit any major abnormality. Abnormalities in DA neurotransmission

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in the nigrostriatal circuit and mitochondrial dysfunction were reported in DJ-1 knockout mice just like for parkin and PINK1 knockout mice [35,52,54,55]. Loss-of-function mutations in a predominantly neuronal P-type ATPase gene, ATP13A2, are another rare cause of early-onset parkinsonism with pyramidal degeneration and dementia [56]. The wild-type protein is believed to localize to the lysosomes, but the unstable truncated ATP13A2 mutants are retained in the endoplasmic reticulum and degraded by the proteasome. To date, no animal models have been reported for ATP13A2.

Pathogenesis of PD No matter what insult initially provokes neurodegeneration, studies of toxic PD models and the functions of genes implicated in inherited forms of PD suggest that misfolding/aggregation of proteins and dysfunction of the ubiquitin–proteasome pathway, mitochondrial dysfunction, and the consequent oxidative stress are the main culprits that elicit degeneration of SNpc dopaminergic neurons [4]. Although the key molecular and cellular events underlying development of PD are divergent, these events may share a feature by converging on neuroinflammatory pathways that over time trigger neurodegeneration (reviewed in [57]).

Neuroinflammation in the Pathogenesis of PD Hallmarks of Neuroinflammation The inflammatory reaction in the brain associated with most acute or chronic neurodegenerative diseases is often termed “neuroinflammation.” Neuropathologic and neuroradiologic studies indicate that neuroinflammation is characterized by a glial response that in certain diseases may be present prior to significant loss of neurons. It was once believed that the blood–brain barrier (BBB) prevented access of immune cells to the brain and, as a result, the immune system and the CNS were relatively independent of each other. However, it has become clear that the permeability of the BBB can be regulated under normal conditions and may increase or become dysregulated in disease states. The brain is fully capable of mounting an inflammatory response. Neuroinflammation consists mainly of an innate immune response involving activation of glial cells and central production of cytokines, chemokines, prostaglandins, complement cascade proteins, and reactive oxygen species and reactive nitrogen species (ROS/RNS) in response to a central or peripheral immune challenge. The features of neuroinflammation include the presence of reactive astrocytes and activated microglial cells in the CNS. In certain neurodegenerative diseases, infiltration of lymphocytes (B and T cells) occurs when there is extensive breakdown of BBB (reviewed in [7]). Neuroinflammation has been appropriately described as a doubleedged sword [58,59]. In acute situations and when short-lived, inflammatory mechanisms limit injury and promote healing; however, when chronically sustained at high levels, neuroinflammation has the capacity to seriously damage viable host tissue.

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Cell Types Involved in Neuroinflammation Microglia, the monocyte-derived resident macrophages of the brain, are the primary effectors of immune surveillance. Microglia play a homeostatic role in CNS and respond to environmental stresses and immunologic challenges by scavenging excess neurotoxins and removing dying cells and cellular debris [60–62]. An initial physical or pathogenic event in the CNS is expected to elicit activation of microglia and secretion of neurotrophic factors such as glial-derived neurotrophic factor family ligands (GDNF or GFLs) that promote neuronal survival, with the purpose of limiting injury, protecting vulnerable neuronal populations, and aiding in repair processes. Activated microglia can also produce ROS and RNS such as nitric oxide (NO) and secrete prostaglandins, chemokines, and cytokines, which can have a deleterious effect on neuronal survival by enhancing oxidative stress and activating cell-death pathways [57]. Astrocytes, another type of glial cell in the CNS, regulate the permeability of the BBB [59]. Specifically, by making intimate contact with other cell types, including neurons, oligodendrocytes, and other astrocytes, the cellular processes of astrocytes contribute to functional coupling between cell types. This tight coupling enables astrocytes to regulate the homeostatic environment (including regulation of the excitatory neurotransmitter glutamate) to ensure proper functioning of the neuronal network. Disruption of this process transiently or permanently may occur during periods of chronic neuroinflammation. The term “reactive gliosis” refers to the upregulation of glial fibrillary acidic protein (GFAP) in astrocytes and may be part of a response to acute injury or neurodegeneration that can lead to permanent scarring and tissue damage (reviewed in [63]). Recently, a study demonstrated that astrocytes can amplify the inflammatory responses of activated microglia, suggesting astrocytes also play a critical role in neuroinflammation [64]. Although more controversial, it is now believed that peripheral immune cells are also involved in neuroinflammation. Several studies implicated that subsets of T cells play a part in neurodegeneration. Specifically, MPTP exposure results in T-lymphocyte infiltration into the brain [65], and extensive and selective nigral infiltration of CD8 T-cytotoxic and CD4 T-helper cells after MPTP injection has also been demonstrated [66]. The mechanisms involved in such cell-specific and regionspecific T-cell recruitment are still unknown but might involve early microglial cell activation and innate neuroinflammatory processes that could modify the local microenvironment.

Triggers of Neuroinflammation Neuroinflammation may be triggered by protein aggregation and formation of inclusions arising from mutations (e.g., α-synuclein) or disruption of the ubiquitin– proteasome system, immunologic challenges (bacterial or viral infections), neuronal injury (brain trauma or stroke), or other epigenetic factors. Chronic inflammatory syndromes (rheumatoid arthritis, Crohn’s disease, and multiple sclerosis) and environmental toxins (pesticides and particulate matter) are the most notable epigenetic factors likely to trigger neuroinflammation. These insults can increase the permeability

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of the BBB to allow infiltration of lymphocytes and macrophages into the brain parenchyma. The immune reactions initiated by viruses and bacteria may contribute to latent vulnerabilities, which could become manifest with future immunologic challenges. Depending on context, duration, and type of inflammatory response, inflammation may be detrimental or beneficial to the individual. During trauma to the CNS, acute inflammatory responses are mounted to limit injury and to aid in neuronal repair. The downgraded response of resident microglia is an evolved response that permits the CNS to respond to injury in a way that will result in minimal brain damage [7].

Evidence of Neuroinflammation in PD Data from postmortem studies provided the first evidence for neuroinflammatory processes in PD. In 1988, McGeer et al. [67] reported the presence of activated microglial cells within the substantia nigra of patients with PD at postmortem. These cells were identified by their immunoreactivity to human leukocyte antigen DR (HLA-DR), a cell-surface receptor belonging to the MHC class II [67]. This key finding was confirmed by other investigators using additional markers, such as HLA-DP, HLA-DQ, HLA-DR (CR3/43), CD68, and ferritin [68,69]. The astrocytic reaction is another well-known neuropathologic characteristic of the substantia nigra in PD. Gliofibrillary acidic protein (GFAP) and glutathione peroxidase are used as astrocytic markers. By quantitative analysis, a 30% increase in the density of astroglial cells in the substantia nigra of patients at postmortem was detected [70]. Lymphocytes might also participate in the inflammatory reaction in the brains of PD patients. Infiltration of CD8 and CD4 T cells but not B cells into the brain was also detected in postmortem human PD specimens, indicating T-cell–mediated dopaminergic toxicity in PD pathogenesis [66]. The presence of neuroinflammatory processes at postmortem has also been confirmed on a molecular basis. Mogi et al. [71–76] reported an increase in concentrations of tumor necrosis factor (TNF), β2-microglobulin, epidermal growth factor (EGF), transforming growth factor α (TGFα), TGFβ1, and interleukins (IL) 1β, 6, and 2 in the striatum of PD patients. TNF and its receptors, IL-1β, and interferon (IFN) γ were also detected in the substantia nigra of patients [77,78]. Studies of biologic fluids (serum or cerebrospinal fluid) also support a role for neuroinflammatory processes in PD. Expression of IL-2 [79], TNF, and IL-6 [80] is increased in the serum of patients with this neurodegenerative disease. Proinflammatory changes have also been reported in the cerebrospinal fluid of PD patients. TNF [76], IL-1β [81], and IL-6 [81,82] are found in samples from patients. Both the cellular and molecular changes seen in the brains of PD patients have been reproduced in several animal models. Demonstration that the blockade of these changes protects against neuronal loss in animals has provided strong evidence to suggest that neuroinflammatory processes are involved in the death of dopaminergic neurons. A glial reaction involving astrocytes and microglial cells and lymphocytic infiltration has been described in several animal models of PD. Such models include

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intracerebral 6-hydroxydopamine injection and peripheral injection of complex 1 inhibitors such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), rotenone, and annonacin. Activated microglial cells have been identified in the brains of rats with unilateral lesions of the nigrostriatal pathway caused by both intrastriatal and medial forebrain bundle injection of 6-hydroxydopamine [83–85]. Injection of lipopolysaccharide (LPS), a gram-negative bacteriotoxin that activates microglial cells, can selectively kill dopaminergic neurons in animals after intranigral injection or systemic injection. Ling et al. [86] reported that injection of LPS into gravid female rats caused their offspring to have fewer dopaminergic neurons as adults, a high proportion of which were abnormal, and higher TNF concentrations in the striatum compared to controls. Microglial activation has also been reported in mice that overexpress human α-synuclein [87], and nigral LPS injections exacerbate wild-type human and A53T mutant α-synuclein–mediated degeneration [87,88]. Therefore, microglial activation is a common feature of animal models in which parkinsonism is induced by neurotoxins or manipulations of genes involved in inherited forms of the disease. Moreover, chronic peripheral inflammation caused by repeated peripheral exposure to LPS increased vulnerability to inflammation-induced degeneration in mice lacking Parkin or Rgs10 (regulator of G-protein signaling 10), whereas the genetic deficiency was insufficient to induce nigral degeneration [89,90]. Therefore, multiple-hit models involving exposure to two different neurotoxins as well as models in which a genetic deficiency is combined with a neurotoxic or endotoxic insult have also given researchers an opportunity to study the interplay between genetics and environment in PD-like pathogenesis. Imaging studies have also enabled clinical researchers to investigate the extent to which neuroinflammation is present in PD patients. Positron emission tomography (PET) scan analysis with the marker PK-11195 (a ligand of the peripheral binding site of benzodiazepine indicative of microglial activation) indicates the presence of neuroinflammatory processes in PD [91,92]. Although not diagnostic of PD, binding of PK-11195 is increased in the pons, basal ganglia, and frontal and temporal cortical regions of patients with PD compared to age-matched controls, suggesting the presence of activated microglia in areas involved in the disease. Several genetic studies have analyzed the relationship between a given genetic polymorphism in neuroinflammation-associated genes and the risk of PD. The strongest genetic evidence implicating TNF in the initiation and progression of PD is that of a single nucleotide polymorphism (1031 C) or SNP in the TNF promoter that drives transcriptional activity and results in higher-than-normal TNF production. This SNP is overrepresented in a cohort of Japanese patients with early-onset PD relative to its frequency in patients with late-onset PD and unaffected controls [93]. Importantly, this 1031 C polymorphism has been independently associated with PD risk in an additional study [94]. A second polymorphism in the TNF gene promoter (-308 G/A) that results in elevated serum TNF levels has also been found to be overrepresented in early-onset sporadic PD [95,96]. Lastly, TNFR1 polymorphisms TNFR1-609 and TNFRI36 have been found to be significantly decreased in PD patients [95]; similarly, studies of SNPs in the IL-1β and IL-1α family also support the role of neuroinflammation in PD (reviewed in [1]).

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Epidemiologic studies provide the most convincing and compelling evidence to support the claim that inflammatory mechanisms are likely to contribute to PD [97–100]. Specifically, a large prospective study of hospital workers indicated that the incidence of idiopathic PD in chronic users of over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs), which scavenge free oxygen radicals and inhibit cyclo-oxygenase (COX) activity, was 46% lower than that of age-matched non-users [98]. Similar findings were reported for chronic users of the nonselective COX inhibitor ibuprofen in a follow-up study involving a large (~180,000) cohort of US men and women [97]. Inhibition of COX-mediated DA oxidation [101] and inhibition of microglial-derived toxic mediator production are likely to be among the mechanisms that contribute to the decreased incidence of PD in chronic NSAID users. Not all studies support the protective effects of aspirin or other NSAIDs on DA neurons in animal models of PD; epidemiologic data exploring the effectiveness of NSAIDs in the prevention of PD have been reviewed recently [102]. These findings are consistent with numerous studies demonstrating that certain neuron–glia interactions can lead to neuronal death [103–108]. Importantly, the PD risk-lowering effects of NSAIDs implicate neuroinflammatory processes in DA neuron loss and development of PD in humans. Although the protective effects of NSAIDs are likely to be primarily mediated by COX inhibition, multiple mechanisms, including the Rho kinase pathway [109,110], may also be involved in mediating the beneficial effects of NSAIDs. Together, these findings raise the possibility that early intervention with NSAIDs or similar anti-inflammatory therapy may be neuroprotective and could delay or prevent the onset of PD.

Molecular Pathways Involved in Neuroinflammation of PD Innate immune response associated with gliosis, in particular microglial cell activation, is an important neuropathologic feature of PD. After appropriate activation, microglial cells are capable of antimicrobial activity as well as cell toxicity through the production and release of toxic oxygen-derived and nitrogen-derived products, which are generated in a process known as the respiratory or oxidative burst. This toxic mechanism for phagocytic cells relies on the regulated induction of several enzymatic systems, among which NADPH oxidase, inducible nitric oxide synthase (iNOS), and myeloperoxidase (MPO) bring about the production of toxic amounts of superoxide (O2) and nitric oxide (NO) free radicals, and hypochlorous acid (HOCl, derived from hydrogen peroxide and chloride anion), respectively [1]. The expression of these biocatalytic systems within the substantia nigra is substantially increased in both patients with PD at postmortem and animal models of the disease [111–114]. Under pathologic conditions, microglial cells become activated and express iNOS, leading to the production and release of NO free radicals. These cells also have upregulated expression and activation of NADPH oxidase, leading to the production of high amounts of O2 free radicals. In turn, O2 and NO free radicals might react to generate the highly reactive peroxynitrite ONOO, which can cause oxidative

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damage to several proteins within neuronal cells, such as tyrosine hydroxylase [115] and α-synuclein [116]. Nonreactive nitrites (NO2), the main end product of the free radical NO and the concentration of which is increased in parkinsonism [117], can be oxidized by MPO into reactive NO2 free radicals, which then contributes to protein nitrosylation [118]. The enzyme MPO produces the non-radical oxidant HOCl, which can damage macromolecules directly (by amine conversion into chloramines, phenol, and unsaturated bond chlorination) or indirectly (by OH free-radical fueling) [119]. Overall, a toxic oxidative environment can be created by activated glial cells in the vicinity of dopaminergic neurons, which may account for most of the deleterious effects of neuroinflammation in parkinsonian syndromes. The finding that MPO is mostly expressed by reactive astrocytes in both PD and MPTP-treated mouse tissue suggests that this glial cell population, usually associated with protective and repair properties, can contribute to the elaboration of the deleterious inflammatory network in PD to some extent [1,111]. The microglial-dependent inflammatory reaction might contribute to the brain recruitment of activated CD4 T cells close to dopaminergic neurons [66]. These T cells might express and release several inflammatory factors, such as TNF, IFN-γ, and Fas ligand. Lymphocyte-derived Fas ligand mediates T-cell–induced dopaminergic neuron injury. Fas ligand-derived CD4 T cells might have a deleterious effect on dopaminergic neurons directly (by activating an intracellular death pathway coupled with Fas receptor expressed on the cell surface of dopaminergic neurons) or indirectly (by activating Fas receptor expressed on activated microglial and reactive astrocytic glial cells), thereby stimulating their activation and the release of additional inflammatory factors. COX2, a key enzyme responsible for prostaglandin synthesis during inflammation and a direct target of NSAIDs such as aspirin and ibuprofen, is another important inflammatory component potentially involved in neurodegeneration in PD. Genetic or pharmacologic inactivation of COX2 afforded neuroprotection in mice and rats exposed to MPTP and in rats exposed to 6-hydroxydopamine [101,120,121]. The expression of COX2 is inducible, particularly under inflammatory circumstances, and it can be abundant in macrophages and other cell types, including neuronal and glial cells in the CNS. The finding that increased COX2 expression associated with parkinsonism is limited to dopaminergic neurons suggests that this at-risk neuronal population might participate directly in the inflammatory processes and, consequently, in their own degeneration [101,122]. COX2 cytotoxicity in PD could also be from oxidative damage mechanisms through the formation of ROS generated during the peroxidase catalysis of prostaglandin G2 to prostaglandin H2. As for the regulatory mechanisms of COX2 induction in dopaminergic neurons, activation of the c-Jun N-terminal kinase (JNK) signaling pathway is crucial [101,122]. Genetic or pharmacologic inhibition of the JNK transduction pathway can be as efficient as COX2 ablation in protecting dopaminergic neurons against degeneration in rodent models [123,124]. Inflammatory cytokines are also important mediators of harmful inflammation. Among them, TNF in particular has received much attention with regard to neuroinflammatory processes in PD. In principle, two mechanisms could account for its neurotoxicity: either a direct mechanism through receptor binding on dopaminergic

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neurons or an indirect mechanism through glial cell activation and expression of inflammatory factors. TNF is a type II transmembrane protein and a member of the TNF superfamily of ligands (reviewed in [125]) that is produced in response to a variety of insults. It is active in cell-associated and soluble forms, the latter of which is released following proteolytic cleavage by TNF-cleaving enzyme (TACE). Exogenous signals, such as those arising from exposure to bacterial and viral proteins, potently induce inflammatory responses within the CNS. TNF expression is also induced by cell-intrinsic stimuli relating to physical damage, such as protein aggregation and abnormal release and/or uptake of neurotransmitter [126,127]. TNF interacts with two cognate receptors: p55 (TNFR1) and p75 (TNFR2). These receptors are expressed on neurons, astrocytes, and microglia throughout the CNS [128]. TNF signaling through TNFR1 and TNFR2 can elicit a variety of cellular responses depending on many factors, including the metabolic state of the cell and the adaptor proteins present in the cell. These differences then influence the activation of a number of intracellular signaling pathways, including nuclear factor kappa-B (NF-κB), p38, JNK, and the ceramide/sphingomyelinase signaling pathway, resulting in a number of responses, including inflammation, proliferation, cell migration, apoptosis, and necrosis (reviewed in [129]). An apoptotic signaling cascade is initiated when the ligand-bound TNF receptor associates with the TNF receptorassociated death domain (TRADD), which results in recruitment of Fas, internalization, and subsequent activation of caspase-8 and then apoptosis. TNF can activate the NF-κB signaling pathway. NF-κB signaling is initiated when phosphorylation of its inhibitory subunit (IκB) occurs, which leads to the dissociation and eventual degradation of IκB [130]. NF-κB subsequently translocates to the nucleus, where it regulates gene transcription by binding to specific DNA sequences and, depending on binding sequence composition, acts as either a transcriptional activator or repressor [130]. In contrast to the activation of the TRADD domain, the activation of NF-κB signaling by TNF receptor stimulation is hypothesized to promote prosurvival signal cascades [131], indicating the ability of TNF to induce both pro-life and pro-death cellular outcomes. Another major signaling cascade activated by TNF engagement with its cognate receptors is the JNK pathway. Activation of this pathway can result in the enhanced activity of several transcription factors, including activator protein-1 (AP-1) and specificity protein-1 (SP-1), via JNK-mediated phosphorylation [132]. These transcription factors can then go on to either positively or negatively regulate gene expression based on cofactor expression profiles and binding site composition within promoter regions of target genes. JNK can also promote both cell survival by regulating c-Jun and cell death by regulating c-myc and p53 activity [133,134]. Ultimately, the activity of any or all of these downstream pathways is believed to depend upon the cell type-specific expression of TNF receptor coupling proteins and points of crosstalk between the NF-κB and JNK pathways [135,136]. Through this diverse signaling network, TNF regulates numerous physiologically important processes in the CNS, including neuronal development, cell survival, synaptic transmission, and neuronal ionic homeostasis [137–139]. TNF might have a direct damaging effect on dopaminergic neurons by activating an intracellular death pathway coupled with TNFR1 expressed on the cell surface of these

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neurons. Activation of death-associated signaling pathways (JNK, NF-κB, and p38 mitogen-activated protein (MAP) kinase pathways) possibly linked to Fas and TNFR1 within dopaminergic neurons has been reported [122–124,140]. Pathways transduced by activation of TNF R1 are linked to the induced expression of COX2 within dopaminergic neurons. However, these cytokines might strongly induce NADPH oxidase, and also stimulate the expression of iNOS within microglial (and possibly astrocytic) cells through the expression and activation of the low-affinity receptor of immunoglobulin E (CD23). This process might lead to the production of toxic amounts of free radicals. In turn, these free radicals could potentiate the expression and release of TNF by adjacent microglial cells, thereby amplifying further the inflammatory reaction. Strong evidence for a role for TNF in PD has been provided in the 6-hydroxydopamine model in rats, in which intranigral infusion of TNF inhibitors or lentivirus-encoding dominant-negative TNF attenuated dopaminergic neurodegeneration [141,142].

Is Anti-Inflammatory Therapy the Answer for PD Prevention? The link between neuroinflammation and PD has become more and more convincing due to the accumulation of proof-of-principle preclinical studies, imaging studies, and postmortem tissue analyses that strongly implicate inflammatory processes in the progressive degeneration of the nigrostriatal pathway. However, it remains to be determined whether anti-inflammatory therapy in humans could have a beneficial effect in preventing or slowing progression of PD. Possible reasons for the failure of past clinical trials with anti-inflammatory compounds may include the advanced state of the patients enrolled in the studies, the dosing regimens chosen for the trials, or simply the wrong anti-inflammatory compound; therefore, further clinical investigations are needed before dismissing the possibility of potential long-term benefits of antiinflammatory drugs. Because the innate inflammatory response often represents a beneficial event [143], long-term global inhibition of inflammatory responses is unlikely to be a desirable or effective strategy to treat the inflammatory condition associated with neurodegenerative diseases [59,144]. Instead, selective targeting of inflammatory mediators and molecular players of cell-death pathways should be considered in the future development of therapies to prevent or attenuate neuronal loss in patients with PD.

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  [97] Chen H, Jacobs E, Schwarzschild MA, McCullough ML, Calle EE, Thun MJ, et  al. Nonsteroidal antiinflammatory drug use and the risk of Parkinson’s disease. Ann Neurol 2005;59:988–9.   [98] Chen H, Zhang SM, Hernan MA, Schwarzschild MA, Willett WC, Colditz GA, et al. Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease. Arch Neurol 2003;60(8):1059–64.   [99] McGeer PL, McGeer EG. Glial cell reactions in neurodegenerative diseases: pathophysiology and therapeutic interventions. Alzheimer Dis Assoc Disord 1998;12(Suppl. 2):S1–6. [100] Samii A, Etminan M, Wiens MO, Jafari S. NSAID use and the risk of Parkinson’s disease: systematic review and meta-analysis of observational studies. Drugs Aging 2009;26(9):769–79. [101] Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot S, et  al. Cyclooxygenase-2 is instrumental in Parkinson’s disease neurodegeneration. Proc Natl Acad Sci USA 2003;100(9):5473–8. [102] Esposito E, Di Matteo V, Benigno A, Pierucci M, Crescimanno G, Di Giovanni G. Non-steroidal anti-inflammatory drugs in Parkinson’s disease. Exp Neurol 2007; 205(2):295–312. [103] Barcia C, Fernandez Barreiro A, Poza M, Herrero MT. Parkinson’s disease and inflammatory changes. Neurotox Res 2003;5(6):411–8. [104] Herrera AJ, Tomas-Camardiel M, Venero JL, Cano J, Machado A. Inflammatory process as a determinant factor for the degeneration of substantia nigra dopaminergic neurons. J Neural Transm 2005;112(1):111–9. [105] Hirsch EC, Breidert T, Rousselet E, Hunot S, Hartmann A, Michel PP. The role of glial reaction and inflammation in Parkinson’s disease. Ann N Y Acad Sci 2003;991:214–28. [106] Mrak RE, Griffin WS. Glia and their cytokines in progression of neurodegeneration. Neurobiol Aging 2005;26(3):349–54. [107] Wersinger C, Sidhu A. Inflammation and Parkinson’s disease. Curr Drug Targets Inflamm Allergy 2002;1(3):221–42. [108] Zhang L, Dawson VL, Dawson TM. Role of nitric oxide in Parkinson’s disease. Pharmacol Ther 2006;109(1–2):33–41. [109] Tang BL, Liou YC. Novel modulators of amyloid-beta precursor protein processing. J Neurochem 2007;100(2):314–23. [110] Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, et  al. Nonsteroidal antiinflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science 2003;302(5648):1215–17. [111] Choi DK, Pennathur S, Perier C, Tieu K, Teismann P, Wu DC, et al. Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice. J Neurosci 2005;25(28):6594–600. [112] Hunot S, Boissiere F, Faucheux B, Brugg B, Mouatt-Prigent A, Agid Y, et  al. Nitric oxide synthase and neuronal vulnerability in Parkinson’s disease. Neuroscience 1996; 72(2):355–63. [113] Knott C, Stern G, Wilkin GP. Inflammatory regulators in Parkinson’s disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2. Mol Cell Neurosci 2000;16(6):724–39. [114] Wu DC, Teismann P, Tieu K, Vila M, Jackson-Lewis V, Ischiropoulos H, et  al. NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Proc Natl Acad Sci USA 2003;100(10): 6145–50.

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[115] Ara J, Przedborski S, Naini AB, Jackson-Lewis V, Trifiletti RR, Horwitz J, et  al. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP). Proc Natl Acad Sci USA 1998;95(13):7659–63. [116] Przedborski S, Chen Q, Vila M, Giasson BI, Djaldatti R, Vukosavic S, et al. Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. J Neurochem 2001;76(2):637–40. [117] Qureshi GA, Baig S, Bednar I, Sodersten P, Forsberg G, Siden A. Increased cerebrospinal fluid concentration of nitrite in Parkinson’s disease. Neuroreport 1995;6(12):1642–4. [118] van der Vliet A, Eiserich JP, Halliwell B, Cross CE. Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity. J Biol Chem 1997;272(12):7617–25. [119] Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 1998;92(9):3007–17. [120] Feng Z, Li D, Fung PC, Pei Z, Ramsden DB, Ho SL. COX-2-deficient mice are less prone to MPTP-neurotoxicity than wild-type mice. Neuroreport 2003;14(15):1927–9. [121] Sanchez-Pernaute R, Ferree A, Cooper O, Yu M, Brownell AL, Isacson O. Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson’s disease. J Neuroinflammation 2004;1(1):6. [122] Hunot S, Vila M, Teismann P, Davis RJ, Hirsch EC, Przedborski S, et  al. JNKmediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson’s disease. Proc Natl Acad Sci USA 2004;101(2):665–70. [123] Saporito MS, Brown EM, Miller MS, Carswell S. CEP-1347/KT-7515, an inhibitor of c-jun N-terminal kinase activation, attenuates the 1-methyl-4-phenyl tetrahydropyridine-mediated loss of nigrostriatal dopaminergic neurons in vivo. J Pharmacol Exp Ther 1999;288(2):421–7. [124] Xia XG, Harding T, Weller M, Bieneman A, Uney JB, Schulz JB. Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson’s disease. Proc Natl Acad Sci USA 2001;98(18):10433–8. [125] Tansey MG, Szymkowski DE. The TNF superfamily in 2009: new pathways, new indications, and new drugs. Drug Discov Today 2009;14(23–24):1082–8. [126] Kaushal V, Schlichter LC. Mechanisms of microglia-mediated neurotoxicity in a new model of the stroke penumbra. J Neurosci 2008;28(9):2221–30. [127] Park KM, Bowers WJ. Tumor necrosis factor-alpha mediated signaling in neuronal homeostasis and dysfunction. Cell Signal 2010;22(7):977–83. [128] Kinouchi K, Brown G, Pasternak G, Donner DB. Identification and characterization of receptors for tumor necrosis factor-alpha in the brain. Biochem Biophys Res Commun 1991;181(3):1532–8. [129] McCoy MK, Tansey MG. TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation 2008;5:45. [130] Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004;25(6):280–8. [131] Marchetti L, Klein M, Schlett K, Pfizenmaier K, Eisel UL. Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway. J Biol Chem 2004;279(31):32869–81.

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[132] Benasciutti E, Pages G, Kenzior O, Folk W, Blasi F, Crippa MP. MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription. Blood 2004;104(1):256–62. [133] Milne DM, Campbell LE, Campbell DG, Meek DW. p53 is phosphorylated in vitro and in vivo by an ultraviolet radiation-induced protein kinase characteristic of the c-Jun kinase, JNK1. J Biol Chem 1995;270(10):5511–18. [134] Noguchi K, Kitanaka C, Yamana H, Kokubu A, Mochizuki T, Kuchino Y. Regulation of c-Myc through phosphorylation at Ser-62 and Ser-71 by c-Jun N-terminal kinase. J Biol Chem 1999;274(46):32580–7. [135] Papa S, Bubici C, Zazzeroni F, Pham CG, Kuntzen C, Knabb JR, et  al. The NF-kappaB-mediated control of the JNK cascade in the antagonism of programmed cell death in health and disease. Cell Death Differ 2006;13(5):712–29. [136] Tang F, Tang G, Xiang J, Dai Q, Rosner MR, Lin A. The absence of NF-kappaBmediated inhibition of c-Jun N-terminal kinase activation contributes to tumor necrosis factor alpha-induced apoptosis. Mol Cell Biol 2002;22(24):8571–9. [137] Butler MP, O’Connor JJ, Moynagh PN. Dissection of tumor-necrosis factor-alpha inhibition of long-term potentiation (LTP) reveals a p38 mitogen-activated protein kinasedependent mechanism which maps to early-but not late-phase LTP. Neuroscience 2004;124(2):319–26. [138] Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 2002; 296(5573):1634–5. [139] Cunningham AJ, Murray CA, O’Neill LA, Lynch MA, O’Connor JJ. Interleukin-1 beta (IL-1 beta) and tumour necrosis factor (TNF) inhibit long-term potentiation in the rat dentate gyrus in vitro. Neurosci Lett 1996;203(1):17–20. [140] Karunakaran S, Saeed U, Mishra M, Valli RK, Joshi SD, Meka DP, et  al. Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. J Neurosci 2008;28(47):12500–9. [141] McCoy MK, Martinez TN, Ruhn KA, Szymkowski DE, Smith CG, Botterman BR, et  al. Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson’s disease. J Neurosci 2006;26(37):9365–75. [142] McCoy MK, Ruhn KA, Martinez TN, McAlpine FE, Blesch A, Tansey MG. Intranigral lentiviral delivery of dominant-negative TNF attenuates neurodegeneration and behavioral deficits in hemiparkinsonian rats. Mol Ther 2008;16(9):1572–9. [143] Wyss-Coray T. Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Nat Med 2006;12(9):1005–15. [144] Marchetti B, Abbracchio MP. To be or not to be (inflamed)—is that the question in anti-inflammatory drug therapy of neurodegenerative disorders? Trends Pharmacol Sci 2005;26(10):517–25.

19 Neuroinflammation and Pediatric Lupus

Marisa Klein-Gitelman1, Hermine Brunner2 1 

Division of Rheumatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL (MKG) 2  Division of Rheumatology, Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio (HB)

Introduction Lupus is the archetypical autoimmune disease. The pathophysiologic mechanisms that result in lupus and specific organ injury appear to be multifactorial and seem to require both genetic risk and environmental stimulation. Pediatric-onset lupus appears at a time during brain maturation as well as physical, psychological, and emotional changes. The impact of lupus and in particular neuropsychiatric disease is dependent on the disease manifestation, disease severity, family characteristics and environment, and response to treatment. We will review the clinical aspects of neuropsychiatric lupus (NPSLE), the possible mechanisms of injury, and current treatment options available.

Manifestations of NPSLE Neurologic involvement in systemic lupus is well recognized. Psychosis and seizures are included in the original classification criteria of lupus; however, many other neurologic manifestations of lupus have been recognized and reported [1]. The American College of Rheumatology (ACR) determined that it was important to develop specific definitions for NPSLE and reported a series of 19 neurologic definitions seen frequently in lupus [2]. The prevalence of NPSLE among adult lupus populations has been reported to be up to 80%, while several pediatric studies report that up to 95% of children with lupus have NPSLE [3,4]. Compared to adults, the presentation of pediatric NPSLE appears to be more complex and varied and may be related to developmental issues [1]. One of the main concerns when assessing for NPSLE is the use of corticosteroids to treat the disease. Corticosteroids are known to have a variety of neuropsychiatric adverse reactions. Given the presence of NPSLE even at the time of diagnosis, it is unlikely that corticosteroids are causal; however, the SALUD study reports that the prolonged use of corticosteroids and the presence of specific autoantibodies, lack of educational opportunity, depression, and diabetes are risk factors for NPSLE in the adult population [5]. Studies of potential risk factors of pediatric NPSLE are lacking; however, one might assume that pediatric NPSLE and adult NPSLE are the Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00019-8 © 2011 Elsevier Inc. All rights reserved.

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sequelae of the same pathophysiologic mechanisms but that the manifestations and course of pediatric NPSLE are complicated by ongoing development and growth of the brain. Furthermore, it is important to recognize that adolescent- and childhoodonset lupus results in a more aggressive disease with worse outcomes. The most recent evidence for this was determined using a nested matched case-control study within LUMINA, a multiethnic U.S. cohort [6]. In this study, neurologic involvement in systemic lupus was significantly more frequent in the adolescent group compared to the adult group (38.7% versus 12.5%; P  0.020). This was also true for kidney disease (P  0.009). There was also a trend toward adolescents with lupus having a higher frequency of neuropsychiatric damage than adults with lupus (29% versus 19.6%). The effect of neurologic involvement for pediatric patients is complicated further in that children with a chronic as well as neurologic disease are at a key point in psychosocial development. Disruption of this process alters the development of personal coping skills and individuation for many adolescents. The case definitions as defined by the ACR can be organized by central nervous system (CNS) versus peripheral nervous system. Overall, peripheral nervous system features are less common than CNS lupus and include myelopathy, mononeuropathy (single or multiplex), plexopathy, polyneuropathy, acute inflammatory demyelinating polyradiculoneuropathy (Guillain–Barré syndrome), and cranial neuropathy. Central nervous case definitions of lupus include acute confusional state, cognitive dysfunction, demyelinating syndromes, anxiety disorder, headache, aseptic meningitis, mood disorder, movement disorder (i.e., chorea), psychosis, seizure, autonomic disorder, and cerebrovascular disease. Isolated peripheral autonomic system disease or peripheral nervous system injury due to a vascular infarct is also possible, but very rare. Of note, diffuse injury to the CNS is far more common than a focal injury in NPSLE. The case definitions can also be classified by type of injury or known versus unknown pathophysiologic mechanism. The mechanisms for some injuries are well described in this book, including demyelination injury to the CNS or peripheral nervous system as well as noninfectious meningitis, Guillain–Barré disease, and transverse myelitis. Other well-described injuries include hemorrhagic and thrombotic vascular injury, seizure, and movement disorder. Some of the disease presentations are associated with specific autoantibodies. However, the pathophysiologic mechanisms for the most common symptom, headache, remain unknown. Other disease manifestations whose pathophysiologic mechanisms are yet to be elucidated include cognitive behavioral and mood disorders [2]. When patients have mood disorders, it is unclear whether symptoms such as anxiety and depression are due to the burden of having lupus itself or are sequelae of the medications used to treat lupus. Cognitive behavioral manifestations of NPSLE are the most difficult to diagnose, understand, and treat. Thought disorders, or cognitive dysfunctional states, are difficult to recognize in general and likely even more difficult to diagnose in the setting of a systemic disease. The symptoms of cognitive dysfunction may be covert or unrecognized by the patient, family, and medical providers, yet often have a significant impact on daily life. In retrospect, cognitive dysfunction is often insidious and persistent and is not particularly associated with evidence of clinical or serologic lupus disease activity. Besides headaches, cognitive dysfunction is among the most common manifestations of NPSLE [7].

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As difficult as it is to diagnose pediatric NPSLE, it is perhaps even more challenging to assess clinically relevant change of pediatric NPSLE. Change in psychosis or dementia has been documented and is easily recognized. However, change in cognitive performance can be protracted, making it difficult to capture in a clinical setting. There are only few studies of pediatric cognitive function in lupus; however, these all support the notion that there are problems with attention, memory, language (both vocabulary and verbal fluency), visual-spatial processing, processing speed, and executive function, which provides the ability to organize and plan in pediatric NPSLE [8,9]. It is well known that maturation in these cognitive domains is challenging even for healthy children and adolescents and is influenced by socioeconomic status [10] and social and school support systems. Thus, the pediatric lupus patient has a brain that is in the midst of physiologic maturation, a process that ends in early adulthood [11]. The brain in this time frame is perhaps more vulnerable to injury. This idea is supported by the presence of cognitive dysfunction in other chronic childhood illnesses such as sickle cell disease, juvenile diabetes, malignancy, and organ transplantation [12–15]. It is difficult to resolve the differences in pathophysiology of these other diseases with a common outcome; however, if the end result of these disease processes impairs neurocognitive maturation, then one would expect difficulties in acquiring new cognitive skills. There are data in pediatric lupus that demonstrate lower test scores in reading comprehension, attention, working memory, and executive function [16]. There are also data that suggest that children with systemic lupus have difficulty acquiring new skills and may develop problems with attention [17]. Thus, children with lupus may have environmental challenges that alter function, superimposed on disease and medication use at a time when the brain is vulnerable due to the developmental changes occurring during childhood and adolescence.

Proposed Etiology of NPSLE Neurobehavioral Syndromes The pathophysiologic mechanisms of injury in lupus are well described in a variety of organ systems. There are data on the pathophysiologic mechanisms of neuronal injury at the cellular level; however, mechanisms that cause cognitive changes remain unclear. It is reasonable to speculate that these injuries are, as many other lupus-related pathologic findings, multifactorial. Pathologic mechanisms include autoantibodymediated injury, neuronal changes due to inflammatory cytokines, and microangiopathy. The ongoing presence of the latter leads to atherosclerotic injury as well. True vasculitis is uncommon with NPSLE. In a series of postmortem examinations of the brains of lupus patients, the major injury seen was vasculopathy with microinfarcts. Many of the smaller vessels had fibrin thrombi within the lumen. The vessels had intimal proliferation and a perivascular pleomorphic infiltrate. Another hypothesis of brain injury is disruption of the blood–brain barrier. This hypothesis has been attached to several avenues of research. One avenue has been analysis of the cerebrospinal fluid (CSF) of lupus patients for the presence of inflammatory cytokines and autoantibodies. Studies have demonstrated increased levels of ICAM-1, TFG-β, interferon alpha, and interleukins (ILs) 6, 8, and 10 [18–20].

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In an interesting study by George-Chandy et al. [21], patients who had two or more manifestations/abnormal tests of NPSLE (psychosis, aseptic meningitis, transverse myelitis, seizures, severely abnormal cognitive tests, oligoclonal bands in CSF, or pathologic magnetic resonance imaging (MRI) findings as defined by the researchers) also had significantly high intrathecal levels of a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) compared to lupus subjects without NPSLE. Another pathway that has been explored more recently in CSF is the coagulation pathway. On the basis that coagulation factors are altered in the setting of inflammation, Kwiecinski et al. [22] studied fibrinolytic factors in the CSF of lupus patients with and without NPSLE in comparison to controls. Urokinase plasminogen activator, tissue plasminogen activator, d-dimer, and plasminogen activator inhibitor-1 were measured in 56 lupus patients, 33 lupus patients with NPSLE, 5 lupus patients with antiphospholipid antibodies, and 53 healthy controls. In this cohort, plasminogen activator inhibitor-1 levels were significantly elevated in the NPSLE patients versus lupus patients without NPSLE (P < 0.05) and versus controls (P < 0.001). Similarly, other inflammatory cytokines (IL-6, IL-8) and markers of brain damage (glial fibrillary acidic protein, neurofilament triplet protein, and tau protein) were elevated, which may be due to changes in the blood–brain barrier; however, these findings are not specific to NPSLE, as similar finding have been reported with other neurologic diseases There is speculation that anticoagulation might be helpful in this setting, irrespective of the presence or absence of antiphospholipid antibodies. It remains to be determined whether these changes in cytokine levels and markers of brain damage are an epiphenomenon or whether they are the cause of NPSLE. The presence of inflammatory cytokines and related inflammatory proteins does not prove that they are causal in the injury seen in the brain, nor is there evidence that the levels vary with fluxes in cognitive function, especially because results across studies have not been consistent. Other possible mechanisms are related to changes in nitric oxide or the presence of specific autoantibodies causing injury at the cellular level. The latter mechanism of injury is thought to be important for the development of lupus psychosis; Matus et al. [23] demonstrated that anti-ribosomal-P binds a neuronal membrane protein and can initiate cellular damage. The presence of ribosomal-P in the CSF is less common than once suspected in NPSLE psychosis [24], and this antibody may be just one of several that has the ability to induce neuronal damage. The theory that autoantibodies are important in the pathophysiologic mechanism causing NPSLE has led to a very interesting animal model of NPSLE. Special subtypes of anti-DNA antibodies cross-react with N-methyl-d-aspartate (NMDA) receptors once they cross the blood–brain barrier. NMDA receptors bind the neurotransmitter glutamate, which is a key excitatory neurotransmitter in the brain. Exposure of cells to high levels of this molecule leads to an excitotoxic cell death. Disturbances in the glutamate pathway are not specific to autoimmune diseases, and it is a common injury pathway for traumatic, metabolic, dementing, and psychiatric diseases. The NMDA receptors have a high concentration in the hippocampus and amygdala. The hippocampus is an important area for learning and memory (cognitive function), while the amygdala is an important area for fear and conditioning

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(emotions). The Diamond lab has made significant advances in our understanding of antibody-mediated injury in the brain using a mouse model. First, antibodies were identified that were cross-reactive between DNA and NMDA receptors. These receptors were located in the mouse hippocampus, causing apoptotic neuronal cell death independent of complement or antibody-dependent cell-mediated cytotoxicity [25]. When mice were immunized with the identified consensus peptide, mice developed positive serology but no evidence of neurocognitive change. It was hypothesized that there needed to be a change in the integrity of the blood–brain barrier, such as seen in infection, which was associated with a rise in IL-1 and TNF. The mice received lipopolysaccharide (LPS), and subsequent to immunization, antibodies were found in the hippocampus and the mice performed poorly on the standard neurocognitive tests given. There was no evidence of progression of the injury once the effect of LPS resolved [26]. Interestingly, when the blood–brain barrier was disrupted using epinephrine, antibodies and injury were found in the amygdala and resulted in a change in conditioning behavior and not in memory tasks as with LPS [27]. These results suggest that antibody-mediated injury to the brain is dependent on both antibody specificity and location of injury, which may also be a result of the type of blood– brain barrier disruption. There is evidence that anti-NMDA receptor antibodies are associated with human NPSLE. This includes a demonstration of the antibody from the postmortem brain of a patient with severe NPSLE [28]. Emmer et al. [29] studied the amygdala of 21 patients with lupus, 37 patients with NPSLE, and 12 healthy controls using diffusion-weighted imaging and calculated the apparent diffusion coefficients, a measure of tissue integrity. There were no differences between patients with SLE and NPSLE; however, all lupus patients had significantly lower diffusion coefficients than healthy controls (P  0.019), suggesting impaired neuronal activity. More interestingly, patients who tested positive for anti-NMDA receptor antibodies had even lower coefficients than lupus patients without these antibodies (P  0.029) or healthy controls (P  0.001). Despite its relatively small size, this study supports the theory that anti-NMDA receptor antibodies play a role in NPSLE development. Studies are under way to confirm the findings in larger groups of lupus patients and to measure CSF and serologic levels of anti-NR2 antibodies in lupus patients. Levy et al. (ACR annual meeting presentation) [30] described an ongoing study to measure the presence of anti-NMDA antibodies in the serum of children with lupus. As previously mentioned, there is a significantly higher frequency of anti-double-stranded DNA antibodies at presentation of pediatric lupus. It will be interesting to learn whether this is also found for anti-NMDA antibodies, since children have been found to have more serologic activity than their adult counterparts. To date, however, the role of the NMDA autoantibody as a causal mediator of injury in the pathophysiology of NPSLE remains unclear. The NMDA receptor is not the only excitatory receptor in the brain. Gamma-amino butyric acid-A (GABA) receptor is another excitatory receptor in the brain associated with neurologic disease states. In an interesting study, Mathieu et al. [31] took advantage of a technique used to study GABA receptors that had previously been employed in investigations in seizures, alcoholism, panic disorder, and schizophrenia. GABA receptors have specific binding sites for medications, including benzodiazepines.

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Iomazenil is a ligand with high affinity for GABA cerebral receptors without intrinsic pharmacologic effect at doses needed to detect labeled drug by single photon emission computed tomography (SPECT) imaging (123I-Iomazenil). Nine NPSLE patients with recurrent, episodic memory loss were imaged and found to have diffuse and/or focal decrease in cerebral GABA receptor density. The results were unique to this population; in one patient, it was the only abnormality associated with cognitive dysfunction. This type of imaging has the potential to follow changes in receptor density and possibly changes in function. Antiphospholipid antibodies are another set of autoantibodies that have a clear pathophysiologic role in NPSLE in both adults and children, although NPSLE can occur in the absence of antiphospholipid antibodies [5,24,32–35]. Antiphospholipid antibodies bind phospholipids and/or phospholipid-binding proteins. These antibodies are associated with the increased tendency to form thromboses. Antiphospholipid antibody-mediated thrombi can occur in large or small vessels, leading to the welldescribed antiphospholipid syndrome. Thrombi result in ischemia and injury to the vessel wall, resulting in the bland vasculopathy described in the postmortem lupus brain. Antiphospholipid antibodies also have the potential to directly bind to and injure neurons. These pathophysiologic processes are associated with cognitive changes in lupus patients and also with chorea, besides leading to strokes. Drug therapy has been implicated in neuropsychiatric changes in lupus. Corticosteroids most specifically have been associated with episodes of psychosis and changes in mood. Steroids may unmask an underlying psychiatric disorder such as depression or bipolar disease that requires disease-specific medication to manage symptoms. It is difficult, however, to propose drugs as a cause of NPSLE, as psychosis, mood disturbance, and cognitive impairment are often present at diagnosis (i.e., prior to therapy) and NPSLE features improve with therapy. Attempts to link steroids to NPSLE have failed to find a convincing association with either the length of therapy or cumulative dose of steroids; however, corticosteroid exposure is likely associated with brain atrophy. It is critical to note that corticosteroids are the first-line therapy for NPSLE symptoms, often leading to a remarkable patient recovery.

Diagnosis of NPSLE As previously delineated, the diagnosis of overt neurologic disease (i.e., stroke, seizure, neuropathy) in the setting of lupus is straightforward Table 19.1. There are specific diagnostic evaluations that are required to consider an appropriate differential diagnosis. The cornerstone to evaluation is the history and physical examination, which should direct laboratory and imaging evaluations, resulting in a diagnosis. The evaluation of less objective disease manifestations becomes muddled. The patient may present with an acute confusional state, psychosis, altered mood, or other cognitive challenge. The attribution of symptoms to lupus is a diagnosis of exclusion, and all other possibilities must be considered. In the differential diagnosis of altered mental status are states that require immediate intervention such as infection, recreational drug use, trauma causing a bleed or concussive state, or acute metabolic

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Table 19.1  Differential Diagnosis Symptoms Associated with New Onset of Cognitive Dysfunction

Alternative Diagnostic Considerations

Tests to Resolve Differential Diagnosis

Abrupt onset

Substance abuse, poisoning

Toxicology screen, including heavy metals

No or minimal recognizable precipitants

Medication side effect

Metabolic studies

Temporal association between onset, exacerbation, and recovery of psychiatric symptoms with course of medical condition

Brain injury

Imaging

Presence of atypical features (e.g., severe weight loss and mild depressive symptoms)

Endocrinopathy

Endocrine studies Thyroid function Glucose Cultures for infection including viral, bacterial, and fungal studies, CSF studies Metabolic panel and nutritional screening

Infection

Nutritional or metabolic abnormalities

state, including organ failure, that needs to be addressed urgently. Infections in the United States and Western Europe are usually bacterial in origin; however, this may change with the use of biologic therapies. Fungal and mycobacterial infections are more common in other areas of the world. Progressive multifocal encephalopathy in patient with lupus treated with rituximab and other therapies has been reported. It is important to have increased vigilance for this serious diagnosis, which presents with altered mental status [36,37]. Thrombotic thrombocytopenic purpura is a life-threatening illness that can present with altered mental status, thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction, and fever, not unlike the presentation of lupus or a lupus flare. This condition is fatal if a timely diagnosis is not established and if treatment initiation is postponed. Another problem that can present with altered mental status or seizures is posterior reversible encephalopathy syndrome (PRES), which is due to increased cerebrovascular permeability and brain edema associated with renal disease, severe hypertension, or recent use of cyclophosphamide. Patients with PRES have typical findings on MRI and require stabilization of blood pressure. All patients need to be assessed for use of drugs or toxins that cause altered mental states, and exclusion of thyroid disease and diabetic coma is mandatory. Finally, there is the consideration of a primary psychiatric disorder.

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The evaluation of a patient with possible NPSLE should include a complete metabolic profile, complete blood count, blood culture, toxicology screen, heavy metal screen, CSF analysis for protein, cell count, bacterial, viral, and fungal studies and cultures, immediate imaging to consider thrombotic or hemorrhagic injury, as well as abscess, and lupus serologic studies. NPSLE often presents in the setting of minimal serologic disease activity. Lastly, the patient needs a psychiatric assessment if initial studies do not point to a specific diagnosis. Patients with lupus have the same risks as the general population for psychiatric disease, as well as an additional risk related to living with a chronic illness. Although these problems are frequently comorbid, there is no evidence to demonstrate that lupus specifically increases risk or causes psychiatric disorders, noting the exception of the role of chronic illness on mental health issues.

Diagnosis of Cognitive Dysfunction Syndromes in Children The first challenge of making a diagnosis of cognitive dysfunction syndromes in NPSLE is recognizing the presence of symptoms that can be ascribed to developmental issues, environment, or medication or drugs, as well as the possibility that the symptoms are subtle and slowly progressive, making it more difficult to recognize. The next challenge is objectively testing for dysfunction in a setting where the pathophysiologic mechanisms remain unclear. Thus, the clinician is left with making a diagnosis based on the following: (1) diagnosis of lupus based on history, physical examination, and laboratory data and (2) clinical neurologic evaluation, which evaluates the patient for clinical neurocognitive dysfunction and the available tests to support the diagnosis, including serology, imaging, and formal neuropsychological tests that support the clinician’s impression that the patient has NPSLE. As previously noted, several studies have demonstrated neurocognitive dysfunction in children. Wyckoff et al. [16] described significant neurocognitive abnormalities in a small cohort of pediatric lupus patients with concerns about declining school performance and memory. Papero et al. [38] compared a group of pediatric lupus patients to a matched group of children with juvenile arthritis and found significant differences. Brunner et al. [8] studied 27 children without prior history of cognitive problems, including attention-deficit disorder, chronic headache, and developmental delay. These lupus patients had formal neuropsychiatric testing, and 59% (16 patients) had a performance in at least one domain two standard deviations below the normal range or two average scores one to two standard deviations below the normal range. The most significant differences were in visuoconstructive processing and memory. Most recently, Muscal et al. [9] reported data on 39 pediatric lupus patients at a time of general wellness, without a history of NPSLE and early in their disease course. Data on 24 patients were obtained retrospectively, while data on 15 subjects were collected prospectively. The patient group was mostly African-American and Hispanic, with average intelligence scores. Most patients had positive antiphospholipid studies and received aspirin therapy. Patients with neurocognitive dysfunction were identified by

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the same method as in the Brunner study. The data demonstrated that 70.8% of the patients on retrospective chart review and 46.7% of the patients studied prospectively had neurocognitive dysfunction. Areas of dysfunction included executive function, visual memory, and visual-spatial planning. Of note, 33% of the retrospective patient group had depressive symptoms based on the presence of referrals on chart review. Muscal et al. also studied neuroimages obtained from this study population. They also documented a high frequency of volume loss and white matter hyperintensities consistent with previous adult studies. Currently, formal neurocognitive evaluation is the gold standard for diagnosing cognitive dysfunction in lupus; however, there is no specific way to determine that the changes are specifically due to lupus, as has been highlighted by Breitbach et al. [10] in adult studies. One hundred twenty-eight pediatric rheumatologists responded to a recent survey to determine when neurocognitive testing is appropriate. Almost all of the respondents (98%) supported formal testing if a particular child with lupus had a decline in academic performance or complained of cognitive difficulties that were observed by the patient or parents. Most (79%) considered testing when a patient had an overt neurologic injury such as a stroke or psychosis, and many (63%) considered testing if there was a significant change in mood. However, there was no agreement to what tests are specifically appropriate, and there are problems with access to testing due to low numbers of pediatric neuropsychologists and the length of time required to complete testing. When access is available, the testing is not often covered by insurance, making the process very costly and therefore not financially feasible for many families. Given these problems, a short validated battery of tests for children would be a critical tool for both the practitioner and the patient. The ACR battery for adults cannot be directly applied to children, as many tests are designed based on age and developmental stage. Thus, the CARRA Neuropsychiatric Lupus Working Group designed a potential battery of tests that will be analyzed for validity (Table 19.2).

Computer-Based Neurocognitive Testing Although validation of a pediatric battery of neurocognitive tests is very helpful, it does not resolve the problems of access and costs for pediatric patients. Thus, there has been an effort to find a computer-based, short test that is accessible and inexpensive. One of the important features of such a test should be the ability to reassess a child routinely without losing validity so that changes can be picked up early and interventions made to maintain the patient’s quality of life and long-term outcomes. The development of adult and pediatric versions of the Automated Neuropsychological Assessment Metrics (ANAM) is likely to fulfill this need. The adult version has been used to test cognitive abilities of adults down to children aged 13 years, and the pediatric version allows testing of children as young as 10 years. The test takes 30–40 min to complete and covers areas of attention and concentration, mental flexibility, spatial processing, processing efficiency, arousal/fatigue level, learning, recall, and working memory.

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Table 19.2  Proposed Pediatric Neuropsychological Test Battery Test

Time (min)

Result

Wechsler Abbreviated Scales of Intelligence vocabulary and matrix reasoning subtests

30

General intelligence

Wechsler Intelligence Scale for Children (WISC) IV coding and symbol search subtests

10

Psychomotor speed

WISC-IV digit span and letter–number sequencing subtest

20

Verbal working memory

Conners CPT II

15

Attention, processing speed

Woodcock-Johnson II Achievement: LetterWord Identification, Reading Fluency, Calculations, and Math Fluency subtests

30

Academic skills

Wide Range Assessment of Memory and Learning-Story Memory, Verbal Learning, Picture Memory and Design Memory subtests

30

Verbal and visual memory and learning

Stroop Color and Word Test

 5

Attention, cognitive flexibility, and response inhibition

Delis-Kaplan Executive Function System Verbal and Design Fluency subtests

10

Speed/executive functioning

Brunner et al. [8] published a study that is an initial validation of the PedANAM. In this study, 27 children with lupus who had no previous diagnosis of cognitive dysfunction were tested for cognitive ability by standard psychometrics and the PedANAM. Sixteen children (59%) had neurocognitive dysfunction, as defined by poor results on standardized testing with a Z-score of –2 in a minimum of one task or between –1 and –2 in a minimum of two tasks. The patients had poor scores in several areas on the PedANAM. Three tasks were significant: the coefficient of variation of time required for a correct response with math processing and with spatial processing and the accuracy of response with a continuous performance test. The ability to these tests to predict neurocognitive dysfunction based on psychometric test results was evaluated by the area under the receiver operating curves (AUC). For the listed tasks, the AUC ranged from 0.75 to 0.84. When all three tasks were evaluated together, the AUC improved to 0.96, suggesting that the PedANAM has potential as a screening tool for NPSLE.

Imaging for Diagnosing Neurocognitive Dysfunction MRI is another computer-based technology that may lead to our ability to make a diagnosis of neurocognitive dysfunction in lupus and, perhaps, investigate anatomic/

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pathologic correlations. In the past, imaging has not been specifically helpful in the diagnosis of cognitive abnormalities in lupus patients. Patients with neuropsychiatric disease often lack abnormalities on conventional MRI other than brain atrophy, which has not correlated with NPSLE symptoms. Brain atrophy in cerebral (73.3%) and cere­ bellar (67.7%) areas was found in a small study of children with lupus without NPSLE symptoms. This group of children did have significant abnormalities when tested psychometrically; however, the MRI findings did not specifically correlate with psychometric results [9]. Some patients have small areas of cortical or subcortical hyperdense areas that do not correlate with clinical findings and are not sensitive to changes in clinical state. Magnetization transfer imaging (MTI) and fluid-attenuated inversion recovery (FLAIR) imaging have not enhanced the ability of neuroradiologists to find abnormalities that can be related to clinical disease. Techniques that are more physiologic, such as positron emission tomography and single photon emission computed tomography, have also been employed. Although these techniques can demonstrate changes in blood flow or metabolism, they have poor anatomic resolution and correlate poorly with specific cognitive changes. Furthermore, there are problems with access to the procedure and significant radiation exposure as well as the sensitivity of the test to change over time. Magnetic resonance spectrometry (MRS) is another technique that assesses metabolic changes in the brain, and lupus subjects with active neurocognitive dysfunction have metabolic changes in both gray and white matter [39]. Unfortunately, none of the MRS metabolites, or their combination, can distinguish between acute and chronic NPSLE. More recently, diffusion-weighted imaging and diffusion tensor imaging (DTI) have been applied to the study of lupus patients with neurocognitive changes [40,41]. These techniques can provide quantitative measurements of white matter changes, suggesting decreased white matter functionality with NPSLE. Studies of morphometry have also described changes in the lupus brain. Appenzeller et al. [42] used voxel-based morphometry to study gray and white matter changes in patients with lupus compared to healthy controls. There was a significant reduction in volume and progressive atrophy in the lupus brain that correlated with disease duration, presence of antiphospholipid antibodies, and severity of cognitive dysfunction. Corticosteroid dose was associated with gray changes only in lupus patients studied. Furthermore, functional MRI can demonstrate changes in brain activation during specifically designed tasks. The images take advantage of changes in blood flow (relative oxygen level of blood hemoglobin) between inactive states and then during a specific task that requires attention or memory, for example. Patients with a specific disease need to be compared to normal controls to understand differences in brain activation. Functional imaging has been used in a variety of settings, most notably in the study of dementia. Recently, DiFrancesco et al. [43] studied functional images of children with lupus with and without evidence of cognitive dysfunction on neuropsychological evaluation and a third group of well children. The children first completed tasks of visuoconstructive processing, memory, attention, executive function, and psychomotor speed using standard psychometric tests. The children then performed tasks of attention, language, and working memory during functional imaging. The test of

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attention was a continuous performance task where the patient is asked to press a button when the same number is flashed consecutively on a screen in a string of numbers shown to the patient. The test of language was the presentation of a noun, with the request to think of as many verbs as possible that could be matched to the noun. The task of working memory was called an N-Back task. The patient is given a diamond with numbers 1–4 on each corner. The same diamond is seen on the screen. When the task starts, the subject sees a string of numbers flashing in the appropriate corners. He or she is asked to press the number seen two times previously after the first two numbers are given and to continue to press the number seen two times previously throughout the task. Two major differences in lupus patient brain activations were found. First, the lupus groups used a larger brain area to perform tasks. Second, the lupus groups had persistence of brain activity when the tasks were over compared to the healthy controls. Thus, these patients used more activation to perform a task and then were unable to suppress activity as quickly as normal subjects. The lupus group with normal neuropsychiatric testing demonstrated these differences to a lesser degree. This suggests that there may be connectivity problems, and the use of functional MRI in association with diffusion techniques may give us further insight. At the present time, neither MTI, MRS, DTI, nor fMRI can be used as a lupusspecific diagnostic test for individual patients with lupus. All these novel imaging modalities allow only for the comparison of groups of patients.

Treatment of NPSLE There is no established treatment protocol for patients with NPSLE. To date, no interventional trials have occurred, and treatments are based on observations of patients who are treated in an uncontrolled manner. As with other forms of lupus, the goal is to alleviate symptoms, often using immunosuppressive medications while managing NPSLE symptoms as if they were present without an underlying diagnosis of lupus. For clear, specific problems such as stroke or seizure, the patient should be treated according to the appropriate paradigm while assessment for lupus disease activity occurs. For patients with altered sensorium, the practitioner must consider infection, ingestion, injury, or metabolic causes initially. Once these causes have been excluded, primary psychiatric disease and lupus cerebritis can be considered. Again, while the patient is undergoing evaluation, psychosis needs to be treated as a primary psychosis and depression as if it were idiopathic or primary depression. There are no specific differences in the evaluation of the pediatric patient in this setting. Once the practitioner has determined that lupus is the most likely cause, a decision to initiate immunosuppression is based on the specific diagnosis and severity. Patients with cerebritis, vasculitis, or myelopathy will receive high-dose steroids and, if severe, intravenous intermittent cyclophosphamide initially. Some centers use azathioprine as an alternative therapy. Patients with antiphospholipid syndrome may need anticoagulation, plasmapheresis, and immunosuppression, depending on severity. These patients can present with thrombotic thrombocytopenic purpura and will need all modalities of treatment.

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For patients with cognitive dysfunction, treatment of the underlying disease is often associated with improved cognition. However, cognitive dysfunction can occur even when other clinical or serologic features of lupus appear to be quiescent. For these patients, psychiatric assessment is imperative, and treatment may require cognitive therapy, specific school and home interventions, and medications to improve specific abnormalities such as attention or mood.

References   [1] Hermosillo-Romo D, Brey RL. Neuropsychiatric involvement in systemic lupus erythematosus. Curr Rheumatol Rep 2002;4(4):337–44.   [2] ACR. The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes. Arthritis Rheum 1999;42(4):599–608.   [3] Sibbitt WL Jr, Brandt JR, Johnson CR, Maldonado ME, Patel SR, Ford CC, et al. The incidence and prevalence of neuropsychiatric syndromes in pediatric onset systemic lupus erythematosus. J Rheumatol 2002;29(7):1536–42.   [4] Olfat MO, Al-Mayouf SM, Muzaffer MA. Pattern of neuropsychiatric manifestations and outcome in juvenile systemic lupus erythematosus. Clin Rheumatol 2004;23(5): 395–9.   [5] McLaurin EY, Holliday SL, Williams P, Brey RL. Predictors of cognitive dysfunction in patients with systemic lupus erythematosus. Neurology 2005;64(2):297–303.   [6] Tucker LB, Uribe AG, Fernandez M, et  al. Adolescent onset of lupus results in more aggressive disease and worse outcomes: results of a nested matched case-control study within LUINA, a multiethnic US cohort (LUMINA LVII). Lupus 2008;17:314–22.   [7] Hanly JG, Fisk JD, McCurdy G, et  al. Neuropsychiatric syndromes in patients with systemic lupus erythematosus and rheumatoid arthritis. J Rheumatol 2005;32(8): 1459–60.   [8] Brunner HI, Ruth NM, German A, et  al. Initial validation of the Pediatric Automated Neuropsychological Assessment Metrics for childhood-onset systemic lupus erythematosus. Arthritis Rheum 2007;57(7):1174–82.   [9] Muscal E, Bloom DR, Hunter JV, Myones BL. Neurocognitive deficits and neuroimaging abnormalities are prevalent in children with lupus: clinical and research experiences at a US pediatric institution. Lupus 2010;19:268–79. [10] Breitbach SA, Alexander RW, Daltroy LH, et al. Determinants of cognitive performance in systemic lupus erythematosus. J Clin Exp Neuropsychol 1998;20(2):157–66. [11] Luna B, Garver KE, Urban TA, et al. Maturation of cognitive processes from late childhood to adulthood. Child Dev 2004;75(5):1357–72. [12] Noll RB, Stith L, Gartstein MA, et  al. Neuropsychological functioning of youths with sickle cell disease: comparison with non-chronically ill peers. J Pediatr Psychol 2001;26(2):69–78. [13] Brismar T, Maurex L, Cooray G, et al. Predictors of cognitive impairment in type 1 diabetes. Psychoneuroendocrinology 2007;32(8–10):1041–51. [14] Shah AJ, Epport K, Azen C, et  al. Progressive declines in neurocognitive function among survivors of hematopoietic stem cell transplantation for pediatric hematologic malignancies. J Pediatr Hematol Oncol 2008;30(6):411–8. [15] Gelb S, Shapiro RJ, Hill A, Thornton WL. Cognitive outcome following kidney transplantation. Nephrol Dial Transplant 2008;23(3):1032–8.

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[16] Wyckoff PM, Miller LC, Tucker LB, Schaller JG. Neuropsychological assessment of children and adolescents with systemic lupus erythematosus. Lupus 1995;4(3):217–20. [17] Klein-Gitelman MS, Zelko F, Kress A, Hunter S, Wagner-Weiner L. Comparison of neuro-cognitive function in children with pediatric systemic lupus erythematosus (pSLE) and their peers—second year follow up. Arthritis Rheum 2002;46:S216. [18] Katsumata Y, Harigai M, Kawaguchi Y, et  al. Diagnostic reliability of cerebral spinal fluid tests for acute confusional state (delirium) in patients with systemic lupus erythematosus: interleukin 6 (IL-6), IL-8, interferon-alpha, IgG index and Q-albumin. J Rheumatol 2007;34(10):2010–7. [19] Fragoso-Loyo H, Richaud-Patin Y, Orozco-Narvaez A, et al. Interleukin-6 and chemokines in the neuropsychiatric manifestations of systemic lupus erythematosus. Arthritis Rheum 2007;56(4):1242–50. [20] Santer DM, Yoshio T, Minota S, et  al. Potent induction of IFH-alpha and chemokines by autoantibodies in the cerebrospinal fluid of patients with neuropsychiatric lupus. J Immunol 2009;182(2):1192–201. [21] George-Chandy A, Trysberg E, Eriksson K. Raised intrathecal levels of APRIL and BAFF in patients with systemic lupus erythematosus: relationship to neuropsychiatric symptoms. Arthritis Res Ther 2008;10(4):R97. [22] Kwiechinski J, Klak M, Trysberg E, et al. Relationship between elevated cerebrospinal fluid levels of plasminogen activator inhibitor 1 and neuronal destruction in patients with neuropsychiatric systemic lupus erythematosus. Arthritis Rheum 2009;60(7):2094–101. [23] Matus S, Burgos PV, Bravo-Zehnder M, et al. Antiribosomal-P protein antibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis. J Exp Med 2007;204(13):3221–34. [24] Hanly JG, Urowitz MB, Siannis F, et al. Autoantibodies and neuropsychiatric events at the time of systemic lupus erythematosus diagnosis: results from an international inception cohort study. Arthritis Rheum 2008;58(3):843–53. [25] DeGiorgio LA, Konstantinov KN, Lee SC, et al. A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus. Nat Med 2001;7(11):1189–93. [26] Kowal C, DeGiorgio LA, Nakaoka T, et al. Cognition and immunity; antibody impairs memory. Immunity 2004;21(2):178–88. [27] Huerta PT, Kowal C, DeGiorgio LA, et  al. Immunity and behavior: antibodies alter emotion. Proc Natl Acad Sci USA 2006;103(3):678–83. [28] Kowal C, Degiorgio LA, Lee JY, et al. Human lupus autoantibodies against NMDA receptors mediate cognitive impairment. Proc Natl Acad Sci USA 2006;103(52):19854–9. [29] Emmer BJ, van der Grond J, Steup-Beekman GM, et  al. Selective involvement of the amygdala in systemic lupus erythematosus. PLoS Med 2006;3(12):2285–90. [30] Levy DM. Presentation at the American College of Rheumatology National Meeting, Philadelphia. November 2009. [31] Mathieu A, Vacca A, Serra A, et  al. Defective cerebral gamma-aminobutyric acid-A receptor density in patients with systemic lupus erythematosus and central nervous system involvement: an observational study. Lupus 2010;20:1–9. [32] Brey RL, SL Holliday, Saklad AR, et al. Neuropsychiatric syndromes in lupus: prevalence using standardized definitions. Neurology 2002;58(8):1214–20. [33] Hanly JG, Hong C, Smith S, et al. A prospective analysis of cognitive function and anticardiolipin antibodies in systemic lupus erythematosus. Arthritis Rheum 1999;42(4):728–34. [34] Menon S, Jameson-Shortall E, Newman SP, et  al. A longitudinal study of anticardiolipin antibody levels and cognitive functioning in systemic lupus erythematosus. Arthritis Rheum 1999;42(4):735–41.

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[35] Harel L, Sandborg C, Lee T, von Scheven E. Neuropsychiatric manifestations in pediatric systemic lupus erythematosus and association with antiphospholipid antibodies. J Rheumatol 2006;33(9):1873–7. [36] Molloy ES, Calabrese LH. Progressive multifocal leukoencephalopathy in patients with rheumatic diseases: are patients with systemic lupus erythematosus at particular risk? Autoimmun Rev 2008;8(2):144–6. [37] Carson KR, Evens AM, Richey EA, et al. Progressive multifocal leukoencephalopathy after rituximab therapy in HIV-negative patients: a report of 57 cases from the Research on Adverse Drug Events and Reports project. Blood 2009;113(20):4834–40. [38] Papero PH, Bluestein HG, White P, Lipnick RN. Neuropsychologic deficits and antineuronal antibodies in pediatric systemic lupus erythematosus. Clin Exp Rheumatol 1990;8:417–24. [39] Mortilla M, Ermini M, Nistri M, Dal Pozzo G, Falcini F. Brain study using magnetic resonance imaging and proton MR spectroscopy in pediatric onset systemic lupus erythematosus. Clin Exp Rheumatol 2003;21(1):129–35. [40] Moritani T, Hiwatashi A, Shrier DA, Wang HZ, Numaguchi Y, Westesson PL. CNS vasculitis and vasculopathy: efficacy and usefulness of diffusion-weighted echoplanar MR imaging. Clin Imaging 2004;28(4):261–70. [41] Padovan M, Locaputo A, Rizzo N, Govoni M, Trotta F. The evaluation of neuropsychiatric lupus erythematosus by functional neuroimaging. Preliminary results. Reumatismo 2004;56(1):24–30. [42] Appenzeller S, Bonilha L, Rio PA, et al. Longitudinal analysis of gray and white matter loss in patients with systemic lupus erythematosus. NeuroImage 2007;34:694–701. [43] DiFrancesco M, Holland S, Ris D, Adler C, DeBello M, Altaye M, et  al. Functional magnetic resonance imaging of cognitive function in childhood-onset systemic lupus erythematosus: a pilot study. Arthritis Rheum 2007;56(12):4151–63.

20 Central Nervous System Vasculitis

Ludovico D’Incerti1, Francesco Deleo2, Gabriele Di Comite3 1 

Department of Neuroradiology, IRCCS Foundation Neurological Institute C. Besta, Milano, Italy 2  Division of Epilepsy, Clinic and Experimental Neurophysiology, IRCCS Foundation Neurological Institute C. Besta, Milano, Italy 3  Department of Immunology, National Institute of Neuroscience, Tokyo, Japan

Introduction to Vasculitides Vasculitides represent a group of highly heterogeneous disorders characterized by inflammation in the wall of blood vessels. Vasculitis, an immune-mediated mechanism, can occur as a primary process or can be secondary to an underlying condition, including infections, malignancies, connective tissue diseases, and drug use. Different immune processes develop according to etiology, and the resulting clinical pictures can vary broadly. Clinical manifestations range from mild single-organ involvement to systemic severe disease. When vasculitis is limited to a specific target organ, it most commonly affects the nervous system, the skin, the kidney, or the eye. In contrast, in systemic vasculitides, potentially life-threatening multiorgan involvement occurs [1]. The classification of vasculitides has been extremely challenging over the decades and several attempts have been made to classify them according to etiology, the clinical picture, or the underlying immunopathologic mechanism. In this regard, a major advance was achieved in 1990 when the American College of Rheumatology (ACR) published a set of classification criteria, grouping primary vasculitides according to the size of affected vessels. They identified three groups: small-vessel, mediumvessel, and large-vessel vasculitides [2]. This classification system is currently broadly accepted since vessels of different sizes are characterized by different molecular structures and, therefore, are targeted by different immunopathologic mechanisms. This eventually results in different clinical syndromes. In this chapter we will give an insight into the relevant clinical, pathogenetic, diagnostic, and therapeutic aspects of the central nervous system (CNS) involvement in primary and secondary vasculitides. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00020-4 © 2011 Elsevier Inc. All rights reserved.

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The CNS in Primary Vasculitides Primary Angiitis of the CNS Primary angiitis of the central nervous system (PACNS) is a rare disorder characterized by inflammation of small and medium vessels of the brain parenchyma, the spinal cord, or leptomeninges in the absence of systemic involvement and without evidence of underlying causative conditions. The term “PACNS” includes several subsets, differing in terms of histopathologic features, prognosis, and therapy [3]. The best-characterized syndrome, granulomatous angiitis of the CNS (GACNS), affects about 20% of patients with PACNS [4]. This disorder has a male predominance and may occur at any age. GACNS is usually characterized by a long chronic course, and acute cases have been rarely described. The clinical presentation varies broadly since any area of the CNS can be affected. The brain can be involved by focal or diffuse lesions, and meninges and the spinal cord are potential targets. Possible clinical manifestations include chronic headache, diffuse encephalopathy with behavioral and cognitive changes, stroke or recurrent transient ischemic attack (TIA), seizures, cranial or peripheral nerve dysfunctions, ataxia, and myelopathy [4]. When a diagnosis of PACNS is suspected, a careful history and physical examination should be performed to look for systemic vasculitis and signs of infections or other underlying diseases. If systemic vasculitis or infections are suspected, serologic tests should be performed accordingly. An essential tool in diagnosis is cerebrospinal fluid (CSF) examination, especially to rule out infections. CSF is abnormal in almost 90% of patients with PACNS, showing modest pleocytosis, normal glucose, elevated protein levels, and occasionally oligoclonal bands and increased IgG [5]. Culture tests and polymerase chain reaction (PCR) analysis can be extremely valuable to exclude infections. Neuroimaging is extremely important, although not sufficient, to reach a diagnosis of PACNS. Magnetic resonance imaging (MRI) has been shown to be more sensitive than computed tomography (CT). Typical, but not specific, findings include multiple and often bilateral infarcts in the cortex, the white matter, or the leptomeninges, with or without contrast enhancement (Figure 20.1) [6–8]. Cerebral angiography has little sensitivity in PACNS (10–20% in histologically proven cases) due to the size of affected vessels and poor specificity. Typical findings include the alternation of areas of smooth-walled segmental narrowing and areas of vascular dilation and occlusions, involving multiple cerebral arteries [9–11]. The same findings can be observed in the setting of atherosclerosis, infections, and vasospastic diseases, so the differential diagnosis can be extremely challenging. A definite diagnosis of GACNS is obtained only through biopsy, disclosing a picture of granulomatous angiitis involving small and medium arteries of the leptomeninges or the cortex, in association with Langerhans giant cells and/or lymphocytic vessel infiltration. Symptoms are related to ischemia of the territories supplied by affected vessels, which undergo occlusion because of necrotizing inflammation with thrombosis [12]. Most patients with PACNS are not diagnosed as having GACNS because their picture is less defined. This broad group includes patients with typical clinical and radiologic features of PACNS, without histologic demonstration of granulomatosis.

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Figure 20.1  Primary angiitis of the CNS responsive to immunosuppressive treatment. MRI shows alterations before (A–C) and after (D–F) treatment with immunosuppressant drugs. (A) Transverse T2-weighted image and (B) FLAIR images demonstrating multiple cortical and white matter signal hyperintensities in frontal and temporal areas. (C) Post-contrast T1-weighted image showing multiple foci of inhomogeneous enhancement in the cortex and in the subcortical white matter. Signal hyperintensity on T2-weighted image is consistent with brain edema, related to ischemic changes caused by arterial thrombosis and/or venular congestion. Cortical and subcortical enhancement is related to leptomeningeal and peripheral white matter inflammatory changes. (D and E) Incomplete regression of signal abnormalities on T2-weighted and FLAIR images, with expansion of the lateral ventricles. Cortical and subcortical hyperintensities consistent with gliotic changes are evident in frontal and temporal areas. In (D), the target of a brain biopsy is seen in left anterior insular white matter (black arrow). (F) Punctate enhancing lesions in right frontal subcortical white matter persist on post-contrast T1-weighted image (white arrow).

A recent paper described three morphologic patterns of vasculitis, with the granulomatous one being the most frequent (58%). The other patterns were purely lymphocytic (28%) and acute necrotizing with transmural fibrinoid vessel wall disruption (14%) [13]. Other patients diagnosed as having PACNS can present with atypical clinical features (such as mass lesions; Figure 20.2) but abnormal CSF analysis or evidence of vasculitis at biopsy or with lesions at unusual sites, such as the spinal cord [3]. A clinical entity to be distinguished from PACNS is reversible cerebral vasoconstriction syndrome (RCVS), representing its major differential diagnosis. Several studies on PACNS published in the past decades included patients with RCVS, thus generating some confusion. RCVS differs from PACNS in that it has a female

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Figure 20.2  Tumor-like lesion in PACNS. (A) Coronal FLAIR and (B) post-contrast T1-weighted image. Inhomogeneous contrast enhancement and perifocal edema mimicking brain tumor in the right temporoparietal area. (C and D) MRI after immunosuppressive therapy demonstrating regression of signal abnormalities and of the mass effect.

predominance, it is characterized by the acute onset of headaches with or without neurologic deficit, and it shows prolonged but reversible cerebral vasoconstriction [14]. Angiography is extremely useful in distinguishing PACNS from RCVS, since angiographic findings in the latter condition are reversible within days to weeks [15]. If PACNS is suspected, an additional cerebral angiogram after some weeks is advisable. RVCS is the most likely diagnosis when the lesions resolve spontaneously or after a short period of glucocorticoid therapy and when CSF analysis and biopsy are normal. Multiple bilateral asymmetrical infarcts, without contrast enhancement, in the gray and white matter can be observed on MRI also in RCVS, since vascular occlusion can be severe and long-lasting [3]. No large trials are available for the treatment of PACNS and RCVS due to the low incidence of the diseases and the difficulty in achieving a definite diagnosis. In clinical practice, treatment is largely extrapolated from the protocols adopted in systemic vasculitides. Corticosteroids and immunosuppressant drugs represent the mainstay of treatment, and cyclophosphamide is largely used in such patients to induce remission of disease activity. Empirically, after 3–6 months of induction with cyclophosphamide, it is possible to switch to less toxic drugs, including azathioprine, methotrexate, or mycophenolate mofetil. Serial MRI and CSF examinations at 3- to 4-month intervals

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are required to monitor response to treatment (see Figure 20.1D–F and Figure 20.2C,D). In patients with RCVS, immunosuppressant and high-dose corticosteroids should be avoided, since an inflammatory process is absent. Spontaneous remission has been observed, but most commonly calcium channel blockers, short-term corticosteroids, and magnesium sulfate are used to try to reverse vasoconstriction [3].

CNS Involvement in Systemic Vasculitides Overall, involvement of the CNS and peripheral nervous system (PNS) is common in primary systemic vasculitides. In contrast, such involvement is only occasionally observed in the setting of infectious diseases or malignancy or as a consequence of drug use [16]. When neurologic manifestations are the presenting symptoms, diagnosis can be extremely challenging. The pattern of PNS involvement in primary and secondary vasculitides is almost invariable, since it is related to occlusion of the vasa nervorum due to necrotizing small-vessel vasculitis. The ensuing picture is that of sensory axonal polyneuropathy or mononeuritis multiplex [17]. The range of possible manifestations of CNS involvement is much wider, since several mechanisms can be implicated. Intracranial vascular disease can be related to small-vessel vasculitis, affecting arterioles and venules of the hemispheres and spinal cord. This is a rare event in primary systemic vasculitides (Figure 20.3); it is instead the hallmark of PACNS and of secondary CNS vasculitis. Instead, thrombosis of dural sinuses, stenosis, and aneurysms are a consequence of medium- and large-artery involvement. Alternative mechanisms of CNS damage include granulomatous meningitis and cytokine-mediated damage presenting with encephalopathy. Moreover, extracranial vasculitis may induce CNS dysfunction, as in the case of carotid stenosis, vena cava syndrome, and renovascular hypertension. Vasculitis of the PNS is far more common than vasculitis of the CNS. This suggests that sophisticated mechanisms are at work to minimize local inflammation in the CNS and that they are probably missing in the PNS.

Large-Vessel Vasculitides The aorta and its major branches are commonly referred to as large vessels. Giant cell arteritis (GCA; also named temporal arteritis) and Takayasu’s arteritis (TA) are the members of this group of vasculitides. They share a histologic pattern characterized by granulomatous inflammation of the vessel wall. Typical features are transmural infiltration with lymphomononuclear and giant cells localizing between the intima and media tunica, intimal hyperplasia leading to lumen occlusion, and internal elastica lamina disruption resulting in aneurysm formation [18]. GCA has a prevalence of 2–20 per 100,000 people older than 50 years. The external carotid artery and its branches are the most commonly affected vessels. Occlusion of the superficial temporal artery, the occipital artery, the facial and lingual artery, and the intraorbital branches induces ischemia of the supplied territories and, consequently, determines the typical symptoms of GCA, which include headache, scalp tenderness, jaw or tongue claudication, and visual disturbances. In addition, fever and increased acute phase reactants reflect systemic inflammation [19].

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Figure 20.3  Small-vessel vasculitis of the CNS. (A, B, D, and E) Transversal T2-weighted images and (C) coronal FLAIR images in two patients. Nonspecific multiple foci of T2 hyperintensity (arrows) are observed in the cerebellar white matter and in the brain stem (A–C), as well as in the cerebral hemispheres (D and E).

Diagnosis is achieved by observing the typical symptoms and by temporal artery biopsy. Neurologic complications of GCA derive from the involvement of vessels supplying blood to the CNS or to cranial and peripheral nerves. In one report recruiting 80 patients with biopsy-proven active temporal arteritis, optic nerve involvement was the most frequent neurologic complication, observed in 23 patients. Cerebral ischemia was described in four patients and was always reported in the distribution of the vertebrobasilar circulation [20]. Lesions affecting the optic nerve are usually classified according to the anatomic part involved. They include anterior and posterior ischemic optic neuropathy and retinal ischemia. Anterior ischemic optic neuropathy is the most common event, with an overall incidence varying between 6% and 70% according to the series [21]. Anterior ischemic optic neuropathy is caused by occlusion of the medial posterior ciliary arteries and might be related to alternative disorders, which must be considered in the differential diagnosis. Ischemic events are central retinal and cilioretinal artery occlusions, occurring in around 14% of patients, and posterior ischemic optic neuropathy, occurring in 7% of cases [21]. Ischemic optic neuropathy is an ophthalmologic emergency, since it may lead to irreversible visual loss. Therefore, it requires prompt treatment with high doses of intravenous corticosteroids. In a multicenter report including 170 biopsy-proven GCA patients,

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the overall frequency of neuro-ophthalmologic manifestations was 50%. Interestingly, patients with ocular involvement showed higher levels of acute phase reactants and a higher rate of headache, myalgias, and systemic symptoms than other patients, suggesting a sustained systemic inflammatory process. Among those patients, permanent visual loss of varying degree was the most common symptom, observed in 98% of cases. The degree of visual impairment mainly depends on how soon the diagnosis is made and, consequently, how early an appropriate treatment is started. In the 85/170 patients with neuro-ophthalmologic manifestations, symptoms following in frequency visual loss were amaurosis fugax (30%), diplopia (6%), and eye pain (8%). Amaurosis fugax typically precedes permanent visual loss, and therefore its early recognition is crucial to avoid permanent damage [21]. In a historical Mayo Clinic series, bilateral visual loss was reported in 30% of GCA patients. The usual interval between the involvement of the two eyes was 1 day to 1 week, with the longest interval being 4 weeks [22]. The early funduscopic appearance in acute ischemic optic neuropathy consists of slight pallor and edema of the optic disk, with scattered cotton–wool patches and small hemorrhages. Later, optic atrophy occurs [19]. Although rare, stroke is another possible neurologic complication of GCA; it has been reported in 1–3% of patients [22]. Cerebral infarction is the most frequent manifestation (58%), followed by subarachnoid hemorrhage (24%) and cerebral hemorrhage (18%) [18]. Fatal cases due to brain stem infarction or to dissection of the intracranial portion of the vertebral artery have been described [23,24]. The mainstay of treatment in GCA is corticosteroids, which always lead to a prompt response in terms of symptoms and acute phase reactants. A starting daily dose of oral prednisone between 0.75 and 1 mg/kg of body weight is the standard of treatment, followed by gradual tapering in 12–18 months. If patients have visual disturbances, an initial dose of intravenous methylprednisolone, 500–1000 mg once a day for 3–5 days, is recommended to avoid permanent visual loss. Most commonly corticosteroids alone are sufficient to maintain the control of disease activity. In refractory cases immunosuppressant drugs can be added, namely methotrexate and azathioprine [25,26]. The anti-TNF-alpha monoclonal antibody infliximab was tested in a small clinical trial but failed to improve disease control [27]. The association of low-dose aspirin is always advisable [28]. TA patients present with a clinical picture that differs from that of GCA, since different vessels are involved. Clinical manifestations are related to ischemia and include claudication of the upper or lower limbs, angina abdominis, renovascular hypertension, and CNS ischemic symptoms. Diagnosis is achieved by demonstrating vascular stenosis and aneurysms, by means of angiography and color Doppler ultrasound, and by revealing thickening and contrast enhancement of the vessel wall by CT or MRI. The reported incidence of neurologic manifestations ranges from 60% to 80% according to the series [29]. However, it reaches 90% if nonspecific symptoms such as headache and vertigo are considered [30]. Dizziness is the most prevalent symptom related to cerebral ischemia. It may be related either to direct vertebral artery involvement or to stealing phenomena consequent to stenosis of the subclavian or posterior communicating artery [31]. Visual disturbances, resulting from involvement of the common carotid artery leading to decreased ophthalmic circulation,

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have been reported in approximately 40% of patients [30]. The most threatening manifestation of TA is stroke. Its incidence is unknown due to the absence of large epidemiologic studies. In one study describing 27 patients, it was reported in 7 of them (26%); ischemic stroke occurred in 5 patients and hemorrhagic stroke in 2, and TIAs were observed in 2 patients. Of note, among the patients with ischemic stroke, thromboembolic occlusion was noted in half of them. Thromboembolism has been supposed to be the effect of aneurysm formation of the aortic arch and its branches and of cardiac valves, leading to aortic regurgitation [31]. In another study conducted on 18 patients, microembolic signals in the middle cerebral artery were detected by transcranial Doppler sonography in 4 patients [32]. Intracranial artery stenoses due to vasculitis have been rarely detected with conventional angiography. It is therefore likely that stroke might be related to carotid or vertebral artery occlusion or to embolization into intracranial vessels rather than to direct involvement of intracranial vessels by vasculitis. Renovascular hypertension, related to renal artery stenosis with increased renin production, can also play a role, particularly in the development of hemorrhagic stroke [31]. The mainstay of therapy for TA is corticosteroids, although response is extremely variable, since the inflammatory process may be inactive at many sites of vascular stenosis. Acute phase reactants and neuroimaging are used to monitor response. Angioplasty or bypass grafts may be necessary once symptomatic irreversible arterial stenosis has occurred. In restricted cases, particularly those affecting the ascending aorta, surgery is recommended [33].

Medium-Vessel Vasculitides In 1994, the Chapel Hill Consensus Conference on the nomenclature of vasculitides distinguished classic polyarteritis nodosa (PAN) from its counterpart affecting small vessels, currently referred to as microscopic polyangiitis (MPA) or microscopic polyarteritis [34]. Before that date, studies on PAN included both conditions, and therefore the literature is confusing. Classic PAN is an extremely rare condition, and large studies describing this pure entity are missing. It is characterized by inflammation of medium vessels leading to microaneurysms, which can be detected by angiography. There is a strong epidemiologic association with hepatitis B virus (HBV) infection. Common manifestations include renovascular hypertension, related to kidney involvement, livedo reticularis and the typical subcutaneous nodules consequent to skin involvement, and mononeuritis multiplex or polyneuritis [35]. CNS involvement occurs in 20–30% of cases [36]. Most commonly, it develops after 2–3 years of active systemic disease and is accompanied by systemic symptoms, namely fever, malaise, myalgias, and wasting syndrome [37]. Almost always the brain is the target organ, although spinal cord involvement has occasionally been reported. In a historical large series of patients with PAN, 12% had CNS involvement alone and 34% had concurrent CNS and PNS involvement [38]. The most common manifestations of CNS involvement are diffuse encephalopathy, focal deficits, and seizures [36]. Neuroimaging typically shows infarcts in the regions of small peripheral cerebral cortical or subcortical arteries, which is consistent with the involvement of medium and

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small arteries [39]. Hemorrhage is a rare complication of intracranial vasculitis and is usually observed at sites where it is typically induced by hypertension in the general population. Therefore, renovascular hypertension is the possible cause of hemorrhage in patients with PAN [38]. However, in one reported series in which the role of hypertension was reliably excluded, intraparenchymal and subarachnoid hemorrhages were found in association with the typical histologic findings of arteritis resulting from PAN [40,41]. Interestingly, angiography shows that aneurysms are frequent in visceral arteries but uncommon in the intracranial circulation. The treatment of PAN can represent a serious challenge. Oral corticosteroids are the mainstay of therapy, but immunosuppressant drugs, including cyclophosphamide, have to be considered in the most severe and potentially life-threatening cases, including those with CNS manifestations. The frequent association with HBV infection has to be taken into account. The use of antiviral treatment, including lamivudine, vidarabine, and interferon-alpha, followed by plasma exchange is currently advisable [42,43]. Kawasaki disease (KD) is a common disorder among children. It is characterized by systemic inflammation with fever, conjunctivitis, erythema of the lips and the mucosa, cutaneous rash, and lymphadenopathy. It is usually self-limiting, lasting approximately 12 days without therapy. However, it may be complicated by coronary artery aneurysms with myocardial ischemia and vascular obstruction in peripheral arteries. Irritability and lethargy are common manifestations in KD, possibly reflecting CNS involvement. In a report considering 540 cases of KD, neurologic involvement was present in 1.1%. Acute or subacute encephalopathy, seizures, cerebral infarction, ataxia, myositis, aseptic meningitis, and lower facial nerve palsy have been described [44–46]. It may be postulated that the same vasculitic process that affects coronary arteries might be responsible for CNS manifestations. In 6 of 21 children with acute KD, single-photon emission computed tomography (SPECT) imaging demonstrated localized cerebral hypoperfusion without neurologic symptoms [47]. Although MRI scans revealed no abnormalities at the acute stage of the disease, the CNS manifestations associated with KD might have been due to focal impairment of blood flow caused by cerebral vasculitis. Important histologic postmortem findings in children who died of KD showed varying degrees of leptomeningeal thickening, mild endarteritis, and periarteritis [46]. Treatment of KD during the acute phase of the disease includes oral aspirin and intravenous immunoglobulins. Corticosteroids are commonly restricted to patients who fail to respond to the first-line therapy. Only exceptionally are plasma exchange and immunosuppressant drugs required [48].

Small-Vessel Vasculitides This group of disorders is characterized by necrotizing inflammation in the wall of small and medium arteries. The typical histologic features include vascular and perivascular infiltration with lymphomononuclear cells, leukocytoclasia (a term that refers to the presence of debris of dead neutrophils in the vessel wall), and fibrinoid necrosis, related to extensive endothelial activation with thrombosis and necrosis [49].

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This group of disorders includes two immune complex-mediated diseases, namely cryoglobulinemia and Henoch–Schönlein purpura (HSP), and the three disorders associated with the presence of antibodies toward the cytoplasm of neutrophils (antineutrophil cytoplasmic antibody [ANCA]): Wegener’s granulomatosis (WG), Churg– Strauss syndrome (CSS), and MPA. ANCA-associated vasculitides are also referred to as pauci-immune vasculitides due to the absence of immune complexes in histologic sections. Cryoglobulins are immunoglobulins that display the feature of precipitating at a temperature lower than 37°C. They are the hallmark of cryoglobulinemia and are induced by a concomitant hepatitis C virus (HCV) infection in 50–90% of cases. Only in a minority of patients are hematologic malignancies responsible for the formation of cryoglobulins [50]. In HSP, immune complexes are made up of IgA directed toward an unidentified antigen. Epidemiologic and clinical data support a relation with infections of the upper airways. Cryoglobulinemia and HSP are both characterized by skin involvement, in the form of palpable purpura, asymmetric oligoarthritis, and glomerulonephritis, although they occur with different frequency. The exact prevalence of the involvement of the nervous system in mixed cryoglobulinemia is unknown since studies on large cohorts of patients are not available. HCV genotypes 1b and 3 have been reported to induce neuropathic cryoglobulinemia more frequently than other genotypes [51]. In mixed cryoglobulinemic vasculitis, the PNS is commonly affected, whereas CNS involvement has been rarely reported. In a recent study conducted on 18 patients, evoked potentials were abnormal in 83% of them (sensory evoked potentials in 72%, visual evoked potentials in 44%, motor evoked potentials in 39% and brain stem auditory evoked potentials in 22%). Brain MRI revealed abnormal findings in 83% of patients (small T2-weighted hyperintense lesions in 72%, focal or diffuse atrophy in 50%) and cognitive impairment was observed in 22% [52]. Three cases of focal manifestations due to cerebrovascular events and 10 cases of encephalopathy resulting from focal or diffuse angiitis have been previously reported in essential cryoglobulinemia, with MRI showing bilateral white matter lesions. In addition, intracranial hypertension and hydrocephalus have been anecdotally observed [53]. HCV-associated cryoglobulinemic vasculitis should be treated in conjunction with a hepatologist. Treatment with IFN-α2b or pegylated IFN-α2b, both in combination with oral ribavirin, resulted in a complete clinical response in 63%, a sustained virologic response in 58%, and clearance of cryoglobulins in 46% of patients. Corticosteroids and immunosuppressant drugs are necessary when severe involvement, including CNS disease, is present. No large clinical trials are available in patients with cryoglobulinemic vasculitis and, especially in the absence of HCV infection, treatment is mostly extrapolated from the protocols in use in ANCA-associated vasculitis, which will be subsequently discussed. A systematic review of 13 papers reporting on 57 cases of cryoglobulinemia treated with the anti-CD20 monoclonal antibody rituximab reported a clinical response in 80–93% patients but a relapse in 39% of them [54]. Involvement of the nervous system in HSP has been rarely described. A review of 79 patients revealed that headache and mental changes are the most frequent manifestations, since they are observed in 71% of young patients, followed by seizures in 51% of cases and focal neurologic deficits in 14% [55]. When CNS vasculitis is

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present, MRI is the diagnostic tool of choice and it usually shows cerebral angiitis, more frequently over the parieto-occipital cortex [56]. Other mechanisms that may favor headache and mental changes include hypertension, metabolic derangements, and corticosteroid use. Resolution of HSP is usually spontaneous and only occasionally are corticosteroids used. The clinical picture in WG and CSS shows some similarities. In both cases, the inflammatory process starts in the upper and lower airways, in the form of a granulomatous reaction, directed against unidentified antigens. The nose, sinuses, and bronchi are affected. In CSS a long history of asthma and peripheral eosinophilia is typical and eosinophils are abundant in the granulomas. Therefore, allergens have been advocated as possible causative agents. In the following phase, the inflammatory process extends to extra-respiratory organs and ANCA increases in frequency. In this phase, granulomas are no longer present and a true vasculitic process, affecting the kidney, the skin, the PNS, and potentially any other organ, is observed [49]. CNS manifestations in patients with WG have been described in 3–33% of cases according to case series [57–60]. Three alternative pathogenetic mechanisms have been proposed: vasculitis of small vessels of the CNS; spreading of granulomas to the brain from contiguous paranasal structures; and development of granulomas far from upper airway structures [59]. We previously showed that the CNS involvement in WG, and particularly meningitis, typically develops in the phase of respiratory granulomatosis rather than during systemic vasculitis. During this phase, erosive lesions of the skull base occur more extensively, and therefore diffusion of granulomas to the meninges and the cerebral parenchyma is more probable [60]. Of 324 patients with WG described by Nishino et al. [59] in 1993, 90 (28%) had CNS manifestations. They presented with cranial neuropathy in 6% of cases, external ophthalmoplegia in 5%, cerebrovascular events in 4%, seizures in 3%, encephalopathy in 2%, and miscellaneous events in 8%. Pachymeningitis has also been reported. Although rare, it is characteristic of WG as compared with other forms of vasculitis. We previously described two patients with this condition and reviewed the literature [61]. Headache, multiple cranial nerve palsies, seizures, encephalopathy, proptosis, ataxia, and blurred vision are all possible manifestations of pachymeningitis in WG. MRI is the technique of choice to reveal cerebral vasculitis, diffuse or focal dural thickening, and enhancement of inflamed orbital and paranasal mucosa [60,61]. The nervous system is affected in 10–60% of patients with CSS, but almost always the PNS is the target organ. CNS involvement is extremely rare. Cerebrovascular events have been reported in association with cerebral angiitis and infarcts in the subcortical white matter. In a series of 29 patients with CSS and neurologic manifestations, only 3 patients had cerebral infarction, 1 patient had ischemic optic neuropathy, and another patient had bilateral trigeminal neuropathy [62]. Seven cases of subarachnoid hemorrhage and anecdotal reports of intraparenchymal hemorrhage and meningeal involvement have also been described [63–66]. CNS involvement in CSS almost invariably follows by years the onset of asthma and eosinophilia. MPA is characterized by systemic small-vessel vasculitis affecting the kidney, the skin, the joints, and the PNS, resembling the systemic vasculitis observed in WG and CSS. Conversely, the airways are almost always preserved. No granulomas develop

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and the only possible respiratory manifestation is hemorrhagic alveolitis. MPA is an extremely rare condition and few epidemiologic and clinical studies are available. No cases of CNS involvement in MPA have been reported to our knowledge, with the exception of a single case of pachymeningitis and a case presenting as capsular warning syndrome and subsequent stroke [67,68]. To conclude, one study investigating cognitive impairment in patients with ANCA-associated vasculitis without neurologic symptoms found that 30% of subjects had a subclinical neuropsychological impairment, characterized by mild abstract reasoning loss, mental speed reduction, and nonverbal memory impairment. MRI findings in those patients were consistent with the presence of small-vessel subcortical damage of the brain [69]. Treatment of patients with ANCA-associated vasculitides with CNS involvement must be prompt and aggressive. The standard treatment consists of oral or pulse intravenous high-dose cyclophosphamide in association with daily 1 mg/kg oral prednisone. While prednisone is gradually tapered, cyclophosphamide is continued for at least 3 months, resulting in activity remission in 80% of cases. In those cases immunosuppressant treatment can be shifted toward a less toxic drug, such as azathioprine, leflunomide, or methotrexate. In refractory cases cyclophosphamide must be continued for at least 6–9 months and additional treatment should be considered, including plasmapheresis, high-dose intravenous immunoglobulin, and rituximab [54].

Behçet’s Disease Behçet’s disease differs from all other forms of vasculitis in that it potentially affects arteries and veins of any caliber [34]. Possible manifestations include superficial thrombophlebitis and deep vein thrombosis, related to large vein occlusion; aphthous lesions of the mouth and genital mucosa, cutaneous lesions, arthritis, and parenchymal cerebral manifestations are consequent to involvement by small and medium vessels. Moreover, large arteries may be affected, as in the case of the lungs, accompanied by aneurysm formation. When the CNS is affected, the term “neuroBehçet” is used. The prevalence of neurologic manifestations ranges from 2.5% to 65% of patients according to case series [1]. Neurologic manifestations most commonly follow systemic manifestations, particularly mucocutaneous and articular ones. The largest series of patients with neuro-Behçet ever reported includes 200 Turkish patients (155 males and 45 females). Of them, 81% presented with parenchymal CNS involvement, affecting the brain stem in 51% of cases, the spinal cord in 14%, and the hemispheres in 15%. The predilection for the brain stem has been explained by regional hemodynamic properties. Telencephalic structures are drained by superficial and deep venous systems, anastomosing via medullary veins. Conversely, in the brain stem, there are few intraparenchymal anastomotic structures. Therefore, the absence of collateral venous pathways at the mesencephalic, diencephalic, and pontine levels favors ischemic lesions [70]. The clinical picture in patients with neuro-Behçet is typically characterized by pyramidal signs, headache, cranial nerve palsies, hemiparesis, behavioral changes, neuro-ophthalmologic and neuro-otologic manifestations, and sphincter disturbance [70–72]. Only one patient with multiple lesions affecting the parietal cortex, mimicking metastatic disease, has been reported [73].

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Parenchymal involvement is due to inflammation of small arteries and veins and is referred to as primary neuro-Behçet. Secondary neuro-Behçet affects 30% of patients and is related to nonparenchymal disease. Possible mechanisms include dural sinus thrombosis or vena cava syndrome with intracranial hypertension and medium-sized artery involvement with aneurysm formation and stenosis. Venous involvement presents with papilledema, headache, focal neurologic defects, seizures, nerve palsies, and altered consciousness. Artery involvement leads to intracerebral or subarachnoid hemorrhages or infarcts. Secondary neuro-Behçet is associated with a better prognosis than primary neuro-Behçet [70,71]. Approximately 50% of patients with neuroBehçet are moderately to severely disabled after 10 years. Cerebellar symptoms at the onset of the disease, a rapidly progressive course, and the presence of elevated protein levels and pleocytosis in the CSF are unfavorable prognostic factors, whereas initial presentation with headache is associated with a better outcome [74]. Parenchymal involvement can be recognized by MRI, which is more sensitive and specific than CT. Usually lesions are single contrast-enhancing and are located in the basal ganglion region or in the brain stem, extending to the diencephalic structures (Figure 20.4A,B).

Figure 20.4  Behçet’s disease. Transverse (A) and coronal (B) FLAIR and T2-weighted images demonstrating inhomogeneous midbrain hyperintensity. (C and D) Analogous intramedullary signal abnormality. Although this picture is not specific, it is consistent with neuro-Behçet’s disease.

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Alternatively, lesions can be bilateral or scattered in the basal ganglion region, the internal capsule, the hemispheres, or the medulla (see Figure 20.4C,D). The CSF usually shows an elevated cellular and/or protein count. In cases of nonparenchymal involvement, the CSF is normal in terms of cellular and chemical composition but may be under increased pressure [71]. Various histopathologic findings in parenchymal CNS involvement have been described, ranging from perivascular cuffing with lymphocytes or neutrophils to demyelination with vasculitis and multifocal necrosis [70]. Vasculitis of the vasa vasorum is supposed to be the cause of arterial aneurysms. In 46% of patients, a certain degree of cognitive impairment has been reported, unrelated to overt neurologic manifestations. This picture has been more commonly observed in patients with active disease and in those receiving prednisone [75]. The 2008 European League Against Rheumatism (EULAR) recommendations for the treatment of Behçet’s disease support the use of high-dose corticosteroids and immunosuppressant drugs, including cyclophosphamide, azathioprine, methotrexate, and TNF-alpha antagonists, in patients with neuro-Behçet. No standardized protocols, derived from large controlled trials, are available, and clinical practice is largely based on evidence from open trials and observational studies. [76,77]

Secondary CNS Vasculitides CNS vasculitis has been reported as a complication of several conditions, including infections, malignancy, connective tissue disorders, and drug use. When the clinical and radiologic pictures suggest CNS involvement, an appropriate diagnostic procedure should be performed. CSF analysis is crucial to identify an infectious cause. A careful history, an accurate physical examination, and usually total body imaging are required to identify other causes, especially malignancies. In many cases, CNS vasculitis mimics PACNS, so the diagnosis can be extremely challenging.

Infectious Vasculitis CNS vasculitis related to infectious agents most commonly affects small and medium vessels and the clinical and radiologic picture can closely resemble that of PACNS. Systemic manifestations of infection are absent in many case. Therefore, when a diagnosis of PACNS is suspected, it is mandatory to exclude an infectious cause before starting treatment with corticosteroids or immunosuppressant drugs. In particular, human immunodeficiency virus (HIV), varicella zoster virus (VZV), and syphilis have to be taken into account. Epidemiologic features can be extremely useful in this regard.

Varicella Zoster Virus Patients usually become infected with VZV during childhood, when they develop chicken pox. Neurologic manifestations often accompany this event in the form of mild neurologic symptoms, including headache, photophobia, and neck stiffness, consistent with mild symptoms of meningitis. More relevant manifestations are observed

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in only 1% of cases, with cerebellar ataxia being the most frequent. The vast majority of patients with chicken pox fully recover from neurologic complications [78]. Reactivation of the virus may occur, usually with transaxonal spreading to the skin and development of the typical rash. Only occasionally does VZV spread to the CNS and induce vasculitis. CNS vasculitis can both affect immunocompromised and immunocompetent individuals. A history of recent cutaneous rash supports the diagnosis, although CNS symptoms develop in the absence of cutaneous manifestations in a high percentage of patients [79]. VZV can affect both large and small vessels. The former is more common among immunocompetent patients, the latter in immunocompromised patients. Small-vessel vasculitis tends to be more localized and less severe than PACNS, with the exception of patients with acquired immunodeficiency syndrome (AIDS), who may develop diffuse vasculitis. Clinical manifestations include TIA and stroke, also of the spinal cord, when large vessels are affected. These conditions are usually associated with trigeminal nerve involvement. For unknown reasons, the ophthalmic division of the trigeminal nerve (herpes zoster ophthalmicus) is disproportionately affected compared with the maxillary and mandibular divisions. Angiography commonly reveals internal carotid artery involvement [78]. When small vessels are involved, meningoencephalitis with behavior and cognitive changes, myelitis, and ventriculitis are likely to occur [80]. Angiography uncovers segmental and unilateral involvement of vessels, most commonly in the territories of the middle cerebral artery, and MRI shows focal leukoencephalopathy, with small enhancing lesions, located deep in the white matter or at junctions of the gray and white matter [3,78]. Diagnosis includes CSF analysis. Infection is confirmed by the finding of an antibody titer higher in the CSF than in the peripheral blood. Detection of viral DNA in the CSF by PCR is a low-sensitivity tool. In a recent report of 14 patients with VZV-related CNS vasculitis, PCR tested positive in 28% of them; instead, antibodies in the CSF were detected in all cases [79]. The absence of VZV antibodies and DNA in the CSF definitely excludes the diagnosis. VZV DNA detection by PCR is extremely useful to monitor response to antiviral treatment. Recovery is associated with absence of symptoms and of DNA in the CSF.

Human Immunodeficiency Virus Diagnosing CNS disease in patients with AIDS is challenging. Most commonly, neurologic manifestations are related to CNS infection by opportunistic microbes, though 35% of histologic analyses revealed encephalitis, leptomeningitis, and/or vasculitis. Vasculitis most commonly follows the initiation of antiretroviral treatment, reflecting an immune reconstitution inflammatory syndrome. However, it has also been described independently from antiretroviral drug administration. Consistently, in these cases antiviral treatment can successfully treat CNS disease [81]. HIV-related vasculitis includes disease of extracranial large arteries, eventually leading to ischemic stroke, and involvement of intracranial medium-sized arteries, with or without aneurysm formation, and small vessels. The mechanism of HIV-induced vasculitis remains to be elucidated. Previous studies have demonstrated leukocytoclastic vasculitis of the vasa vasorum in extracranial carotid arteries and intimal leukocyte infiltration

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in intracranial medium-sized vessels [82,83]. Patients may be asymptomatic or may develop a broad range of manifestations, according to the size of affected vessels. Stroke and TIA reflect large-artery disease. In a recent series of 67 young patients presenting with ischemic stroke, HIV-related vasculitis was recognized as the causative mechanism in 6 of them [82]. Encephalopathy with cognitive impairment and nonspecific neurologic symptoms are observed in patients with vasculitis of small and medium-sized vessels [84]. MRI in HIV patients with CNS vasculitis usually reveals numerous lesions with gadolinium enhancement in both cerebral hemispheres. A major point to achieve diagnosis is excluding opportunistic infections and HIVrelated malignancies. Therefore, CSF tests with PCR analysis and culture tests are crucial [81]. When CNS vasculitis is related to immune recovery following antiretroviral drug use, treatment with corticosteroids, and occasionally with immunosuppressant drugs, is to be considered [85].

Treponema Pallidum Syphilis is a diffuse, worldwide disease caused by Treponema pallidum. It is by far more prevalent in poorly developed countries. In Western countries it is more commonly observed in young patients with AIDS. T. pallidum can affect the CNS in a minority of patients. Diagnosis of neurosyphilis is achieved when a positive treponemal test in blood and a positive Venereal Disease Research Laboratory (VDRL) test in the CSF are available. CNS involvement includes vasculitis, which is caused by T. pallidum infiltrating vessels in the subarachnoid space. It has been shown that the clinical picture of CNS vasculitis among patients with and without HIV infection is comparable. Meningovascular syphilis is associated with focal neurologic impairment in the territory of the affected vessels of the brain or spinal cord, resulting in thrombosis and ischemic or hemorrhagic stroke. In a series of 13 immunocompetent patients with CNS vasculitis, clinical manifestations included hemiparesis, ataxia, dysphasia and dysarthria, sensory levels due to spinal cord infarct, and diplopia [86]. CT and MRI show areas of ischemic or hemorrhagic stroke and atrophy is commonly associated [87]. Treatment of patients with neurosyphilis includes high-dose intravenous penicillin for at least 2 weeks. In a series of 29 patients treated and followed by Conde-Sendin et al. [86] 28% fully recovered, 55% underwent partial recovery, and 17% had no improvement at all.

Other Microbial Agents Other microbial agents have been reported to cause CNS vasculitis, including Borrelia burgdorferi, Taenia solium, Bartonella spp., Staphylococcus and Streptococcus spp., and Mycobacterium tuberculosis. B. burgdorferi, along with T. pallidum, belongs to the family of Spirochaetacheae. There have been several reports of patients presenting with signs of focal or diffuse CNS dysfunction, in association with MRI findings showing ischemic infarct, focal or diffuse vasculitis, demyelinating lesions, intracerebral hemorrhage, and meningitis [88–93]. In a selected case, angiography showed narrowing of the middle cerebral

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artery [90]. Diagnosis was achieved by detection of specific antibodies in the plasma or CSF and was confirmed by an adequate response to antimicrobial treatment. T. solium is the causative agent of cysticercosis, which is endemic in many developing countries. It presents with neurologic complications in many cases. However, CNS vasculitis is observed in a small proportion of patients, most commonly in the form of arachnoiditis leading to stroke [94]. Basal arachnoiditis can be detected by MRI and vasculitis of the main basal vessels can be demonstrated by angiography [95]. In an angiographic study on 28 patients, 53% of them had evidence of cerebral arteritis, 42% presenting with stroke. The most commonly involved vessels were the middle cerebral artery and the posterior cerebral artery [96]. Parenchymal stroke has also been described as a consequence of small perforating vessel inflammation and, only occasionally, of medium intracranial vessel involvement [97]. Treatment of cysticercosis includes corticosteroids and antiparasitic drugs [94]. Bartonella henselae has been anecdotally reported as a cause of basal skull vasculitis showing a meningitis-like picture [92], whereas Staphylococcus and Streptococcus spp. have been reported to cause medium-vessel vasculitis with the formation of aneurysms, leading to rupture and hemorrhagic stroke, as a consequence of embolism from foci of endocarditis [98].

Vasculitis Complicating Malignancies CNS vasculitis related to malignancy has been most commonly reported in association with Hodgkin’s and non-Hodgkin’s lymphoma, typically with MRI findings consistent with small- and medium-vessel vasculitis and with histologic evidence of granulomatous angiitis. In such patients biopsy is recommended, since CNS infiltration by lymphoproliferative cells should be excluded to define the proper treatment. Treatment of the underlying disease can lead to resolution of the CNS manifestations, although recurrence of vasculitis in the absence of cancer relapse has been described [4,99–103]. Only anecdotally has CNS vasculitis been observed as a complication of solid tumors, including a biopsy-proven case in a patient with breast cancer [104]; one case, diagnosed by MRI and angiography, in a 12-year-old patient with Ewing sarcoma [105]; and one patient with pheochromocytoma with angiographic evidence of vasculitis [106]. In one case a myelodysplastic syndrome was complicated by CNS vasculitis mimicking PACNS [107].

Vasculitis in Connective Tissue Disorders When CNS vasculitis is suspected, connective tissue disorders have to be considered as possible diagnoses. An accurate history, physical examination, autoantibody testing, blood examination including acute phase reactants, and body imaging are necessary to rule out connective tissue disorders. Rheumatoid arthritis (RA) is typically characterized by polyarthritis affecting small and large joints of the limbs in a symmetrical manner, often associated with the finding of the rheumatoid factor or autoantibodies against cyclic citrullinated

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peptides. In a relevant proportion of patients, inflammation can involve other organs and systemic vasculitis is possible, usually referred to as rheumatoid vasculitis. Typical manifestations include small-vessel vasculitis of the skin, causing digital gangrene and palpable purpura, mononeuritis multiplex or polyneuritis, serositis, and occasionally renal involvement. The CNS is rarely involved. CNS vasculitis frequently develops in patients with a long history of RA, irrespective of disease activity, and is associated with a high mortality rate despite treatment. The clinical picture is extremely variable, reflecting encephalopathy related to diffuse vasculitis, or being characterized by focal signs of hemorrhagic stroke. Seizures, dementia, cognitive and behavior disturbances, hemiparesis, ataxia, dysphasia, and nerve palsy are all possible manifestations. MRI and cerebral angiography findings are consistent with small- and medium-vessel vasculitis, and CSF examination can show high levels of the rheumatoid factor. Corticosteroids and immunosuppressant drugs are the mainstay of treatment [108–112]. Systemic lupus erythematosus (SLE) is the prototype of autoantibody-mediated diseases. It presents an extremely broad range of clinical pictures, from mild localized disease to life-threatening systemic involvement. The systemic injury may result from the direct action of autoantibodies toward the cellular surface, as in the case of hemolytic anemia; from vascular injury due to immune complex deposition, which is the mechanism responsible for glomerulonephritis, cutaneous manifestations, arthritis, and serositis; and from direct cytokine action, as in diffuse encephalopathy. CNS involvement in SLE is relatively frequent and several pathogenetic mechanisms have been postulated, including thrombosis due to antiphospholipid antibodies, noninflammatory small-vessel vasculopathy, local production of cytokines, and direct injury related to autoantibodies toward the neuron surface. Pathologic studies demonstrated in most cases the absence of inflammatory abnormalities in CNS specimens from patients with neuro-SLE, suggesting that vasculitis is not a main factor in the development of these syndromes. Small vessels may appear thickened, with thrombi and signs of hyalinization [16]. However, single cases of CNS vasculitis have been reported (Figure 20.5), mostly presenting with encephalopathy and cognitive disturbances. In these cases histology revealed a picture consistent with diffuse immune complex-mediated small-vessel vasculitis [113–116]. Anti-endothelium and antiphospholipid antibodies have been advocated as the possible cause of CNS vasculitis [116]. Treatment consists of intravenous corticosteroids and immunosuppressant drugs, including intrathecal methotrexate plus dexamethasone [117]. Sjögren syndrome is characterized by impairment of lacrimal and salivary gland function due to chronic lymphomononuclear infiltration, resulting in the so-called sicca syndrome (keratoconjunctivitis and xerostomia). CNS involvement is not uncommon and Sjögren syndrome can mimic multiple sclerosis. A cohort of 82 patients with Sjögren syndrome and neurologic manifestations has been previously described. In 68% of them the CNS was affected, with spinal cord involvement in 35% of cases, brain involvement in 40%, and optic neuropathy in 16%. MRI mainly showed white matter or spinal cord T2-weighted hyperintensities, and 30% of patients had oligoclonal bands on CSF examination [118].

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Figure 20.5  Systemic lupus erythematosus. (A and B) T2-weighted images demonstrating multiple focal hyperintensities from periventricular and subcortical white matter and from basal ganglia. (C) FLAIR image also demonstrates involvement of the right inferior parietal cortex. (D) Enhancement in the left caudate nucleus is observed in post-contrast T1-weighted image.

Sarcoidosis is a systemic granulomatous disease mainly affecting young and middle-aged individuals. Most patients present with bilateral hilar lymphadenopathy, pulmonary interstitial infiltrates, and ocular and skin lesions. Less commonly the heart, the liver, the spleen, the salivary glands, the muscles, the bones, the kidneys, and the CNS are involved. A total of 5–15% of patients with systemic sarcoidosis show neurologic involvement, and the symptoms may be observed at presentation. The CNS is most commonly involved, and only rarely as the single organ. When the meninges are involved, MRI discloses thickening and enhancement of the basilar leptomeninges, and CSF examination shows an elevated cellular immunoglobulin and protein count. Meningeal thickening may be responsible for aqueductal stenosis, leading to hydrocephalus, diabetes insipidus, and cranial nerve palsies, especially of VII, VIII, VI, and I nerves. Parenchymal localizations present as multiple or diffuse enhancing or nonenhancing lesions, which can result in focal symptoms, myelitis, encephalopathy, and dementia [119,120].

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Drug-Induced Vasculitis Vasculitis is not an uncommon side effect of drug use, although CNS vasculitis has been rarely reported [121]. Intravenous and intranasal use of cocaine has been associated with a few cases of meningeal vasculitis, clinically resembling infectious meningitis. MRI, angiographic, and histologic findings were consistent with small- and medium-vessel vasculitis and CSF analysis showed neutrophilic pleocytosis [122]. Cocaine use has also been shown to be associated with the development of ANCA and airway granulomatosis resembling WG, which in turn can lead to meningeal vasculitis [60]. Interestingly, propylthiouracil, a drug that not uncommonly induces ANCAassociated vasculitis, has also been reported to cause meningeal and CNS vasculitis [123,124]. This suggests a common underlying immunopathologic mechanism among cocaine and propylthiouracil users, driving the formation of ANCA and the spread of vasculitis to the CNS. Other drugs have been anecdotally reported to induce cerebral vasculitis, including gemcitabine [125], efalizumab [126], methimazole [127], TNF-alpha antagonists (namely etanercept, infliximab, and adalimumab [128]), Ecstasy [129], carbamazepine [130], and others.

Conclusions Overall, CNS vasculitis is not uncommon in the setting of primary systemic vasculitides or as a complication of other disorders, including connective tissue diseases, malignancies, and infections. The clinical manifestations can be extremely heterogeneous and can overlap among different conditions. Neurologists and rheumatologists should be aware of the broad range of clinical pictures they might encounter in clinical practice and should be familiar with the diagnostic tools available for differential diagnosis so that they can promptly treat patients and prevent permanent sensorimotor or cognitive function loss.

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21 Systemic Lupus Erythematosus—

Vasculopathy/Vasculitis, Susac Syndrome, and Myasthenia Gravis

Magdalena Olszanecka-Glinianowicz1, Antoni Hrycek2, Paweł Cies´lik2 1 

Department of Pathophysiology, Medical University of Silesia, Katowice, Poland 2  Department of Internal, Autoimmune and Metabolic Diseases, Medical University of Silesia, Katowice, Poland

Introduction The development of systemic connective tissue diseases, formerly referred to as collagen tissue diseases, is related to an immune system dysfunction and continuous overproduction of pathogenic antibodies with formation of immune complexes. Genetic, environmental, infectious, and endocrine factors, as well as the process of aging, contribute to the development of these diseases. The incidence of systemic connective tissue diseases is differentiated. In clinical practice, rheumatoid arthritis and systemic lupus erythematosus (SLE) are the most frequent entities. It is noteworthy that relatively rare diseases from this group account for most diagnostic difficulties [1]. SLE is an autoimmune disease with no predilection to involve specific organs. It is characterized by the presence of anti-native DNA and anti-SM (Smith) antibodies, which may contribute to a sustained pro-inflammatory state [2,3]. The signs and symptoms of SLE are diverse and nonspecific. A likely presentation of SLE includes fatigue, malaise, oral ulcers, arthralgia, photosensitive skin rashes, lymphadenopathy, pleuritic chest pains, dry eyes and mouth, Raynaud’s phenomenon, and mild hair loss. Moreover, patients may present with dysfunction of major organs, including kidneys and lungs [4]. Severe neurologic symptoms related to central, peripheral, and autonomic nervous system disturbances are also frequently observed. More often these patients are diagnosed with headache, polyneuropathy, and cerebrovascular disease. It was shown that antiphospholipid syndrome (APS) increases the risk of nervous system involvement [5]. SLE subjects commonly (prevalence from 17% to 75% depending on different selection of patient and methods of assessment) present with psychiatric abnormalities such as cognitive dysfunction, acute confusional state (delirium), anxiety disorders, mood disorders, and psychosis. Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00021-6 © 2011 Elsevier Inc. All rights reserved.

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It is suggested that a possible mechanism of the development of these diseases is vascular injury by pathogenic antibodies [6]. Vasculopathy is a typical pathology in SLE, reported in 10–40% patients. It occurs more often in women (80%) than in men and may precede the development of a full-blown SLE [4,7,8]. Signs and symptoms of various autoimmune diseases may coexist with the previously diagnosed SLE. Such a pathologic link is observed in 30% of SLE patients [9–11]. The most common concomitant diseases include Sjögren syndrome (13% of cases), followed by rheumatoid arthritis and thrombocytopenia (both 6% of cases), and finally APS and hypothyroidism (both 4% of cases) [9]. Nevertheless, occasionally the diagnosis of another autoimmune disease, like myasthenia gravis (MG), is documented in SLE. As it was described earlier, vasculitis is the main pathomechanism of the nervous system injury that develops in SLE patients. Vascular damage in humans develops due to inflammatory and non-inflammatory mechanisms. This damage may be induced by environmental factors (toxic agents, medications, microorganisms) or cancers (as a component of paraneoplastic syndrome), or may be directly associated with an active immune process. The differentiation of the type of vascular damage is very difficult, sometimes impossible, and requires in-depth immune, histopathologic, and imaging diagnostic approaches and professional clinical experience. It may play a key role in the selection of treatment strategy and establishment of prognosis. Therefore, the identification of the etiology, pathomechanisms of the clinical and histopathologic settings, and SLE-associated vascular complications is of great clinical significance. A typical lupus vasculopathy includes vasculitis with inflammation and vascular wall necrosis, followed by thrombus formation in the lumen and finally occlusion of the affected artery [12–14]. Based on the intensity of the selected features, SLE vasculopathy is divided into several types, such as noncomplicated vascular deposits of immune complexes, non-inflammatory necrotic vasculopathy, thrombotic microangiopathy, and true lupus vasculitis [12,13]. Leukocytoclastic inflammation constitutes more than 60% of all lupus vasculitis cases, vasculitis with cryoglobulinemia 30%, and systemic vasculitis resembling polyarteritis nodosa about 5% of cases [8,13,15–17]. The remaining 5% of clinical syndromes of SLE vasculitis include thrombocytopenia with thrombotic purpura, venous thrombosis, APS, and urticarial vasculitis [8,13]. Lupus vasculopathy usually involves cutaneous vessels, renal glomeruli, coronary and brain vessels, the brain, lung alveoli, and less often the gastrointestinal tract [18]. Cutaneous lupus vasculopathy in SLE occurs most often and is reported in 94% of patients with lupus vasculitis [8,19,20], while the vessels of internal organs are affected in only 18% of SLE vasculitis patients [8]. The typical renal presentation of vasculitis is focal segmental glomerulonephritis with fibrinoid necrosis. In lungs vasculitis appears as necrotic alveolar capillaritis predisposing to pulmonary hemorrhage [18]. Brain vasculitis occurs in only about 10% of SLE patients and is associated with highly variable clinical symptoms—from a mild cognitive dysfunction to severe psychosis and convulsions, local ischemia, and strokes [18,21]. The peripheral nervous system may also be affected by lupus vasculopathy, leading to multifocal inflammatory mononeuropathies.

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Selected Etiologic Factors Vasculitis in SLE may be induced by many factors. Some drugs, such as penicillin, allopurinol, thiazides, pyrazolones, retinoids, streptokinase, cytokines, monoclonal antibodies, chinolones, hydantoin, carbamazepine, and other anticonvulsants, may play a role in the induction of inflammatory vascular lesions in SLE. These drug molecules may act as haptens, altering the antigen properties and stimulating the formation of an autoantigen [18]. Vasculitis may be preceded by the direct attack of microorganisms on the blood vessel wall or may be caused by an infected thrombotic mass [22]. It seems that hepatitis C virus takes part in the development of vasculitis with cryoglobulinemia [18,23]. The relationship between blood cryoglobulins and hepatitis C infection remains unexplained. Also, other viruses and bacteria such as cytomegalovirus and Staphylococcus may induce SLE vasculitis. The following mechanisms of infection-induced SLE vasculitis have been suggested: direct attack on the vascular wall by a virus, directly inducing an inflammation; activation of endothelial cells and their lesion; and binding of bacterial antigens to the basement membranes and binding to IgG specifically, which in turn induces an immune response and an inflammatory process [22].

Pathophysiology of Vasculopathy and Vasculitis in SLE As mentioned previously, SLE vasculopathy may be of inflammatory or thrombotic origin [8]. Both mechanisms involve the immune system and endothelium [8,24]. It seems that endothelial cell activation and pronounced expression of adhesive molecules are the key factors in the pathogenesis of this disease [19,24]. Cellular adhesion molecules are classified into three groups: selectins, integrins, and immunoglobulin superfamily (IGSF) [25]. E-selectin expression is limited to the activated endothelial cells and is stimulated by interleukin (IL) 1 and tumor necrosis factor (TNF) α [25]. E-selectin influences the initial adhesion of non-activated leukocytes (neutrophils, monocytes, and immune memory T lymphocytes) to the activated endothelial cells, enables leukocyte rolling, and possibly plays a role in intracellular transmission [26]. The leukocyte surface integrins include the lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4). These integrins bind to the IGSF receptor group, which includes adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). The expression of both adhesion molecules on the endothelium surface requires induction by inflammatory cytokines: TNF-α, IL-1, and interferon (IFN) γ [25]. The adhesion molecule VCAM-1 reacts with the VLA-4 chain expressed by monocytes, lymphocytes, basophils, eosinophils, macrophages, and glomerular parietal epithelial cells [26]. E-selectin, ICAM-1, and VCAM-1, with their ligands (i.e., sialyl Lewis X [sLeX], LFA-1, and VLA-4), are probably the predominant adhesion molecules locally participating in SLE inflammation. It has been demonstrated that LFA-1

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expression is increased on peripheral blood lymphocytes in SLE patients. Increased levels of circulating LFA-1 and VLA-4 have also been reported in vasculitis patients [25]. The anti-dsDNA and IgG class of anti-endothelial cell antibodies (AECA) have been reported to increase the expression of cellular adhesion molecules. Antibodies and immune complexes induce the cytokine synthesis and increase the expression of endothelial cell adhesion molecules, with granulocyte aggregation as a secondary effect [26]. The induction of ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells (HUVEC) with the use of purified IgG from SLE patients has been demonstrated. In active SLE patients a strict endothelial cell lymphocyte adhesion has not been confirmed; however, the results of studies dealing with this issue were inconclusive. Moreover, T lymphocytes of patients with active SLE and lymphopenia were characterized by the IL-1–stimulated decreased adhesion potency to HUVEC. It is postulated that cells of potent adherence may be eliminated from the bloodstream, which could explain the lymphocyte count decrease in active SLE [25]. Cellular adhesion molecules enable leukocyte adhesion and rolling along the endothelial cell surface and the control of leukocyte permeation to tissues affected by inflammation [25,26]. Enlargement of the granulocyte cell surface and enhancement of the expression of endothelial adhesion molecules determine the adhesion of these cells to the vascular wall and their secondary permeation to the extravascular space [26]. The adhesion molecules are also a molecular signal for the immune memory lymphocytes toward a certain antigen and enable a constant lymphocyte flow through tissues, where a certain antigen has been discovered for the first time [25]. Increased levels of soluble forms of adhesion molecules and IL-6 were reported in the plasma of SLE patients, indirectly indicating the activation of endothelial cells [27]. These molecules are enzymatically released from the cytokine-activated cell surface. Several studies demonstrated a correlation between their levels and disease activity [25,26]. In SLE patients, plasma sVCAM-1, sE-selectin, and sICAM-1 levels are increased. It has been shown that sVCAM-1 levels correlate with SLE activity [26]. The sVCAM-1 serum levels were increased in the active forms of lupus nephritis, neuropsychiatric SLE, and deep vein thrombosis associated with APS in SLE patients. The sVCAM-1 titer correlated with the severity of thrombotic complications and thrombotic microangiopathy with renal failure. It has also been demonstrated that the levels of all soluble adhesion molecules, such as sVCAM-1, sICAM-1, and sE-selectin, correlated with the antiphospholipid (aPL) antibodies titer. A relation between sVCAM-1 and soluble thrombomodulin has been demonstrated [25]. The sE-selectin and sICAM-1 levels are increased, reflecting the multiorgan damage [28]. There are suggestions that soluble adhesion molecules may play a protective role in SLE, like sE-selectin may protect against the development of lupus nephritis. Several authors have suggested that it could exert some physiologic effects by influencing the interactions between endothelial cells and circulating leukocytes; other studies, however, demonstrated that sE-selectin is rather an inactive decomposition product. Similar findings refer to the role of sP-selectin [25]. Activated endothelial cells are able to bind various proteins and cells to the vessel wall. This process is at first limited to postcapillary venules, which are often affected in small-vessel disease. However, localization of vasculitis in arterial branches is

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most probably the result of compression forces. The location of damage may also depend on the hydrostatic pressure values and local blood circulation disorders [19]. The common hypothesis for SLE vasculopathy concerns the endothelial deposition of circulating immune complexes [22], followed by activation of the complement cascade, most probably with C5b-9 complex formation, and destruction of vascular basal membranes [18]. However, there are also other mechanisms causing the immune response to antigens primarily located in the vessel wall. For example, the nucleohistone adhesion to the vessel basement membrane in lupus nephritis results in secondary antibody binding and the local formation of complex [22]. Moreover, the clearance of immune complexes is decreased and the tissue factor expression is upregulated, which also may contribute to SLE vasculopathy [29]. Most probably, the C1q complement component and its receptor in the endothelial cellular membrane also take part in endothelial activation/damage [21,24]. Another complement system component, C5a, may induce endothelial release of heparan sulfuric acid, which produces a pro-thrombotic effect in lupus vasculitis [21]. There are many autoantibodies and circulating immune complexes in SLE that directly or indirectly affect endothelial cells, causing chronic vessel wall damage [24,29]. The activation and damage of endothelial cells and the associated monocyte adhesion are caused particularly by antibodies against endothelial cells and cell membrane phospholipids [30]. Systemic vasculitis in SLE is the proatherogenic condition and is characterized by leukocyte activation and increased production of cytokines and other inflammatory mediators [24,29,31]. The role of antibodies in the pathogenesis of SLE vascular damage cannot be overlooked. In over 80% of SLE subjects, AECA are detected [32,33]. It is suggested that their presence is typical for vasculitis and vascular thrombosis and lupus nephritis. These antibodies rarely cause a simple cytotoxic effect [27]. AECA belong to the G, M, and A immunoglobin classes [32] and bind to antigens through the F(ab)2 region [1]. The structure of these antigens has not been fully determined [18,22,34]. It has been demonstrated that they are constitutive surface proteins of 25–200 kDa molecular mass [27], different from class II human leukocyte antigens (HLA) and blood group antigens [18]. The list of endothelial cell AECA antigens includes heparin-like compounds, DNA and DNA–histone complexes, PO and L6 ribosome protein, elongation factor 1, adenyl cyclase-associated protein, profilin II, plasminogen activator inhibitor, fibronectin, and β2 glycoprotein-I [21,32]. AECA are not specific to endothelial cells, as antigen determinants for them are also present on fibroblasts, leukocytes, and blood monocytes [18]. These antigens induce a proinflammatory and pro-adhesive endothelial cell phenotype through the NF-κB activation pathway [35]. The AECA-induced endothelial cell injury is associated with increased monocyte adhesion and enhanced expression of adhesion molecules such as E-selectin, ICAM-1, and VCAM-1, and oversecretion of chemoattractants and cytokines such as monocyte chemoattractant protein-1 (MCP-1) and IL-1, IL-6, and IL-8 [21,27,32,33,36]. AECA may activate the complement, and most probably this mechanism is involved in the pathogenesis of vasculitis [18,27]. It was demonstrated that plasma of patients with systemic vasculitis displayed AECA antibody-associated cytotoxicity, which occurs uniquely in the presence of lymphocytes. The monoclonal AECA were

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increasing IL-6 secretion, which may accelerate the lymphocyte infiltration rate, and causing vascular damage. AECA of IgG class probably activate leukocytes, which infiltrate the blood vessel wall. The in vitro experiments demonstrated enhanced leukocyte adhesion to HUVEC, related to higher expression of adhesion molecules. This creates conditions for maintaining cell adhesion to the vessel wall and for proinflammatory endothelial cell activation, manifested by adhesion molecule expression and increased cytokine secretion [27]. AECA may also induce endothelial cell apoptosis independently of Fc receptor [33,37]. An AECA antigen that presumably determines the apoptosis process is heatshock protein 60 (Hsp60) [37]. A cross-reaction between AECA and Hsp60 has been demonstrated in patients with SLE vasculitis [38]. AECA also participate in thrombus formation in SLE patients. They may react with various components of endothelial cells and the extracellular matrix, including proteoglycan molecules, bound with negatively charged glycosaminoglycans (GAG) (i.e., heparan sulfate and hyaluronate) [39]. AECA-dependent endothelial cell activation may increase the release of plasminogen activator inhibitor 1 (PAI-1), platelet activating factor [36], and thrombomodulin and enhance von Willebrand factor synthesis [32,33], in this way inducing pro-thrombotic lesions of blood vessels. It has been suggested that soluble thrombomodulin is a marker of endothelial damage in the microcirculation [25]. Moreover, it was suggested that AECA probably take part in the pathogenesis of urticarial vasculitis with the C1q deficiency and the IgG anti-C1q antibody presence. These antibodies may specifically induce angioedema, joint pain, eye inflammation, glomerulonephritis, and chronic obstructive pulmonary disease [18]. In turn, the anti-neutrophil cytoplasmic autoantibodies (ANCA) belong to a heterogeneous family and are reported in all immunoglobin classes directly against cytoplasmic antigens contained in azurophilic granules of neutrophils and monocyte lysosomes [7,40,41]. According to indirect fluorescent labeling images, there are cytoplasmic (cANCA) and perinuclear ANCA (pANCA), and atypical, cANCA and pANCA, forms [7,40–43]. cANCA antibody activity is directed against the proteinase 3 (PR3), while pANCA antibody activity is directed against myeloperoxidase (MPO). However, they also react with other proteins [7,42–44]. Initially, it was thought that the presence of ANCA was associated only with primary systemic vasculitis. An association of cANCA (PR3-ANCA) with Wegener’s granulomatosis, and that of pANCA (MPO-ANCA) with microscopic polyangiitis, and crescentic glomerulonephritis, has been demonstrated [40,41]. However, ANCA antibodies were also detected in secondary vasculitides associated with systemic connective tissue diseases, including SLE (15–20% of patients). In SLE usually pANCA have been reported [7]. ANCA-associated vasculitis is characterized by focal necrotic foci of capillaries, venules, and sometimes arterioles [45]. It is suggested that necrosis development may be the result of microcirculation sequestration of activated neutrophils and monocytes [45,46]. Various cytokines, chemokines, bacterial lipopolysaccharides, and intestinal toxins activated cells act to increase the membrane expression of azurophilic granules, which contain the ANCA-specific antigens PR3 or MPO [45]. At this stage ANCA

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complexes with PR3 or MPO would activate neutrophil adhesion to endothelial cells, with consequent extravascular permeation, vessel damage, and apoptosis development [7,41,46]. A direct involvement of integrin and cytokine receptors and a protective effect of β2-integrin antibodies in the endothelial damage have been demonstrated [46]. It has also been shown that the IgG, PR3-ANCA antibodies stimulate neutrophil degranulation and lysosome enzyme release, and also highly reactive forms of oxygen and nitric oxide [7,45]. Lysosome enzymes probably cause the vascular basement membrane damage. PR3 release takes part in proteoglycan and elastin degradation, endothelial cell damage, and apoptosis induction [45]. The activating effect of PR3 and MPO on T-helper CD4 lymphocyte proliferation has also been postulated. Probably ANCA also take part in the induction of inflammation and inhibition of PR3 binding with the α1-antitrypsin inhibitor, as a result decreasing the antigen and immune complex decomposition [7]. The role of ANCA in systemic vasculitis in SLE patients is unclear, as the relation between the presence of these antibodies and SLE-specific organ complications has not been demonstrated [40]. The following antibodies that may participate in the vascular damage in SLE are anti–double-stranded DNA antibodies (anti-dsDNA). They possess an anti-endothelial activity against certain antigens on the endothelial surface [27]. However, their direct cytotoxic effect on endothelial cells has not been demonstrated [18]. The synthesis of IL-1 and IL-6 induced by anti-dsDNA has been demonstrated, which indirectly indicates endothelial cell activation [32]. The anti-DNA–histone complexes have a similar effect [27]. Also, circulating DNA fragments contribute to SLE vasculitis pathogenesis. The stimulation of endothelial cell ICAM-1 expression and the increase in mRNA IL-6, IL-8, TNF-α, and IFN-γ synthesis, with participation of DNA fragments, have been shown [47]. The aPL antibodies are responsible for vascular damage in APS. One of the secondary forms of APS is observed in SLE patients [48]. It has been postulated that aPL antibodies impair endothelial function and promote thrombus formation during infection or pregnancy [30]. It has been also suggested that endothelial damage, regardless of the origin, exposes endothelial cell phospholipids, which enables the adhesion of aPL antibodies [19]. These antibodies cause further vascular endothelial damage and increase the arterial and venous thrombosis risk and proliferative heart valve lesions. The antigen specificity of aPL antibodies includes the negatively charged cell membrane phospholipids (i.e., cardiolipin [CL], diphosphate glycerol, phosphatidyl ethanol amine, phosphatidyl ethanol serine). Lysophosphatidylcholine (LPC) may also induce an immune reaction, leading to vascular inflammatory lesions and other SLE symptoms. The β2 glycoprotein-I and other plasma proteins may change the phospholipid antigenicity through neo-antigen formation [48]. The pathogenetic action of aPL antibodies is variable. When binding with membrane phospholipids, aPL may inhibit reactions catalyzed by them in the coagulation cascade, for example, by inhibiting C and S protein activation. These antibodies may also activate endothelial cell thrombin formation. The binding of aPL antibodies with platelet membrane phospholipid binding protein predisposes to platelet activation and adhesion, with consequent thrombus formation. These antibodies probably activate the complement system. As a result, the aPL antibodies demonstrate pro-adhesive,

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pro-inflammatory, and pro-thrombotic effects on endothelial cells. The activity of aPL antibodies may also form a link to early atherosclerosis in SLE patients [30]. As mentioned before, vasculopathy and vasculitis may play important roles in organ damage and symptoms of numerous autoimmune diseases. One of the rare vasculopathies caused probably at least by aPL antibodies is Susac syndrome, which is characterized by a triad of symptoms: encephalopathy, obliteration of branches of the retinal artery, and hearing loss.

Susac Syndrome as an Example of a Rare Autoimmune Vasculopathy This is one of the rare autoimmune diseases providing great diagnostic difficulties. So far only about 100 cases have been reported worldwide. Perhaps the real incidence of Susac syndrome is higher, however, as it can be misdiagnosed as, for instance, multiple sclerosis, migraine, SLE, encephalitis, Ménière’s disease, ischemic stroke of embolic etiology, aseptic meningitis, Lyme disease, Creutzfeldt–Jakob disease, schizophrenia, and so forth [49–51]. In addition, the differential diagnosis of Susac syndrome should include not only vasculitis associated with APS or SLE but also Wegener’s granulomatosis, as hearing loss may be the first symptom of the disease [52,53]. This entity was diagnosed for the first time in 1974 by Professor Susac in patients with these symptoms [49,54]. He thought initially that the lesions were a form of granulocytic vasculitis, but this disease was associated with vision disorders and hearing loss. As these disorders affected only small precapillary arterioles, he named them “microangiopathy,” probably caused by the immune disorders. Professor Susac encountered further cases of this syndrome in 1986 and presented one of them during the Neuroophthalmic Symposium, when Dr. William F. Hoyt was the first to use the term “Susac syndrome” [50]. Mass et al. [55] proposed another term, “RED-M” (retinopathy, encephalopathy, and deafness related to microangiopathy). Schwitter et al. [56], to describe these disorders, used the name SICRET (small infarcts of cochlea, retinal, and encephalic tissues), whereas Petty et al. [57] described them as retinocochleocerebral vasculopathy. Up to 1994 Susac syndrome was reported in 20 young women aged 21–41 years, and therefore it seemed that it occurred only in females. However, since then it has been reported in men as well. The disease affects females 3 times more often than males. No age-related increase in incidence has been observed: it has been reported both in children (recently in a 9-year-old girl) and in the elderly, aged 70 years [58–60]. Factors contributing to the syndrome and its pathogenesis are obscure. One of the hypotheses assumes that a viral infection (with an unknown pathogen) can be a cause of an autoimmune reaction leading to microvascular injury. Antiphospholipid antibodies, factor V Leiden mutation, and protein S deficiency have been observed [50,57,61,62]. It still has not been explained why, in the majority of patients, the pathologic process is restricted to blood vessels in the brain, eyes, and ears.

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However, some patients develop systemic symptoms, and changes can be observed also in small blood vessels in muscle tissue biopsies, which suggests a systemic character of the disease, with a predisposition toward the characteristic triad [59]. Some authors assume that Susac syndrome results from the vasoconstrictive syndrome, but this hypothesis seems rather unlikely. Symptoms have been observed during pregnancy and hormone therapy, which suggests a contribution of sex hormones to the pathogenesis [50,62]. As mentioned, Susac syndrome is characterized by a triad of symptoms, but at the onset of the disease the majority of patients do not develop a full triad; they may manifest later, after several weeks or even years. This makes the diagnosis difficult in the initial phase. Neuropsychiatric disorders are observed in 75% of cases in the initial phase of the disease. Hearing and vision disorders are observed in only 10% of patients [50,62]. Encephalopathy in Susac syndrome may present a diverse course and can manifest as severe headaches (sometimes migraine-like), personality changes, confusion, memory impairment, and psychiatric disorders. Due to serious psychiatric manifestations, sometimes with productive symptoms, some patients with Susac syndrome can be referred to psychiatric departments. However, the accompanying multifocal neurologic symptoms usually make it possible to distinguish this syndrome from true psychiatric disease. In some cases, the only way to make the appropriate diagnosis is magnetic resonance imaging (MRI) of the central nervous system, which can visualize ischemic lesions of the corpus callosum, the amygdaloid body, basal ganglia, the hypothalamus, and meninges [54,61,63,64]. Cranial nerves in Susac syndrome are not affected, and deafness is the result of direct cochlear injury. In a similar way, vertigo results from injury to the semicircular canals. Visualization imaging is still useless for detecting microinfarcts in these structures [65]. Occlusion of the retinal artery branches is most difficult to diagnose, because vision disorders (i.e., reduced vision acuity and restriction of a vision field) can be mild and thus less noticeable to the patient than other symptoms. Besides, these disorders rarely occur in the initial phase and develop only with longer duration of the disease [63,66]. The clinical course of Susac syndrome can be active, variable, and self-limiting. The active phase of the disease varies in duration, usually lasts several months, and after resolution leaves various cognitive and functional defects. Some patients recover with only mild persistent symptoms and signs, while others develop profound cognitive deficits, gait disorders, and deafness. In the majority of cases serious vision injury is not observed. The duration of remission may range from several months to 10–20 years [62]. Detailed laboratory tests fail to demonstrate typical abnormalities of autoimmune diseases, clotting disorders, and infectious diseases. In some patients aPL antibodies, factor V Leiden mutation, and protein S deficiency have been found. There are no elevated protein levels in cerebrospinal fluid. Sometimes moderate pleocytosis, usually accompanied by lymphocytosis, is detected. Occasionally elevated IgG levels with increased synthesis of these antibodies and oligoclonal bands can lead to the misdiagnosis of multiple sclerosis [57,62].

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The changes in the electroencephalogram recording are also nonspecific [51]. The results of cerebral arteriography are almost always normal, because the involvement of precapillary arterioles (<100 μm) cannot be visualized with this technique. Fluorescein angiography is very useful in the diagnosis of Susac syndrome. This test frequently reveals an occlusion in arterial branches of the retina, as well as pathognomonic multifocal fluorescence in arterial branches [66,67]. Loss of hearing in an audiometric test is usually observed in low and middle frequencies. Vestibular dysfunction is evaluated with a caloric test [65,68,69]. As mentioned previously, MRI of the central nervous system is a very useful technique in the diagnosis of Susac syndrome for detecting ischemic lesions located in the corpus callosum (central fibers with sparing of peripheral fibers), amygdaloid body (almost always), basal ganglia and hypothalamus (70%), and meninges (33%). Of interest, no close relationship between the severity of encephalopathy and the intensity of lesions detectable on MRI has been observed. The resolution of clinical symptoms of encephalopathy is followed by the disappearance of typical lesions in the white matter, but an evident atrophy remains. Small cerebral cortex microinfarcts are not visible on MRI [70– 72], but their presence there can be conceived, as they have been described in biopsy specimens of the cerebral cortex, the white matter, and meninges, together with mild perivascular inflammatory lesions [61]. Currently no widely accepted therapeutic algorithm for Susac syndrome is available. In the case series, glucocorticosteroids or cyclophosphamide or immunoglobulin monotherapy was effective. In some patients concurrent use of these agents in various combinations was also successful. Plasmapheresis and hyperbaric oxygen have also been used [50,54,57,61,63,67,73,74]. There are also suggestions that treatment of this syndrome may involve monoclonal antibodies against CD20 antigen (present in maturing B lymphocytes) used in patients with malignant lymphomas (rituximab). A prospective 6-year follow-up of nine patients with Susac syndrome demonstrated that glucocorticosteroids were effective in the treatment of encephalopathy; however, dose reduction resulted in the return of symptoms. Glucocorticosteroid therapy did not improve hearing and did not prevent further occlusions of retinal artery branches. On the other hand, anticoagulants yielded a positive result, preventing relapses of encephalopathy and renal vessel occlusion [75]. Fox et al. [76] reported a significant hearing improvement and reduction of lesions in the central nervous system visualized by MRI shortly after intravenous administration of gamma-globulin and methylprednisolone. Experts on Susac syndrome stated [77,78] that therapy should be initiated as soon as the diagnosis is made to avoid dementia, deafness, and blindness. Initially, therapy should be given intravenously, followed by a long-term oral medication. Therapy should not be restricted to glucocorticosteroids. In summary, Susac syndrome is a rarely diagnosed vasculopathy of unknown pathogenesis. It seems that in some cases aPL antibodies may be a cause of vessel damage. Only MRI of the central nervous system may confirm the diagnosis of Susac syndrome. This syndrome should be included in the differential diagnosis in all cases of unexplained encephalopathy [79].

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Neurologic Symptoms Related to Vasculopathy and Vasculitis As described earlier, the autoimmune disease had many neuropsychiatric manifestations related to the localization of the vessels affected by vasculopathy. The precise mechanism of the cerebral vessel damage remains unknown. However, it was revealed that the presence of aPL antibodies increased the risk of neurologic complications in autoimmune diseases. The pathomechanism of aPL antibodies’ action in the development of vasculopathy and vasculitis was described earlier. aPL can also bind to glial cells, myelin, and neurons and impair their function [80–82]. Some authors suggested that aPL antibodies may play an important role in cognitive and behavioral deficits in patients with SLE [83]. It seems that a cause of these disturbances may be changes in microcirculation and impairment in the function of neurotransmitters connected with the action of aPL [84,85]. The occurrence of aPL antibodies is not only characteristic for Susac syndrome, but also for 40% of patients with Sneddon syndrome. This syndrome is characterized by stroke or transient ischemic attack (TIA) and livedo reticularis [86]. The clinical course of Sneddon syndrome is progressive. Patients frequently present with cognitive deterioration and disabilities [87]. MRI often shows leukoaraiosis and small lacunar infarcts [88]. The pathogenesis of this syndrome is unclear; aPL antibodies may participate in only some cases, as about 60% patients are aPL negative. The autoimmune disease should be suspected in each case of ischemic stroke that occurs in children and adults younger than 45 years of age, especially in women. Only about 20% of these subjects are aPL positive [89–91]. Among SLE subjects, convulsions caused by ischemic injury of the brain and development of an epileptic focus frequently are observed [92]. The rate of convulsions is higher in aPL-positive SLE subjects [93]. Rasmussen’s encephalitis is an example of an autoimmune disorder of the central nervous system associated with refractory epilepsy [92]. It was suggested that an important role in the pathogenesis of this disturbance is played by antibodies against GluR3 glutamate receptor, GluRepsilon 2 subunit, and antimitochondrial antibodies [94,95]. SLE was also uncommonly associated with peripheral neuropathy such as Guillain– Barré syndrome and mononeuritis multiplex. Guillain–Barré syndrome is an acute demyelinating disorder, mostly of the motor nerves. The clinical symptoms include progressive muscle weakness initiating in the lower limbs that may lead to paralysis of entire limbs and sometimes also the respiratory muscles and dysfunction of the autonomic system manifested by tachycardia, orthostatic hypotension, and sphincter dysfunction. The pathogenesis of this syndrome is unclear. In some patients antibodies against various phospholipids and nuclear antigens are detected, but their production is the consequence of myelin damage rather than the cause of demyelination [96]. The mononeuritis multiplex—distal, asymmetric, axonal polyneuropathy—is the effect of immune and vascular mechanisms. The autoantibodies and immune complex deposits induce inflammation and lesion of the nerves. On the other hand, vasculitis and thrombosis of the nerve vessels may directly cause the nerve injury [97]. Besides SLE, the mononeuritis multiplex is rarely associated with polyarteritis nodosa and Churg–Strauss syndrome.

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SLE and Myasthenia Gravis [98] Another uncommon disturbance of the peripheral nervous system associated with SLE is MG. MG is one of the autoimmune diseases that may develop prior to or after the onset of clinically overt SLE. In some patients with MG thymectomy was reported as the triggering factor of SLE [9,99–105]. Immune disorders associated with polyclonal activation of B lymphocytes are believed to play a central role in the pathogenesis of SLE. It has been recently highlighted that the α chemokine subfamily (CXC) is involved in the pathogenesis of both disorders [105,106]. These chemokines participate in chemoattraction of numerous immunoreactive cells. Some of these chemokines act as activators of monocytes, dendritic cells, T, B, and NK cells, eosinophils, and basophils. They also participate in angiogenesis [107], which is particularly important in connective tissue diseases [108,109]. Probably the CXCL13 chemokine plays a major role in the autoimmune response in MG. Studies on animal models proved that its interaction with B lymphocytes may result in subsequent SLE development [105]. Unlike SLE, MG is the organ-specific autoimmune disease predominantly associated with activation of T lymphocytes and the presence of specific autoantibodies against acetylcholine receptor [104,110], specific tyrosine kinase receptors (MuSK), and the muscle proteins [111]. These antibodies could directly cause the disease by impairing myoneural junction function, which in turn reduces muscular strength (causing fatigue). As the thymus is the site of T-lymphocyte production, any pathologic changes in this organ (hyperplasia, thymoma) inevitably result in cell dysfunction. An increased number and increased activation of autoreactive CD4 T lymphocytes are considered to be the major pathogenic factors of MG [112]. Their interaction with B lymphocytes leads to autoantibody production, which form immune complexes resulting in the immune response that causes organ damage [113]. Under physiologic conditions, regulatory T lymphocytes inhibit the activity of CD4 T lymphocytes, which stops the autoimmune process. It has been suggested that deficiency or dysfunction of regulatory CD4 CD25 T lymphocytes is the pathogenic factor in connective tissue diseases [114] and creates a favorable environment predisposing to the development of MG [112] and SLE [2,113]. SLE rarely occurs concomitantly with MG, so there are only reports of single cases and small series of patients [115] providing evidence for the associations between these two autoimmune diseases. Both diseases demonstrate a higher prevalence in young women and both are characterized by periodic exacerbation and remission in the clinical course, and by the presence of antinuclear antibodies [99]. Moreover, genetic, environmental, hormonal, and immunologic factors were assumed to be present in both diseases [101,116–123]. Common causative factors include bacterial and viral infections, smoking, pesticides, estrogens, various medications, and exposure to ultraviolet light [124]. Thymectomy is an established therapy for patients with thymoma-associated MG and selective erythroid aplasia. However, pure erythroid aplasia may also develop in the thymectomy follow-up [123]. Thymectomy may be the triggering factor not only

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for SLE but also for a number of other autoimmune diseases, including idiopathic portal hypertension, Hashimoto’s disease, cutaneous vessel vasculitis, and APS. SLE after thymectomy manifests mainly as polyarthritis [99]; however, other symptoms, including skin lesions, cytopenia, pleuritis, and an increased body temperature, have also been reported [11]. The role of thymectomy as the triggering factor for SLE in MG patients remains obscure. Thymectomy is likely to initiate the excessive production of autoantibodies in patients predisposed to SLE [124]. However, little is known about the pathologic mechanism behind this phenomenon. In MG patients thymectomy was followed by a slight reduction of the number of T lymphocytes and attenuation of immune tolerance but hyperactivation of B lymphocytes and hypergammaglobulinemia. A variety of autoantibodies, including anti-dsDNA and anticardiolipin, are usually detected [99,122]. In conclusion, the issue of SLE coexisting with MG discussed here is complex and remains unclear. Further research is needed, particularly to evaluate the role of prolactin, the potential regulatory function of which has recently been reported in MG patients [110]. It is well known that suppression of prolactin secretion may be a novel ancillary therapy for patients with moderate SLE [112,116,125,126].

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22 Inflammatory Mechanisms in Guillain–Barré Syndrome Robert N. Schwendimann Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA

Guillain–Barré syndrome (GBS) is a common cause of acute paralysis that occurs worldwide. It was described nearly 100 years ago by the French neurologists Guillain, Barré, and Strohl, who observed two soldiers who developed acute paralysis with areflexia [1]. Both individuals recovered completely. These patients were also found to have cerebrospinal fluid (CSF) abnormalities with elevated protein levels but no increase in cell counts. The history of a rapidly progressive weakness affecting the limbs, with or without sensory findings, associated with acellular CSF that is high in protein, remains the hallmark of the syndrome to this day. Over the past 20 years, other syndromes have been described that may show some variation from the original descriptions but are still considered to be variants of GBS. It is now well known that several different subtypes of this inflammatory polyneuropathy exist and that these subtypes may have different pathophysiologies [2]. In North America and Europe GBS typically presents as acute inflammatory demyelinating polyradiculoneuropathy (AIDP). AIDP is similar to the syndrome that was originally described. It develops as a relatively acute, progressive paresis of the limbs, occasionally in an ascending pattern, often with sensory symptoms and areflexia. Nerve conduction studies show abnormalities associated with a demyelinating type of neuropathy with prolonged distal latencies, prolonged F-wave latencies, and variable changes in sensory nerve action potentials early in the clinical course. Multifocal slowing of nerve conduction and partial conduction block may be a later finding [3]. The elevated CSF protein level with absence of cells (albumino–cytologic dissociation) characterizes the clinical picture as well. The clinical course typically follows a monophasic pattern, generally with slow improvement of the clinical condition, although many patients are left with fixed neurologic deficits. Treatments are generally supportive, since GBS can result in cranial neuropathies, autonomic disturbances, and respiratory failure. There is evidence that treatment with intravenous immunoglobulin G or plasma exchange is beneficial in the management of AIDP [2]. In Northern China, Japan, and Central and South America the clinical picture may be quite different, in that axonal forms occur in 30–47% of patients. Axonal involvement causing pure motor symptoms is known as acute motor axonal neuropathy Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00022-8 © 2011 Elsevier Inc. All rights reserved.

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(AMAN). When there is both motor and sensory involvement the condition is known as acute motor and sensory axonal neuropathy (AMSAN). There are similarities clinically between these syndromes and AIDP in that they may all involve the cranial nerves and affect all four limbs and respiratory function. Autonomic involvement is more common in AIDP than in AMAN and AMSAN. The results of CSF studies in AMAN and AMSAN are similar to those seen in AIDP. In AMAN and AMSAN, however, electrophysiologic studies are more typical of those seen in axonal neuropathy rather than a demyelinating process. Sensory nerve action potentials and sensory conduction are affected in AMSAN but are normal in AMAN. Pathologically, there is involvement of the axon, while the myelin sheath is intact. Macrophages are observed under the myelin sheath in contact with the axon. Activated complement components may be found around the axon, suggesting the presence of antibodies directed to axonal antigens [2]. Another variation of GBS was described by Miller Fisher (Miller Fisher syndrome [MFS]) in 1956 [4]. This clinical picture consists of a triad of symptoms: ophthalmoplegia, ataxia, and areflexia. The patients described also had elevation of CSF protein levels without cells. They also followed a course of gradual improvement and resolution of clinical symptoms. The similarities between MFS and AIDP clinically strongly suggested that the two conditions were related. There can be overlap between MFS and the clinical features of AIDP. Electrophysiologic findings in MFS are not similar to those in AIDP, but pathologic studies have shown a multi­ focal inflammatory process that resembles AIDP. The diagnosis of GBS is generally well known to neurologists and involves primarily clinical history and findings on examination. Examination of the CSF is important and electrodiagnostic/clinical neurophysiologic studies are helpful in differentiating demyelinating and axonal types. These studies are helpful in further differentiating motor and sensory involvement in the axonal forms. Many patients with GBS describe an antecedent infection, often with gastrointestinal symptoms. Campylobacter jejuni infections have preceded outbreaks of AMAN in China [5]. Various other types of infections occur as well, but C. jejuni and infections with cytomegalovirus are the most common in association with GBS. Other infections with Epstein–Barr virus, Mycoplasma pneumoniae, and rabies and exposure to vaccination for “swine flu” are also described [6–9]. Pathologically, the peripheral nerves show evidence of multifocal infiltrates of mononuclear cells in a patchy distribution in patients with AIDP [10]. Clinical deficits correspond to the distribution of this inflammation. Myelin damage occurs as a result of activated macrophages that invade intact myelin sheaths. Changes in the ultrastructural appearance include retraction of myelin near the nodes of Ranvier, resulting in a widened nodal gap. As the myelin sheath degenerates, there is evidence of ovoid formation and phagocytosis of myelin debris by macrophages. In severe cases, axonal degeneration can be seen, usually at sites of intense inflammatory change [11,12]. The immunologic mechanisms explaining invasion of the nerves by macrophages are not fully known. Activated macrophages may be directed to the area by activated helper T cells that react against specific antigens on the surface of Schwann cells or the myelin sheath. Inflammatory mediators such as matrix

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metalloproteinases and other toxic mediators are released by these activated macrophages and result in Schwann cell injury and invasion of the peripheral nerve. This possibility is supported by evidence that there is T-cell dysregulation in patients with GBS. These changes all support the theory that AIDP immunologically is a cellmediated process [13]. Another theory of pathogenesis supports humoral immunity as the cause of AIDP, particularly in the early stages of the disease. In early stages, antibodies bind to epitopes on the outer surface of the Schwann cells and induce complement activation that leads to myelin destruction before macrophage invasion. Secondary axonal damage may be secondary to inflammatory mediators and cells [14,15]. There is a difference between the pathologic features of AMAN and those of AIDP. In AMAN, macrophages invade the space between the Schwann cell and the axon, but the myelin sheath remains intact. This causes either blockage of axonal conduction or distal axonal degeneration. Axons may be damaged at the ventral roots in severe cases, which prolongs recovery [16,17]. Over the past decade or so, there has been great interest in studying the immunobiologic mechanisms in GBS, which may lead to new treatments of the disorder [18]. Numerous studies have shown involvement of gangliosides in the pathogenesis of GBS. Gangliosides are N-acetylneuraminic acid (sialic acid)-containing glycosphingolipids that are found concentrated on the surface of neuronal membranes, with their oligosaccharide portions exposed on the cell surface [19]. They appear to reside in clusters that are known as “lipid rafts” or detergent-resistant membranes along with other sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)anchored proteins. These clusters, also known as microdomains, work together to facilitate different membrane-mediated functions such as cell adhesion and signal transduction [20]. About 60% of patients with GBS have antibodies to various gangliosides during the acute phase of the disease, especially GM1, GM1b, GD1a, GD1b, GalNAcGD1a, and ganglioside complexes GD1a and GM1/GD1bGalNAc-GD1a [21,22]. Although cellular immune responses are important in the pathophysiology of GBS, anti-ganglioside antibodies also play an important role in the development of GBS and variants by binding to sites where target antigens are located [22]. There is evidence based on pathologic studies on human specimens and recent experiments that anti-ganglioside antibodies trigger an inappropriate activation of the complement cascade that leads to nerve injury in GBS. Experiments in vitro and ex vivo suggest that the complement activation through the classical pathway leads to the development of GBS and its variants. Activation of this pathway leads to formation of membrane attack complex (C5b-C9), cell opsonization, and phagocytosis. Experimental studies on animals deficient in complement C6 showed that they did not develop membrane attack complex in the presence of anti-GQ1b antibodies, strongly supporting the theory that complement activation is important in this inflammatory process [13,23–25]. Treatments for GBS include the use of plasma exchange and also intravenous immunoglobulin; they often result in rapid resolution of symptoms. This early response to these agents suggests that failure of conduction or functional block occurs without

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pathologic changes of axons and myelin sheaths. A likely cause of the reversible conduction failure or functional block in the absence of structural destruction of nerve components is impairment of some functional molecule on the nerve membrane [26,27]. Voltage-gated sodium channels (VGNaC) are represented in high densities at the nodes of Ranvier and are the most potent molecules that are associated with muscle action potential generation. There is some evidence that dysfunction of these VGNaC is a primary pathophysiologic mechanism in GBS, since transient blockade of these channels can cause conduction slowing or conduction block [28,29]. Increased refractoriness to axonal excitability has been described in AMAN patients with antibodies to GM1, GM1b, or GalNAc-GD1a, followed by rapid recovery of compound muscle action potentials with treatment. AIDP patients without antibodies do not exhibit the same response. These same ganglioside epitopes are believed to be present in high density at the nodes of Ranvier where the VGNaC clusters are present. The interaction of antigen and antibody at these sites may directly or indirectly alter the function of the VGNaC, since anti-ganglioside antibodies have been shown to cause blockade of VGNaC at the nodes of Ranvier. This occurs through a complement-mediated mechanism. Not all studies have confirmed that blockade of VGNaC is related to the presence of antibodies. Most studies do suggest, however, that VGNaC are linked to the ganglioside GM1, and that antibodies to GM1 can cause functional abnormalities in the VGNaC at the nodes and represent a principal factor in the pathogenesis of GBS, especially AMAN [30]. Voltage-gated calcium channels (VGCaC) may also be affected by gangliosides in GBS. Ga1NAc-GD is a molecule targeted by antibodies in the pure motor variant (AMAN). IgG antibodies to this ganglioside can produce complement-independent presynaptic inhibition of acetylcholine (ACh) release at the neuromuscular junction in rat muscle-spinal cord cells grown in tissue culture [31]. Studies have shown that these antibodies cause these changes by their effect on the VGCaC presynaptically. These effects occur with antibody-positive sera from AMAN patients, but not those with AIDP. Since synaptic ACh release is regulated by entry of calcium by way of VGCaC at the presynaptic membrane, it is possible that ganglioside antibodies impair the VGCaC and thus block spontaneous muscle action potentials at the neuromuscular junction. While the inhibition of VGCaC current represents an alternative pathophysiology in GBS, clinical and electrophysiologic studies in patients with positive ganglioside antibodies have not supported failure at the neuromuscular junction [32]. There is a new evidence that antibodies to ganglioside complexes consist of two gangliosides. These gangliosides appear to be expressed at the nodes of Ranvier in the axolemma. In view of the clustering of VGNaC at the same area it is possible that antibodies to the complexes result in a pure motor GBS. It is not known whether the dysfunction of the VGNaC is caused by complement activation or by direct effects on the VGNaC [33]. The Miller Fisher variant of GBS is a syndrome characterized by ophthalmoplegia, ataxia, and areflexia. It typically has a self-limited course and there are very few pathologic studies of autopsy material. Segmental demyelination is present with mild inflammation in sensory and motor spinal nerve roots and in cranial nerves 3, 7, 10,

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and 11 [34]. MFS is often associated with an antecedent infection, often C. jejuni. These infections are associated with production of IgG antibodies against the ganglioside GQ1b that are expressed in abundance in the paranodal myelin sheaths of nerves controlling the extraocular muscles, the neuromuscular junction, and the dorsal root ganglia [35,36]. The presence of anti-GQ1b antibodies is an excellent diagnostic marker in MFS. Effects of the anti-GQ1b antibodies are induced by complement-mediated damage to the perisynaptic Schwann cells and axonal terminals. The antibodies also inhibit presynaptic calcium inflow and interact with proteins associated with the exocytotic apparatus. This interferes with release of neurotransmitter that prevents activation of postsynaptic neurons, causing muscle weakness. Anti-ganglioside antibody binding to paranodal myelin sheaths may also result in ion channel clustering and conduction failure by disrupting the paranodal axo–glial junction [37]. AIDP has long been thought to be a T-cell-mediated disorder based on the presence of lymphocytic inflammation found on biopsy of the nerve [2]. A humoral immune response, however, is also suspected to be involved with surface antigens of Schwann cells. Glycolipids such as GD1b, LM1, or galactocerebroside may be target antigens in AIDP [38–40]. The presence of antibodies has been suggested by findings in experimental allergic neuritis (EAN). EAN can be induced by inoculation of laboratory animals with components of peripheral myelin protein such as P0 and P2. EAN is considered to be the animal model for AIDP [2]. Pathology in EAN shows T-cell and macrophage infiltration as well as demyelination of peripheral nerve similar to that seen in AIDP. Antibodies to various proteins found in myelin may be present in the sera of patients with AIDP, though how these antibodies are involved in AIDP pathophysiology is not known [41]. Why there is conduction failure and demyelination in AIDP is not well understood either. Disruption of VGNaC clusters in spinal roots has been observed in rats immunized with peripheral myelin. In the peripheral nervous system, clustering of VGNaC is regulated by the overlying Schwann cells [42]. Gliomedin and neurofascin-186 (NF-186) are adhesion molecules that are involved in aggregating VGNaC at the nodes of Ranvier. Gliomedin is expressed on Schwann cell microvilli and NF-186 is expressed at the nodal axonal membrane. Studies done on rats with EAN induced by inoculation with crude peripheral myelin showed disruption of the VGNaC that was associated with clinical signs. There was also early disruption of neurofascin and gliomedin occurring prior to VGNaC dispersion and demyelination. This early breakdown of NF-186 and gliomedin was followed by node alterations. Antibodies to both neurofascin and gliomedin were subsequently found in the sera of these animals and were associated with node alterations and degradation of axo–glial units. Pathologic events at the node were not associated with complement deposition. In EAN rats that were immunized with synthetic P2 peptide, there was very little nodal change and no antibodies to neurofascin and gliomedin. The pathophysiology of AIDP appears to be related to multiple possibilities, including humoral factors such as antibodies to neurofascin and gliomedin [43]. Further information regarding these factors may be important in determining new therapies for the AIDP form of GBS. Molecular mimicry is a major mechanism by which anti-ganglioside antibodies arise in GBS. A widely studied infectious agent in GBS is C. jejuni. It has been

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reported in the past that GBS in patients who have C. jejuni infections tend to have a more severe illness. Lipopolysaccharides found on certain strains of C. jejuni resemble GM1 and GD1a and produce molecular mimicry [35]. The lipopolysaccharide of C. jejuni has a structure that is ganglioside-like. This suggests that in AMAN and MFS, molecular mimicry plays a major role in development of the disease. Effects of the anti-ganglioside antibodies include the complement-mediated disruption of Na channels that results in reduction of sodium current and an anti-latroxine-like effect on motor endplates that results in a excessive release of ACh followed by depletion and destruction of perisynaptic Schwann cells and nerve terminals [28,44,45]. It appears that inflammatory cytokines are important in initiating, enhancing, and perpetuating the pathogenic events that occur in GBS [46,47]. Increased levels of the pro-inflammatory cytokine, interleukin-12 (IL-12), and receptor IL-12R1 were found in the acute phase of AIDP, corresponding to disease severity. IL-12 is a proinflammatory cytokine that has potent effects on immunoregulatory activities and in determination of the differentiation and generation of Th1 cells. Studies in EAN have shown that upregulated IL-12 mRNA expression was present at the height of the inflammatory process. Mice deficient in IL-12p40 develop a less severe form of EAN [48]. Tumor necrosis factor-alpha (TNF-α) is also a key mediator of regulatory processes in autoimmune demyelinating diseases. It is also considered to be important in the pathogenesis of GBS. TNF-α was initially characterized by its inflammatory effect; later it was found to exert an anti-inflammatory effect. This contradictory activity is explained by its binding to its two receptors, TNFR1 and TNFR2, with distinct effects. A recent study by Deng et al. suggests that IL-12 promotes disease development in AIDP, while TNF-α may play a dual role in the pathogenesis of AMAN. These findings may have therapeutic implications [48]. Interferon-gamma (IFN-γ) also appears to be important in the development of GBS. A recent study demonstrated that T cells from patients with GBS produced IFN-γ in response to stimulation with GM1 ganglioside [49]. IFN-γ also has been shown to be involved in the conversion of CD4CD25 to regulatory T cells in patients with GBS. It is thought that an increase in CD4CD25 regulatory T cells at the acute stage of GBS might be an effective treatment in GBS [50]. Host susceptibility may also be necessary for the development of clinical syndromes. Some lipopolysaccharides that are found in gram-negative bacteria may stimulate Toll-like receptors (TLR) that cause induction of inflammatory cytokines [51]. Studies of a small group of patients with GBS with certain polymorphisms of the TLR4 gene showed an increased susceptibility to GBS. Investigations of TNF-α promoter polymorphisms in GBS revealed that alleles associated with higher levels of TNF-α were more common in patients with AMAN, but not AIDP [52]. The pathophysiology of GBS is heterogeneous, but there is evidence that complement mediation is important in AIDP, AMAN, MFS, and other variants. Treatment of GBS with drugs that may affect the complement cascade should be considered [14]. When antibody gangliosides are present, one approach to treatment might be to absorb the antibodies on a column with a specific affinity for the ganglioside [53]. Drugs that affect T-cell cytokines or prevent T cells from passing into the endoneurium might be

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considered in cases of AIDP. Drugs like the beta-interferons that are helpful in management of multiple sclerosis might have benefit in the treatment of GBS [54]. Drugs that block sodium channels have been shown to have an axon-protective benefit [55]. Continued progress in understanding the underlying pathogenesis of GBS should lead to improved management of this inflammatory neuropathy.

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[38] Kusunoki S, Mashiko H, Mochizuki N, Chiba A, Arita M, Hitoshi S, et  al. Binding of antibodies against GM1 and GD1b in human peripheral nerve. Muscle Nerve 1997;20:840–5. [39] Yako K, Kusonoki S, Kanazawa I. LM1 in Guillain-Barré syndrome. J Neurol Sci 1999;168:85–9. [40] Saida T, Saida K, Dorfman SH, Silberberg DH, Sumner AJ, Manning MC, et  al. Experimental allergic neuritis induced by sensitization with galactocerebroside. Science 1979;204:1103–6. [41] Schmidt B, Toyka KV, Kiefer R, Full J, Hartung HP, Pollard J. Inflammatory infiltrates in sural nerve biopsies in Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy. Muscle Nerve 1996;19:474–87. [42] Novakovic SD, Levinson SR, Schachner M, Shrager P. Disruption and reorganization of sodium channels in experimental allergic neuritis. Muscle Nerve 1998;21:1019–32. [43] Lonigro A, Devaux J. Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis. Brain 2009;132:260–73. [44] Jacobs BC, Bullens RW, O’Hanlon GM, Ang CW, Willison HJ, Plomp JJ. Detection and prevalence of alpha-latroxin-like effects on serum from patients with Guillain-Barré syndrome. Muscle Nerve 2002;25:549–58. [45] Susuki K, Rasband MN, Tohyama K, Koibuchi K, Okamoto S, Funakoshi K, et  al. Anti-GM1 antibodies cause complement-mediated disruption of sodium channel clusters in peripheral motor nerve fibers. J Neurosci 2007;27:3956–67. [46] Pelidou SH, Zou LP, Deretzi G, Nennesmo I, Wei L, Mix E, et  al. Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats. Scand J Immunol 2000;51:29–35. [47] Zhu J, Bai XF, Mix E, Link H. Cytokine dichotomy in peripheral nervous system influences the outcome of experimental allergic neuritis: dynamics of mRNA expression for IL-beta, IL-6, IL-10, IL-12, TNF-alpha, TNF-beta, and cytolysin. Clin Immunol Immunopathol 1997;84:85–94. [48] Deng H, Yang X, Jin T, Wu J, Hu LS, Chang M, et al. The role of IL-12 and TNF-α in AIDP and AMAN. Eur J Neurol 2008;15:1100–5. [49] McCombe PA, Csurhes PA. T cells from patients with Guillain-Barré syndrome produce interferon-gamma in response to stimulation with the ganglioside GM1. J Clin Neurosci 2010;17:537–8. [50] Huang S, Lei L, Liang S, Wang W. Conversion of peripheral CD4CD25 regulatory T cells by IFN-γ in patients with Guillain-Barré syndrome. J Neuroimmunol 2009;217:80–4. [51] Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76. [52] Nyati KK, Prasad KN, Verma A, Singh AK, Rizwan A, Sinha S, et  al. Association of TLR4 Asp299Gly and Thr399Ile polymorphisms with Guillain-Barré syndrome in Northern Indian population. J Neuroimmunol 2010;218:116–19. [53] Willison HJ, Townson K, Veitch J, Boffey J, Isaacs N, Andersen SM, et  al. Synthetic disialylgalactose immunoadsorbants deplete anti-GQ1b antibodies from autoimmune neuropathy sera. Brain 2004;127:680–91. [54] Pritchard J, Gray IA, Idrissova ZR, Lecky BR, Sutton IJ, Swan AV, et  al. A randomized controlled trial of recombinant interferon-beta 1a in Guillain-Barré syndrome. Neurology 2003;61:1282–4. [55] Bechtold DA, Yue X, Evans RM, Davies M, Gregson NA, Smith KJ. Axonal protection in experimental autoimmune neuritis by the sodium channel blocking agent flecainide. Brain 2005;128:18–28.

23 Neurologic Manifestations of Herpes Zoster

Maria A. Nagel1, Don Gilden1,2, Ravi Mahalingam1, Randall J. Cohrs1 1 

Department of Neurology, University of Colorado School of Medicine, Aurora, CO, USA 2  Department of Microbiology, University of Colorado School of Medicine, Aurora, CO, USA

Introduction Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. Primary infection causes varicella (chicken pox), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia (DRG), and autonomic ganglia along the entire neuraxis. Years later, as cell-mediated immunity to VZV declines with age or immunosuppression (as in organ transplant recipients and patients with cancer or AIDS), VZV reactivates to cause zoster (shingles), often followed by chronic pain (postherpetic neuralgia [PHN]) as well as vasculopathy, meningoencephalitis, myelopathy, cerebellitis, and various ocular disorders, the most serious of which is retinal necrosis (Figure 23.1). VZV reactivation can also produce radicular pain without rash (zoster sine herpete), although it has now become clear that all of the neurologic and ocular complications of VZV reactivation identified earlier can occur without rash. Herein, we present the pathogenesis of primary VZV infection, clinical aspects of VZV reactivation and treatment, vaccination studies, recent advances in the molecular aspects of VZV infection, latency and apoptosis, and animal models for studying varicella biology.

Primary VZV Infection After inhalation of airborne virus particles, mucosal dendritic cells in the nasopharynx become infected. VZV productively infects both immature [1] and mature [2] dendritic cells. In the respiratory mucosa, VZV replicates in both Langerhans and plasmacytoid dendritic cells. Loss of Langerhans cells in biopsied varicella vesicles suggests migration of infected Langerhans cells to regional lymph nodes, resulting in transfer of infectious VZV from the primary infected cell to resident T lymphocytes [3]. VZV-infected CD4 memory T cells isolated from human tonsils expressing cutaneous lymphocyte-associated antigen (CLA) and chemokine receptor type 4 Neuroinflammation. DOI: 10.1016/B978-0-12-384913-7.00023-X © 2011 Elsevier Inc. All rights reserved.

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Latency

Reactivation

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Zoster (shingles)

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Encephalitis postherpetic Myelopathy Cerebellitis neuralgia

Ocular disorders

Figure 23.1  Neurologic disease produced by reactivation of varicella zoster virus.

(CCR4) retain their skin homing phenotype. While infected lymphocytes do not shed significant amounts of virus, they can transfer VZV to permissive cells via cell-tocell contact [4]. Thus, migration of virus-infected mucosal dendritic cells to draining lymph nodes and subsequent infection of memory T cells provides a reasonable conduit for VZV to access skin endothelial cells, where VZV replicates in dermal fibroblasts and keratinocytes [5]. Surface expression of major histocompatibility complex (MHC) class I protein is reduced in VZV-infected fibroblasts [6] and keratinocytes [7] due to virus-induced defects in trafficking of the protein within the cytoplasm of VZV-infected cells [8,9]. Surface expression of MHC class II molecules is reduced in VZV-infected fibroblasts [10] and keratinocytes [7]. VZV-infected dermal cells can evade clearance by cytotoxic T cells, thus increasing their ability to transfer virus to neighboring cells. VZV propagation typically results in the production of “light” particles, which lack virus DNA [11], most likely due to destruction of developing maturing virus particles in endosomes [12]. Since the mannose 6-phosphate-associated pathway leading to VZV degradation in late endosomes is not present in differentiated keratinocytes, infectious cell-free virus is released. In this manner, VZV replication continues and infection spreads until sufficient gamma-interferon is produced to induce an antiviral state in neighboring uninfected cells [10], but only after virus-induced cytokinemediated inflammation has produced the typical maculopapular lesions of varicella [13,14]. After primary infection, VZV becomes latent and reactivates to produce multiple neurologic and ocular disorders, including zoster, PHN, vasculopathy, meningitis, encephalitis, myelopathy, cerebellitis, ocular disorders, and zoster sine herpete.

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Herpes Zoster Zoster is common: nearly 1 million individuals in the United States are affected annually. The incidence of zoster is 5–6.5 per 1000 individuals at age 60, and it increases to 8–11 per 1000 at age 70 [15]. Unlike varicella, which occurs primarily in the spring, there is no seasonal predilection for zoster. Zoster in young adults may be the first manifestation of HIV infection [16]. Interestingly, varicella (chicken pox) in infancy predisposes to zoster earlier in life [17]. Zoster is characterized by dermatomal-distribution pain and rash. VZV is highly infectious and transmission occurs by direct contact with skin lesions or by respiratory aerosols. Immunosuppression increases the risk of disseminated zoster [18]. In most patients, the disappearance of skin lesions is accompanied by decreased pain and complete resolution of pain in 4–6 weeks. Magnetic resonance imaging (MRI) has shown enhancement of ganglia and affected nerve roots [19]. Because VZV becomes latent in ganglia along the entire neuraxis, zoster can develop anywhere on the body. Zoster can affect any cranial [20–31] or spinal nerves at all levels. Zoster paresis (zoster with lower motor neuron-type weakness) occurs in the arm, leg [32,33], diaphragm [34], or abdominal muscles [35]. VZV has been seen to reactivate subclinically (without pain or rash) in astronauts [36], with shedding of infectious virus [37]. Cardinal pathologic features of zoster are inflammation and hemorrhagic necrosis with associated neuritis, localized leptomeningitis, unilateral segmental poliomyelitis, and degeneration of related motor and sensory roots [38,39]. Demyelination occurs in areas with mononuclear cell (MNC) infiltration and microglial proliferation. Intranuclear inclusions, viral antigen, and herpesvirus particles have been detected in acutely infected ganglia [40–43].

Treatment Antiviral drugs (e.g., valacyclovir, 1 g thrice daily for 7–10 days) speed the healing of rash and shorten the duration of acute pain. Treatment of patients within 7 days of onset of the Ramsay Hunt syndrome improves recovery [44–46]. We also use prednisone (60 mg orally for 3–5 days) to reduce the inflammatory response, although double-blind placebo-controlled studies to prove additional efficacy are lacking. In immunocompromised patients, intravenous acyclovir (5–10 mg/kg thrice per day for 5–7 days) is recommended.

Postherpetic Neuralgia PHN is characterized by constant, severe, stabbing or burning dysesthetic pain that persists for at least 3 months and sometimes years after resolution of rash. About 40% of zoster patients over age 60 experience PHN [47,48]. The cause and pathogenesis of PHN are unknown. Two non-mutually exclusive theories are that (1) excitability of ganglionic or even spinal cord neurons is altered and (2) persistent or low-grade

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productive virus infection exists in ganglia. Watson et al. [49] detailed the pathology in a case of severe thoracic PHN for 5 years. The most striking finding was atrophy of the dorsal horn over five segments, with only one ganglion affected by fibrosis and cellular loss and with only the roots at that level involved. Further studies in three cases with severe pain [50] corroborated the dorsal root atrophy and ganglionic changes described earlier; furthermore, a marked inflammatory reaction was seen in the dorsal horn in one acute case, and loss of axons and myelin in the sensory root and peripheral nerve was noted. The presence of inflammatory changes at multiple levels bilaterally affecting roots, ganglia, and nerves raised the possibility of ongoing generalized inflammation as a pathogenetic mechanism in some cases. Further support for the concept that PHN is produced by low-level ganglionitis comes from the detection of VZV DNA and proteins in blood MNCs of many patients with PHN [51–53], and from the favorable response of some PHN patients to antiviral treatment [54–56]. In a recent prospective, open-label phase I/II clinical trial, 15 patients with moderate to severe PHN were treated with intravenous acyclovir for 2 weeks, followed by oral valacyclovir for 1 month; 8 of 15 (53%) patients reported improvement of pain [56].

VZV Vasculopathy VZV vasculopathy results from productive virus infection in large and/or small cerebral arteries. Patients present with headache, fever, mental status changes, transient ischemic attacks (TIAs), and/or focal deficit (stroke). The clinical spectrum includes aneurysms [57] and hemorrhage, arterial ectasia, and dissection [58]. More than one-third of cases of VZV vasculopathy occur without rash [59]. The cerebrospinal fluid (CSF) often contains a mononuclear pleocytosis and oligoclonal bands; the oligoclonal IgG is antibody directed against VZV [60]. Brain imaging usually reveals ischemic and/or hemorrhagic infarcts, more deep-seated than cortical lesions and particularly at gray–white matter junctions, a clue to diagnosis. Cerebral angiography may show focal arterial stenosis or occlusion (Figure 23.2). Macroscopically, lesions at gray–white matter junctions predominate. Virus is present in affected cerebral arteries, as evidenced by the presence of multinucleated giant cells, Cowdry A inclusion bodies, herpesvirus particles seen by electron microscopy, VZV DNA, and VZV antigen [61]. In chronic cases, virus is also found in areas of infarction, usually close to arteries and veins. Two recent studies revealed an increased risk of stroke after zoster. In a study of 7760 patients who had been treated for zoster and 23,380 controls, Kang et al. [62] found that the risk of stroke was 31% higher within a year after zoster and approximately fourfold higher in patients with herpes zoster ophthalmicus (HZO) versus the comparison cohort. Similarly, Lin et al. [63] found that the risk of stroke in 658 patients with HZO was 4.52-fold higher than in 1974 controls. Neurologists may not be surprised, since historically VZV vasculopathy was called HZO with contralateral hemiplegia. These studies are important because the aging population is rapidly increasing. Stroke can now be added to the list of other serious complications of HZO, such as keratitis and PHN.

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Figure 23.2  Characteristic angiographic, imaging, and pathologic abnormalities in VZV vasculopathy. (A) Three-dimensional time-of-flight magnetic resonance angiography of the circle of Willis shows marked narrowing of the left anterior cerebral artery (short arrow) and occlusion of the right anterior cerebral artery (long arrow). (B) Brain MRI scan shows multiple areas of infarction in both hemispheres, primarily involving white matter and gray– white matter junctions (arrows). (C) Diffusion-weighted MRI in a patient with small vessel VZV vasculopathy. Top scan reveals two ischemic lesions in the posterior thalamus, one in the hypothalamus, and a small ischemic lesion (arrow) in the posterior limb of the internal capsule; one week later, the patient became hemiplegic and a repeat MRI (bottom) showed a discrete infarct in the area of the posterior limb of the internal capsule, although the ischemic thalamic and hypothalamic lesions had resolved. (D) Macroscopic changes in brain from a patient who died of chronic VZV vasculopathy; arrows indicate ovoid areas of ischemia/ demyelination of varying size, primarily at gray–white matter junctions.

Confirmation of VZV vasculopathy requires virologic analysis to detect amplifiable VZV DNA or anti-VZV IgG antibodies (or both) in the CSF. The CSF does not always contain polymerase chain reaction (PCR)-amplifiable VZV DNA, but it does contain anti-VZV IgG [64]. The detection of anti-VZV IgG, but not VZV DNA, likely reflects the chronic, protracted course of disease. Testing for both VZV DNA and anti-VZV IgG must be done, and only negative findings on both can exclude the diagnosis of VZV vasculopathy. Also, since VZV vasculopathy can occur without rash, all vasculopathies of unknown etiology should be evaluated for VZV. Rapid

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diagnosis of VZV vasculopathy is important since the mortality without treatment is 25% [65], while treatment with intravenous acyclovir, even after neurologic disease has been present for months, can be curative [66].

VZV Meningoencephalitis VZV may also present as meningitis or a meningoencephalitis [67,68]. Many reported cases of VZV encephalitis may actually be VZV vasculopathy [69]. We recommend that all patients with VZV meningoencephalitis be treated with intravenous acyclovir as described above for VZV vasculopathy.

VZV Myelopathy VZV myelopathy presents in various ways. One form is a self-limiting, monophasic spastic paraparesis, with or without sensory features, and sphincter problems. This socalled post-infectious myelitis usually occurs in immunocompetent patients, days to weeks after acute varicella or zoster. Its pathogenesis is unknown. The CSF usually contains a mild mononuclear pleocytosis, with a normal or slightly elevated protein level. Steroids are used to treat these patients [70], although some improve spontaneously [71]. Rarely, VZV myelitis recurs, even in immunocompetent patients [72]. VZV myelopathy may also present as an insidious, progressive, and sometimes fatal myelitis, mostly in immunocompromised individuals. Indeed, AIDS has commonly and increasingly been associated with VZV myelitis. MRI reveals longitudinal serpiginous enhancing lesions. Diagnosis is confirmed by the presence of VZV DNA or anti-VZV IgG or both in CSF [72]. Pathologic and virologic analyses of the spinal cord from fatal cases have shown frank invasion of VZV in the parenchyma [73] and in some instances spread of virus to adjacent nerve roots [74]. Not surprisingly, some patients respond favorably to antiviral therapy [75–77]. Importantly, VZV myelitis may develop without rash. Early diagnosis and aggressive treatment with intravenous acyclovir have been helpful, even in immunocompromised patients [75]. The benefit of steroids in addition to antiviral agents is unknown. VZV can also produce spinal cord infarction identified by diffusion-weighted MRI and confirmed virologically [78]. Thus, VZV vasculopathy can cause stroke in the spinal cord as well as in the brain.

VZV Cerebellitis Primary VZV infection (chicken pox) and VZV reactivation can also result in cerebellitis with features of gait ataxia and tremor [79,80]. Since VZV DNA and antigen are present in the CSF of these patients, treatment is as recommended for VZV vasculopathy.

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VZV Retinal Necrosis VZV can produce multiple ocular disorders, including both acute retinal necrosis (ARN) and progressive outer retinal necrosis (PORN). ARN develops in both immunocompetent and immunocompromised hosts. Patients present with periorbital pain and floaters with hazy vision and loss of peripheral vision. Treatment is typically intravenous acyclovir, steroids, and aspirin followed by oral acyclovir [81]. Intravitreal injections of foscarnet and oral acyclovir have been used in early, milder cases. PORN is caused mostly by VZV and occurs primarily in AIDS patients with CD4 counts typically less than 10 cells/mm3 of blood [82], as well as in other immunosuppressed individuals [83]. PORN may be preceded by retrobulbar optic neuritis and aseptic meningitis [84], central retinal artery occlusion, or ophthalmic-distribution zoster [85], and may occur together with multifocal vasculopathy or myelitis. Patients present with sudden painless loss of vision, floaters, and constricted visual fields, with resultant retinal detachment. Multifocal, discrete opacified lesions begin in the outer retinal layers peripherally and/or the posterior pole; only late in disease are inner retinal layers involved. Diffuse retinal hemorrhages and whitening with macular involvement bilaterally are characteristic findings. Treatment with intravenous acyclovir has given poor or inconsistent results [86], and even when acyclovir helped, VZV retinopathy recurred when drug was tapered or stopped. PORN patients treated with a combination of ganciclovir and foscarnet or with ganciclovir alone had a better final visual acuity than those treated with acyclovir or foscarnet [87]. The best treatment for PORN in AIDS patients may be prevention with highly active antiretroviral therapy (HAART), which appears to decrease its incidence [88].

Zoster Sine Herpete Zoster sine herpete (radicular pain without rash) is due to reactivation of VZV [89], a concept first supported by the description of dermatomal-distribution radicular pain in areas distinct from pain with rash in zoster patients [90]. Currently, most clinicians regard zoster sine herpete exclusively as the rare occurrence of chronic radicular pain without rash. Other causes of chronic radicular pain include diabetes, lymphoma, cancer, and sarcoidosis. Virologic confirmation of zoster sine herpete is best provided by detection of VZV DNA in CSF. In recent years, the detection of VZV DNA and anti-VZV IgG antibody in patients with meningoencephalitis, vasculopathy, myelitis, cerebellar ataxia, and polyneuritis cranialis, all without rash, has expanded the spectrum of neurologic disease produced by VZV in the absence of rash. Prevalence estimates of VZV-induced pathology without rash await virologic analysis of additional patients with prolonged radicular pain or other neurologic symptoms and signs. Analyses should include tests for anti-VZV IgG, anti-VZV IgM, and PCR-amplifiable VZV DNA in CSF, anti-VZV IgM in serum, as well as examination of blood MNCs for VZV DNA.

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Vaccination Widespread, aggressive VZV vaccination has reduced the total number of varicella cases by ~85% and the number of moderate to severe cases by 95–100% [91]. Like the live varicella vaccine for children, there is now a live zoster vaccine that appears to be safe and effective clinically. The results of a prospective, double-blind, placebo-controlled trial of attenuated VZV vaccine designed to prevent zoster and PHN in men and women over age 60 were recently reported [92]. Otherwise healthy adults age 60 years or older (median 69 years) were vaccinated with placebo or an attenuated Oka/Merck-VZV vaccine containing 18,700–60,000 plaque-forming units (PFUs) of virus, considerably greater than the ~1350 PFUs in the Oka/Merck-VZV vaccine administered to American children since 1995. More than 19,000 subjects in each group were followed closely for 3 years. The incidence of zoster in the placebo group was 11.1 per 1000 person-years, approximating the results of an epidemiologic survey performed a decade ago, which revealed zoster exceeding 10 cases per 1000 person-years in individuals older than 75 years [15]. The effect of zoster vaccine was impressive; compared to placebo, zoster vaccination reduced the incidence of shingles by 51%, the incidence of PHN by 66%, and the burden of illness by 61%. Serious adverse effects and deaths occurred in 1.4% of both vaccine and placebo recipients. In more than 6000 subjects who kept daily diaries of minor adverse effects for 42 days, 48% of vaccine recipients reported injection site erythema, pain, or tenderness, swelling, and itching, compared to 16% of placebo recipients. In the same 6000 subjects, serious adverse effects were significantly more frequent (P  0.03) in vaccine recipients (1.9%) compared to placebo recipients (1.3%), although no specific serious effects emerged. The relative impact of these side effects on elderly (age 70) compared to younger patients was not examined but might be important in future analyses, since the at-risk population over age 70 years is projected to increase substantially in the coming decades. Although the Oka/Merck-VZV vaccine on rare occasions unmasks a childhood immunodeficiency disorder, no cases of disseminated zoster that might have been attributed to zoster vaccine in a person with undiagnosed lymphoma, leukemia, or similar disorders were reported. In 2006, zoster vaccine received US Food and Drug Administration (FDA) approval for healthy VZV-seropositive adults over age 60. Zoster vaccine increases cellmediated immunity to VZV in such individuals, and ideally the boost will last for decades. Since zoster and its attendant neurologic complication of PHN are increasingly common and serious every decade after age 60, zoster vaccine can be highly recommended. The US Census Bureau projects that by the year 2050, there will be more than 21 million Americans 85 years of age or older [93]. Nevertheless, even if every healthy adult in the United States over age 60 years is vaccinated, there would still be ~500,000 zoster cases annually, and about 200,000 of these patients will experience PHN, as well as stroke, blindness, and myelopathy caused by VZV reactivation. Furthermore, zoster vaccine is not approved for immunocompromised individuals, so that neurologic disease produced by VZV reactivation in this population will continue to be a problem.

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Molecular Aspects of VZV Infection and Latency Viral DNA and Virus Gene Expression Primary VZV infection results in virus replication in the nasopharyngeal region where infected T cells transfer virus to the skin, leading to varicella [94]. Nerve fibers innervating sites of active virus replication can transport virus anterograde to neurons in ganglia. When virus DNA has gained access to the neuronal nucleus, the VZV genome either orchestrates expression of genes leading to production and release of newly formed infectious virus or limits its own gene transcription such that no infectious virus is released. During productive virus infection in culture, transcripts from all predicted VZV open reading frames (ORFs) are present [95,96], whereas during latent infection, less than 10% of all VZV ORFs are transcribed. VZV was the first herpesvirus for which the sequence of the entire genome was determined [97]. A search of the National Center for Biotechnology Information database shows the complete DNA sequence of 22 VZV isolates [98]. The VZV genome is stable [99], with a genetic variation of 0.00063 differences per nucleotide [100]. The stability and uniformity of the VZV genome imply that virus from various geographic regions does not undergo significant genetic mutation when propagated in the laboratory. While analysis of the VZV genome is unencumbered by mutations induced by in vitro propagation, the study of VZV latency is strictly dependent upon in vivo-derived samples. VZV is an exclusively human pathogen. Unlike herpes simplex virus type 1 (HSV-1), for which mouse and rabbit models are available to analyze latency and either spontaneous [101,102] or induced [103–105] virus reactivation, there are no animal models of VZV latency and reactivation. Consequently, relevant information on VZV latency has been obtained through analysis of human ganglia removed at autopsy. VZV DNA is detected in all cranial nerve ganglia, DRG, or autonomic ganglia along the entire neuraxis. Within ganglia, VZV is present predominately, if not exclusively, in 1–7% of individual neurons [106–112]. Unlike VZV DNA extracted from virus particles in which the termini of the viral DNA molecule are mostly free [113], the virus genome during latency forms a circle or multiple concatemers [114]. Latent VZV DNA is also associated with cellular histones [115]. In latently infected human ganglia, the VZV DNA copy number does not differ significantly in the left and right trigeminal ganglia from the same person; however, there is considerable variation (37–3500 VZV DNA copies per 100 ng trigeminal ganglia DNA) in the virus burden among individuals [116,117]. Overall DNA sequence analysis of reverse transcriptase (RT)-PCR products generated from latently infected human ganglia demonstrated transcription of VZV genes 21, 29, 62, 63, and 66, but not VZV genes 4, 10, 40, 51, and 61 [118,119]. Subsequent analysis using real-time (quantitative) RT-PCR to determine the prevalence and abundance of VZV gene transcripts identified by DNA sequence analysis in latently infected ganglia [120] showed that in 28 trigeminal ganglia from 14 humans, VZV gene 63 transcripts were detected most often (63%), followed by gene 66 (43%), gene 62 (36%), and gene 29 (21%); no gene 21 transcripts were detected in any ganglia. Quantitative

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analysis also showed that VZV gene 63 RNA was by far the most abundant (3710– 6895 copies per μg mRNA), followed by VZV gene 29, VZV gene 66, and VZV gene 62 (491–594, 85–117, and 38–65 copies per μg mRNA, respectively). Although less than 20% of the ~70 VZV genes have been studied, transcription of VZV ORF 63 appears to be a hallmark of virus latency. While VZV gene 63 transcripts are the most prevalent and abundant in latently infected human ganglia, expression of VZV ORFs 4, 18, 21, 29, 40, 62, and 66 may occur if virus reactivates subclinically in small numbers of neurons. While VZV gene 63 transcripts are consistently detected during latency, detection of immediate-early (IE) 63 protein, the product of VZV ORF 63, is difficult. Immunohistochemistry using rabbit polyclonal antibody applied to latently infected human ganglia detected VZV IE63 predominantly in the cytoplasm of latently infected ganglionic neurons in two of nine subjects; in one subject, IE63 was detected in all sections from four of five thoracic ganglia, and in the second subject in all sections from one trigeminal ganglion [108]. In another study, 6% neurons stained positive for IE63, again mainly in the cytoplasm by immunohistochemistry in DRG from three subjects and detected proteins encoded by VZV ORFs 4, 21, 29, 62, and 63 in latently infected ganglia from three subjects [121]. Subsequent immunohistochemical analysis of latently infected human ganglia detected proteins encoded by VZV ORFs 21, 29, 62, and 63 [122], VZV ORF 62 [123–125], and VZV ORF 66 [120]. While findings in all of these reports await confirmation by independent technology, it is worth noting that the detection of transcripts and the protein encoded by VZV ORF 66, which is a protein kinase that reduces surface MHC class I expression [9], may be relevant in explaining the relatively little evidence of immunologic surveillance in latently infected ganglia [126] for VZV presentation [123,124] and the lack of T cells in latently infected ganglia.

Apoptosis Many viruses, including VZV, can induce apoptosis. VZV induces apoptotic pathways in infected fibroblasts [127] and in peripheral blood MNCs [128]. A study by Hood et al. [129] to compare infection of human foreskin fibroblasts with that of human dorsal root ganglionic cells showed that apoptosis was induced only in fibroblasts, a finding of particular importance since VZV establishes and maintains latency in human neurons for the lifetime of the host. Additional studies suggest that VZV IE63, a gene duplicated in the viral genome and expressed during lytic and latent infection, plays a role in inhibition of apoptosis in VZV-infected neurons [130]. This is consistent with findings in HSV-1 that some viral genes have an anti-apoptotic function [131]. In cultured human dorsal root ganglionic cells infected with VZV deleted in one copy of ORF 63, apoptosis was increased as compared to that after wild-type VZV infection neurons [130], suggesting that VZV IE63 can modulate this process in a dose-dependent manner. Apoptosis proceeds primarily through two distinct pathways: (1) an extrinsic pathway initiated by ligand interaction with death receptors, leading to activation of caspase-8 [132], and (2) an intrinsic pathway activated by an imbalance between

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pro-apoptotic (e.g., Bax and Bak) and anti-apoptotic (e.g., Bcl-2 and Bcl-xL) proteins in mitochondria, resulting in the release of cytochrome c from mitochondria to activate caspase-9 [133]. Both pathways converge through activation of caspase-3, which, along with other effector caspases, cleaves cellular proteins and results in cell death. Our studies of simian varicella virus (SVV)-infected monkey kidney cells in tissue culture revealed SVV-induced apoptosis, as evidenced by nuclear condensation and positive TUNEL staining [134]. Western blotting analysis to further characterize apoptotic mechanisms indicated a significant increase in levels of the cleaved active form of caspase-3 and elevated levels of active caspase-9 in infected cells, whereas active caspase-8 was not detected; expression of anti-apoptotic Bcl-2 was decreased significantly at the mRNA and protein levels in virus-infected cells (Figure 23.3). Immunofluorescence labeling of cells infected with green fluorescent protein (GFP)-tagged SVV revealed co-localization of active forms of caspase-3 and -9 with GFP. Finally, mitochondrial release of the caspase-9 activator cytochrome c into the cytoplasm was also detected. Together, these findings indicated that SVV induces apoptosis in cultured cells primarily through the intrinsic pathway. The intrinsic pathway also appears to be involved in apoptosis produced by VZV infection of human skin (MeWo) cells, in which Bcl-2 mRNA and protein are downregulated [135]. Further studies are needed to identify the mechanisms that induce

Active caspase-3

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β actin SVV hpi

– 24

+ 24

– 40

+ 40

– 48

+ 48

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Figure 23.3  Intrinsic pathway of apoptosis in SVV-infected cells. Western blot analysis of lysates of uninfected () and SVV-infected () Vero cells harvested at multiple times and probed for the active forms of caspase-3 and caspase-9 and for Bcl-2. Blots were reprobed for β-actin. Representative blots from four experiments are shown. Note the increased levels of caspases-3 and -9 and the decreased Bcl-2 expression in infected cells at 64 h post-infection (hpi).

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apoptosis in one cell type but prevent it in another during VZV infection, and to elucidate how VZV modulates cellular mechanisms to enable maintenance of a lifelong latent infection.

Development of Animal Models Since VZV infects only humans, there is a need for a reliable animal model that can reproduce the pattern of disease seen in humans. Any animal model useful in studying varicella latency in humans must fulfill the following criteria: (1) presence of virus nucleic acids in ganglia, but not in non-ganglionic tissues such as lung and liver; (2) restricted transcription of the virus genome; (3) presence of virus exclusively in neurons of ganglia; and (4) ability to reactivate. Inoculation of guinea pigs, rabbits, mice, and rats with VZV by different routes leads to seroconversion in the absence of clinical signs [136,137]. Intramuscular inoculation of guinea pigs with VZV produces a papular exanthem but not vesicles [138]. While VZV spreads to guinea pig ganglia after subcutaneous inoculation [139], the lack of analyses of non-ganglionic tissues from virus-infected guinea pigs for the presence of virus nucleic acids makes it is difficult to evaluate this model to study VZV latency. Subcutaneous inoculation of VZV into adult rats along the spine does not produce chicken pox, although VZV DNA, RNA, and proteins have been detected in neurons from ganglia removed at different time points up to 9 months [140]. Since ganglia were cultured for 3–12 days in that study, it is difficult to rule out VZV reactivation in culture. Transcripts associated with VZV latency (gene 21) but not with active replication (gene 40) were detected in ganglia of newborn rats 5–6 weeks after intraperitoneal inoculation [141]. After footpad inoculation of VZV, neurons as well as non-neuronal cells of DRG were shown to harbor VZV DNA in rats at 1 and 3 months [142], although VZV gene 63 protein was detected in DRG neurons 12–18 months after footpad inoculation [143,144]. Overall, in both rats and mice experimentally infected with VZV, virus is found not only in ganglionic neurons, but also in non-neuronal cells as well as in extraneural tissues. Further and most important, VZV reactivation has not been demonstrated in any rodent species. In marmosets, oral-nasal-conjunctival application of the attenuated vaccine (Oka) strain of VZV results in mild pneumonitis and production of VZV antibody without symptomatic disease [145]. Subcutaneous inoculation of Oka VZV into the breast of a chimpanzee produces viremia and a mild rash restricted to the inoculation site [146], but ganglionic and non-ganglionic tissues were not analyzed in that study. Infectious VZV and virus proteins have been detected in CD4 and CD8 T cells up to 3 weeks after infection of human fetal thymus and liver implants under the kidney capsule or under the skin of severe combined immunodeficiency (SCID-hu) mice [147]. The lack of an intact immune system in these mice, together with external infection of the implanted tissue rather than by viremia, makes it a difficult model for the study of VZV latency. Human tissue xenografts in SCID mice have also been used to study VZV infection and latency. Human fetal DRG implanted under the capsule of the kidney

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become vascularized and maintain organotypic features. VZV infection of DRG xenografts, either by injection of virus-infected fibroblasts into the xenograft or indirectly by inoculation of VZV-infected T cells into the tail vein, results in a burst of VZV replication in the DRG. Within 2 weeks, VZV DNA, VZV proteins, and herpes virions are evident; importantly, cell fusion (syncytial formation) does not develop and the DRG architecture is not destroyed. After acute ganglionic infection, VZV appears to become latent, as evidenced by a low abundance of VZV DNA, an absence of infectious virus, and detection of only VZV gene 63 transcripts, exclusively in neurons [148]. In contrast, a later study that used DRG xenografts to analyze the neuropathogenesis of VZV infection revealed VZV DNA, VZV proteins, and nucleocapsids in both neurons and satellite cells, and numerous viral-induced syncytia [149]. Deletion of VZV glycoprotein I (gI) resulted in loss of neuronal/satellite cell fusion, reduced virus DNA replication, and lack of transition from acute to latent infection; mutational analysis of the VZV gI promoter region revealed neuron-specific transcriptional regulation [150]. Taken together, DRG xenografts maintained in SCID mice provide a unique model to study VZV neuronal interaction.

Simian Varicella Virus Although the true host for natural SVV infection is unknown, epidemic outbreaks in different species of monkeys, including African green, patas, and macaques, were reported in the 1960s at different primate centers in both the United States and the United Kingdom [151]. Clinical, pathologic, immunologic, and virologic features of SVV infection of nonhuman primates closely resemble those of human VZV infection [152,153]. After an incubation period of 1 or more weeks, monkeys become febrile and develop a papulovesicular rash of skin and mucous membranes. Infectious virus can be recovered from blood MNCs [154,155]. Hemorrhagic necrosis, inflammation, and eosinophilic intranuclear inclusions are seen in skin and visceral tissues [156]. SVV becomes latent in neurons of multiple ganglia [157] and reactivates to cause zoster [154]. VZV and SVV glycoproteins share immunologic cross-reactivity, as demonstrated by the protection of SVV-infected monkeys after VZV immunization [158]. The two virus genomes are similar in size, structure, and gene organization [159,160]. Analysis of the complete nucleotide sequence of SVV has revealed an invertible 665-basepair leftward terminal element that is absent in the VZV genome [161–163]. Overall, SVV infection in primates is the counterpart of human VZV infection. Three experimental models of SVV infection have been established. In the first model, intratracheal inoculation of African green or cynomolgus monkeys with 103–104 PFU of SVV results in viremia, and infectious virus can be recovered from blood MNCs from 2 to 11 days after inoculation [164,165]. Monkey ganglia, like other visceral organs, become infected with SVV before the appearance of rash [166]. Histopathologic analysis reveals necrosis and intranuclear inclusions in lung,

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liver, and spleen [167]. SVV-specific antigens and nucleic acid are present in liver, lung, spleen, adrenal gland, kidney, lymph node, and bone marrow and in ganglia at all levels of the neuraxis [153,165,167]. SVV-specific immediate-early, early, and late transcripts are found in skin, lung, liver, and ganglia of acutely infected (11–12 days post-infection) monkeys [167]. SVV DNA and RNA are present in multiple tissues, including ganglia, liver, and blood MNCs, months to years after experimental intratracheal inoculation [168,169]. This model can serve in designing antiviral treatments that maintain the virus in the latent state in patients taking immunosuppressive drugs. In the second model, simulated natural infection in African green or cynomolgus monkeys results in virus latency in ganglia. SVV-seronegative monkeys are exposed to other monkeys previously inoculated intratracheally with SVV [157]. After 10–14 days, monkeys exposed to intratracheally inoculated monkeys develop a mild rash and SVV DNA is detected in skin scrapings of these naturally infected monkeys, confirming that SVV causes the rash. Occasionally, SVV DNA is also detected in blood MNCs of naturally infected monkeys. At 6–8 weeks after the resolution of rash, SVV DNA is present in multiple ganglia along the neuraxis, but not in lung or liver, indicating latent infection. Subclinical reactivation of latent SVV has been observed in an asymptomatic irradiated rhesus macaque, leading to disseminated varicella in a seronegative irradiated monkey from the same colony [170]. In addition, there have been other reports of spontaneous outbreaks of SVV infection in rhesus [171] and pigtailed [172] macaques, some of which had undergone organ transplant procedures including total body irradiation. Latently infected monkeys subjected to stress and experimental immunosuppression undergo SVV reactivation, as confirmed by the development of zoster, the detection of VZV DNA in organs other than ganglia, the presence of SVV RNA specific for late capsid proteins (ORFs 40 and 9) in ganglia, and the presence of SVV antigens in skin, ganglia (including axons), and viscera [173]. This second model can be used to examine viral as well as cellular processes, particularly the inflammatory response, that play a role in the establishment of, maintenance of, and reactivation from latency. With respect to the role of cellular processes, it remains unclear why an age-related decline in T-cell–mediated immunity in humans correlates strongly with zoster incidence, yet VZV-specific T cells are not essential in maintaining latency in ganglia [126]. Although VZV downregulates MHC class I surface expression by its retention in the Golgi compartment [6,8,9], neurons generally do not express MHC class I genes, so that latency and reactivation are probably regulated by an innate immune response involving cytokines or chemokines. The identification of the SVV-specific T-cell response and associated cytokines in ganglia during reactivation will help to identify potential targets for prevention of zoster in humans. In the third model, intrabronchial inoculation of rhesus macaques with SVV produces varicella, viremia, and cell-mediated and humoral immune responses to SVV. Acute SVV viremia resolves, and months later, viral DNA is detectable in ganglia but not in lung or liver. Furthermore, transcripts corresponding to SVV ORFs 21, 61, 62, 63, and 66 but not ORF 40 can be detected during latency in ganglia. SVV ORF 63 protein is found in the cytoplasm of neurons within latently infected ganglia [174].

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Conclusion Continuing clinical, pathologic, and virologic studies of patients suffering from the multiple neurologic complications of VZV reactivation are needed. While VZV vaccination in healthy adults has proven to be highly effective, there is also a need for antiviral drugs that block VZV reactivation in patients taking immunosuppressive drugs. Development of such reagents, as well as better treatments for patients suffering from complications of viral reactivation, awaits further studies to elucidate the still-unclear molecular mechanisms that enable the virus to maintain lifelong infection of a specific cell type and that trigger viral reactivation in a manner presumably involving virus-induced modulation of the cellular immune system. Analyses of viral nucleic acid and gene expression in latently infected human ganglia and the development of a primate model of varicella latency are being used to address these issues.

Acknowledgments This work was supported in part by Public Health Service grants AG006127 and AG032958 from the National Institutes of Health. The authors thank Marina Hoffman for editorial review and Cathy Allen for manuscript preparation.

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