Paradigms of Neural Injury, Volume 30 (Methods in Neurosciences)

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Paradigms of Neural Injury

Paradigms of Neural Injury

This Page Intentionally Left Blank

Paradigms of Neural Injury

Edited by J. Regino Perez-Polo Department of Human Biological Chemistry and Genetics University of Texas Medical Branch Galveston, Texas

ACADEMIC PRESS San Diego New York Boston

London

Sydney Tokyo Toronto

Front cover photograph: Computer-enhanced picture of dissociated embryonic chick sensory neurons differentiated with 10 ng/ml nerve growth factor.

This book is printed on acid-free paper. Copyright 9 1996 by ACADEMIC PRESS, 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 photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX

International Standard Serial Number: 1043-9471 International Standard Book Number: 0-12-185300-4

PRINTED IN THE UNITED STATES OF AMERICA 96 97 98 99 00 01 EB 9 8 7 6 5

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Table of Contents

Contributors Preface Volumes in Series

vii xi xiii

1. Paradigms for Study of Neurotrophin Effects in Oxidant Injury George R. Jackson and J. Regino Perez-Polo

2. Nitric Oxide Toxicity in Central Nervous System Cultures

26

Valina L. Dawson and Ted M. Dawson

3. Development of in Vitro Injury Models for Oligodendroglia

44

A. Espinosa, P. Zhao, and J. de Vellis

4. Glia Models to Study Glial Cell Cytotoxicity

55

Antonia Vernadakis and M. Susan Kentroti

5. Rodent Glioma Models

81

William W. Maggio

6. Peripheral Lesioning of Olfactory System: Expression of Neurotrophin Receptors

97

Christopher P. Turner and J. Regino Perez-Polo

7. Basal Forebrain Cholinergic Lesions and Complete Transection of Septal-Hippocampal Pathway

106

Lawrence R. Williams ,

Neurochemical Lesions: Tools for Functional Assessment of Serotonin Neuronal Systems

115

Joan M. Lakoski, B. Jane Keck, and Ashish Dugar ,

Cerebral Glucose/Energy Metabolism: Valid Techniques in Humans and Animals

124

Siegfried Hoyer

10. Heavy Metal Effects on Glia

135

Evelyn Tiffany-Castiglioni, Marie E. Legare, Lora A. Schneider, Edward D. Harris, Rola Barhoumi, Jan Zmudzki, Yongchang Qian, and Robert C. Burghardt

11. Source, Metabolism, and Function of Cysteine and Glutathione in the Central Nervous System David K. Rassin

167

vi

TABLE OF CONTENTS 12. Magnetic Resonance Spectroscopy of Neural Tissue

178

Richard J. McClure, Kanagasabai Panchalingam, William E. Klunk, and Jay W. Pettegrew

13. Acute Stroke Diagnosis with Magnetic Resonance Imaging

209

Stephen C. Jones, Neng C. Huang, Michael J. Quast, Alejandro D. Perez-Trepechio, Gilbert R. Hillman, and Thomas A. Kent

14. Evaluation of Free Radical-Initiated Oxidant Events within the Nervous System

243

Stephen C. Bondy

15. Exogenous Administration of Cytokines into the Central Nervous System: Analysis of Alterations in Cell Morphology and Molecular Expression

260

M. A. Kahn and J. de Vellis

16. Animal Models to Produce Cortical Cholinergic Dysfunction

275

Reinhard Schliebs and Volker Bigl

17. In Vitro Studies of Liposome-Mediated Gene Transfection

290

K. Yang, J. Regino Perez-Polo, F. Faustinella, G. Taglialatela, and R. L. Hayes

18. Construction and Analysis of Transgenic Mice Expressing Amyloidogenic Fragments of Alzheimer Amyloid Protein Precursor

298

Rachael L. Neve and Frederick M. Boyce

19. Golgi Technique Used to Study Stress and Glucocorticoid Effects on Hippocampal Neuronal Morphology

315

Ana Maria Magarifzos, Eberhard Fuchs, Gabriele Fliigge, and Bruce S. McEwen

Index

327

Contributors

Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

ROLA BARHOUMI (10), Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station, Texas 77845 VOLKER BIGL (16), Paul Flechsig Institute for Brain Research, University of Leipzig, D-04109 Leipzig, Germany STEPHEN C. BONDY (14), Department of Community and Environmental Medicine, Center for Occupational and Environmental Health, University of California, Irvine, Irvine, California 92717 FREDERICK M. BOYCE (18), Department of Neurology, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129 ROBERT C. BURGHARDT(10), Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station, Texas 77845 TED M. DAWSON (2), Departments of Neurology and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287 VALINA L. DAWSON (2), Departments of Neurology and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287 J. DE VELLIS (3, 15), Departments of Neurobiology and Psychiatry, Mental Retardation Research Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 ASHISH DUGAR (8), Departments of Pharmacology and Anesthesia, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 A. ESPINOSA (3), Department of Neurobiochemistry, Mental Retardation Research Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 F. FAUSTINELLA(17), Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 GABRIELE FLC)GGE(19), Division of Neurobiology, German Primate Center, D-37077 G6ttingen, Germany EBERHARD FUCHS (19), Division of Neurobiology, German Primate Center, D-37077 G6ttingen, Germany

vii

viii

CONTRIBUTORS

EDWARD D. HARRIS (10), Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77845 R. L. HAYES (17), Department of Neurosurgery, Health Science Center, University of Texas, Houston, Houston, Texas 77030 GILBERT R. HILLMAN (13), Departments of Pharmacology and Toxicology and Academic Computing, University of Texas Medical Branch, Galveston, Texas 77555 SIEGFRIED HOVER (9), Brain Metabolism Group, Departments of Pathochemistry and General Neurochemistry, University of Heidelberg, 69120 Heidelberg, Germany NENG C. HUANG (13), Departments of Pharmacology and Toxicology, and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555 GEORGE R. JACKSON(1), Department of Neurology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 STEPHEN C. JONES (13), Cerebrovascular Research Laboratory, Department of Biomedical Engineering, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 M. A. KAHN (15), Departments of Neurobiology, Psychiatry, and Biobehavioral Sciences, Mental Retardation Research Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 B. JANE KECK (8), Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 THOMAS A. KENT (13), Departments of Neurology and Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555 M. SUSAN KENTROTI (4), Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262 WILLIAM E. KLUNK (12), Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 JOAN M. LAKOSKI(8), Departments of Pharmacology and Anesthesia, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 MARIE E. LEGARE (10), Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station, Texas 77845 ANA MARfA MAGARIlqOS(19), Neuroendocrinology Laboratory, Rockefeller University, New York, New York 10021

CONTRIBUTORS

ix

WILLIAM W. MAGGIO (5), Division of Neurosurgery, University of Texas Medical Branch, Galveston, Texas 77555 RICHARD J. MCCLURE (12), Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 BRUCE S. MCEWEN (19), Neuroendocrinology Laboratory, Rockefeller University, New York, New York 10021 RACHAEL L. NEVE (18), Department of Genetics, Harvard Medical School, McLean Hospital, Belmont, Massachusetts 02178 KANAGASABAI PANCHALINGAM(12), Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 J. REGINO PEREZ-POLO (1, 6, 17), Department of Human Biological Chemis-

try and Genetics, University of Texas Medical Branch, Galveston, Texas 77555 ALEJANDRO D. PEREZ-TREPECHIO (13), Cerebrovascular Research Laboratory, Department of Biomedical Engineering, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 JAY W. PETTEGREW(12), Departments of Psychiatry and Neurology, Health Service Administration, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 YONGCHANGQIAN (10), Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77845 MICHAEL J. QUAST (13), Department of Radiology, and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555 DAVID K. RASSIN (11), Department of Pediatrics, The University of Texas Medical Branch, Galveston, Texas 77555 REINHARD SCHLIEBS(16), Paul Flechsig Institute for Brain Research, University of Leipzig, D-04109 Leipzig, Germany LORA A. SCHNEIDER (10), Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station, Texas 77845 G. TAGLIALATELA(17), Department of Human Biochemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555 EVELYN TIFFANY-CASTIGLIONI (10), Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station, Texas 77845

CHRISTOPHER P. TURNER(6), Department of Neurology, Veterans Administration Medical Center, San Francisco, California 94121

X

CONTRIBUTORS ANTONIA VERNADAKIS (4), Departments of Psychiatry and Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262 LAWRENCE R. WILLIAMS (7), Amgen Neuroscience, Thousand Oaks, California 91320 K. YANG (17), Department of Neurosurgery, University of Texas, Houston, Health Science Center, Houston, Texas 77030 P. ZHAO (3), Department of Neurobiochemistry, Mental Retardation Research Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 JAN ZMUDZKI(10), Department of Pharmacology and Toxicology, Veterinary Research Institute, 24-100 Pulawy, Poland

Preface

The classic perturbation paradigms for the study of the nervous system date to the turn of the century. They relied on the augmentation of tissue present and the removal of tissues of interest, followed by careful microscopic work dependent on artfully conceived tissue fixation and staining protocols. Over the intervening decades, as neurotransmitters and their agonists and antagonists, growth factors and cytokines, and their antibodies and antisense oligonucleotides proliferated and newer and ever more discerning instruments and techniques were developed, the level of sophistication and physiological acuity of the models has increased, perhaps culminating with the most recent additions of transgenic mice, patch clamp measures of electrical activity and capture of unicellular mRNA samples, and noninvasive imaging of the nervous system. As research reports and interpretations of these paradigms become widespread, there is a tendency to focus on the results obtained and on their biological or clinical significance without a sufficient assessment of the actual paradigm being exploited. The result is that in some instances a technique may be adopted to other systems with sometimes confusing consequences. In the interest of providing a forum for the evaluation of some of the currently used paradigms, we offer in this volume an ecletic collection of a number of lesion and probing paradigms in use today. While it would be impractical to make such a list exhaustive, there is a sufficient variety to provide investigators with some insights into a technique being considered for adoption and, perhaps of more utility, some suggestions as to the power and limitations of such paradigms in general. We trust that the techniques presented will be useful to those selecting paradigms for perturbing neural structures or, perhaps, more wisely deter their inappropriate application. J. REGINO PEREZ-POLO

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Methods in Neurosciences

Editor-in-Chief P. Michael Conn

Volume 1 Gene Probes Edited by P. Michael Conn Volume 2 Cell Culture Edited by P. Michael Conn Volume 3 Quantitative and Qualitative Microscopy Edited by P. Michael Conn Volume 4 Electrophysiology and Microinjection Edited by P. Michael Conn Volume 5 Neuropeptide Technology: Gene Expression and Neuropeptide Receptors Edited by P. Michael Conn Volume 6 Neuropeptide Technology: Synthesis, Assay, Purification, and Processing Edited by P. Michael Conn Volume 7 Lesions and Transplantation Edited by P. Michael Conn Volume 8 Neurotoxins Edited by P. Michael Conn Volume 9 Gene Expression in Neural Tissues Edited by P. Michael Conn Volume 10 Computers and Computations in the Neurosciences Edited by P. Michael Conn Volume 11 Receptors: Model Systems and Specific Receptors Edited by P. Michael Conn Volume 12 Receptors: Molecular Biology, Receptor Subclasses, Localization, and Ligand Design Edited by P. Michael Conn Volume 13 Neuropeptide Analogs, Conjugates, and Fragments Edited by P. Michael Conn Volume 14 Paradigms for the Study of Behavior Edited by P. Michael Conn Volume 15 Photoreceptor Cells Edited by Paul A. Hargrave Volume 16 Neurobiology of Cytokines (Part A) Edited by Errol B. De Souza

xiii

xiv

METHODS IN NEUROSCIENCES Volume 17 Neurobiology of Cytokines (Part B) Edited by Errol B. De Souza Volume 18 Lipid Metabolism in Signaling Systems Edited by John N. Fain Volume 19 Ion Channels of Excitable Membranes Edited by Toshio Narahashi Volume 20 Pulsatility in Neuroendocrine Systems Edited by Jon E. Levine Volume 21 Providing Pharmacological Access to the Brain: Alternate Approaches Edited by Thomas R. Flanagan, Dwaine F. Emerich, and Shelley R. Winn Volume 22 Neurobiology of Steroids Edited by E. Ronald de Kloet and Win Sutanto Volume 23 Peptidases and Neuropeptide Processing Edited by A. lan Smith Volume 24 Neuroimmunology Edited by M. lan Phillips and Dwight Evans Volume 25 Receptor Molecular Biology Edited by Stuart C. Sealfon Volume 26 PCR in Neuroscience Edited by Gobinda Sarkar Volume 27 Measurement and Manipulation of Intracellular Ions Edited by Jacob Kraicer and S. J. Dixon Volume 28 Quantitative Neuroendocrinology Edited by Michael L. Johnson and Johannes D. Veldhuis Volume 29 G Proteins Edited by Patrick C. Roche Volume 30 Paradigms of Neural Injury Edited by J. Regino Perez-Polo Volume 31

Nitric Oxide Synthase: Characterization and Functional Analysis Edited by Mahin D. Maines

[1]

Paradigms for Study of Neurotrophin Effects in Oxidant Injury G e o r g e R. J a c k s o n a n d J. R e g i n o P e r e z - P o l o

Neurotrophins

a n d Cell D e a t h

Naturally occurring cell death, or apoptosis, is a process critical to understanding neural development, injury, and regeneration. Apoptosis is sometimes referred to as "programmed" cell death, a description that unfortunately conveys a sense of preordained order not befitting the more ambiently regulated shaping and pruning of synaptic contacts that take place in the developing nervous system. Thus, neuronal cell death more appropriately may be considered as probabilistic or stochastic, rather than apoptotic (Hamburger and Oppenheim, 1982). Naturally occurring ontogenic cell death serves to establish specificity of synaptic connections. The attrition of neuronal populations to match fields of innervation, resulting in cell death and neurite pruning, may be considered positive regulatory factors in the establishment of specificity in the developing nervous system. (Cowan et al., 1984; Oppenheim, 1985; Hamburger and Oppenheim, 1982). Cell death following injury or during senescence of the nervous system, on the other hand, may have proved life threatening to the organism, particularly in the case of injury to the central nervous system, due to the limited capacity of these neurons to regenerate. Neurotrophins regulate naturally occurring and injury-induced neuronal cell death. In the former case, nerve growth factor (NGF) synthesized in target cells becomes available to some axonal endings and is retrogradely transported to neuronal somata during a critical developmental period (Hendry et al., 1974; Hendry, 1975; Johnson et al., 1987). In this fashion, the survival of neurons that have successfully synapsed with their targets is assured. Those neurons that fail to synapse and take up NGF die. In the case of the superior cervical ganglion, as much as 30% of the total neuronal population is lost during this period of attrition (Hendry and Campbell, 1976). Neurotrophins such as the nerve growth factor protein may also play a role in neuronal cell death that occurs as a consequence of injury. The injury-induced synthesis and secretion of neurotrophic factors are believed to facilitate functional recovery by stimulating axonal process formation (Nieto-Sampedro et al., 1983, 1984; Nieto-Sampedro and Cotman, 1985; Needels et al., 1985, 1986; Whittemore et al., 1985). The NGF-related family of neurotrophins is now known to include brain-derived neurotrophic factor, Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PARADIGMS OF N E U R A L I N J U R Y

neurotrophin 3, and neurotrophin 4/5 (for reviews see Korsching, 1993; Lindsay et al., 1994; Maness et al., 1994). These factors achieve their neurotrophic effects via interaction with a low-affinity NGF receptor, referred to as p75 NCvR, and/or high-affinity tyrosine kinase receptors, referred to as Trk, TrkB, and TrkC (for reviews see Barbacid, 1993; Maness et al., 1994). Unrelated factors that have also been attributed neurotrophic effects include ciliary neurotrophic factor, leukemia inhibitory factor, and basic fibroblast growth factor (Ip and Yancopoulos, 1992; Baird, 1994; Murphy et al., 1993; Thaler et al., 1994). Despite these advances in understanding the physiology of neurotrophins and their receptors, the cellular and molecular mechanisms underlying the survival-promoting activity of neurotrophic factors remain poorly characterized. In contrast to better characterized effects of NGF on neurotransmitter metabolism and neurite formation, there is a dearth of knowledge about the mechanisms whereby NGF enhances cell survival. One approach to understanding cell death in mammals has been to study that occurring in more simple organisms. Genes regulating apoptosis have been characterized extensively in the nematode Caenorhabditis elegans (Ellis et al., 1991). Two C. elegans genes, ced-3 and ced-4, encode proteins that play an active role in cell death. A third gene, ced-9, encodes a protein that is a negative regulator of cell death. The mammalian homolog of ced-9 is the protooncogene bcl-2 (Hengartner and Horvitz, 1994). This gene, originally characterized at the breakpoint of a t(14: 18)-bearing B cell lymphoma, is now known to exert neurotrophic effects. High-level expression of bcl-2 in vitro prevents the death of NGF-deprived sympathetic neurons and of PC12 cells grown in serum-free medium (Garcia et al., 1992; Batistatou et al., 1993; Mah et al., 1993). The protein product of this gene has been ascribed an antioxidant function (Hockenberry et al., 1993; Kane et al., 1993). More recently, the mammalian homolog of ced-3, interleukin-1/~ converting enzyme (Yuan et al., 1993), has been shown to prevent NGF deprivation-induced apoptotic cell death in vitro (Gagliardini et al., 1994). Although a number of hypotheses have been advanced to explain neuronal cell death, definitive explanations for the survival-promoting effects of NGF remain elusive. The observation that inhibitors of transcription or translation can prevent naturally occurring neuronal cell death supports the existence of suicide genes encoding death proteins, or "thanatins" (Martin et al., 1988; Oppenheim et al., 1990; Scott and Davies, 1990); however, only one such protein, cyclin D~, has been demonstrated to be preferentially expressed in dying cells (Freeman et al., 1994). Another explanation for the observation that inhibitors of protein synthesis inhibit apoptosis is that such inhibitors preferentially shunt cysteine toward glutathione synthesis (Ratan et al., 1994).

[1] NEUROTROPHINEFFECTS IN OXIDANT INJURY Excitatory amino acid toxicity has also been implicated in ischemiareperfusion injury (Rothman, 1984; Simon et al., 1984; Drejer et al., 1985; Goldberg et al., 1987; Kochhar et al., 1988; Choi and Rothman, 1990; Lipton and Rosenberg, 1994). Recent evidence for the existence of a redox modulatory site on the NMDA receptor that affects excitotoxicity, as well as other relationships between glutamate and free radicals (Aizenman et al., 1990; Pelligrini-Giampetro et al., 1990), would suggest that interactions between excitotoxins and reactive oxygen species in the genesis of cell injury are more prevalent than previously assumed. The role of DNA fragmentation in apoptosis has been described in studies of glucocorticoid-induced effects on lymphocyte lysis, an event that may be relevant to the ontogeny of thymic autotolerance (Compton et al., 1987; Odaka et al., 1990). Internucleosomal DNA fragmentation has also been characterized in apoptosis associated with neuronal cell death, including that seen in NGF-deprived sympathetic neurons (Dipasquale et al., 1991; Deckwerth and Johnson, 1993; Diana et al., 1993).

Reactive

Oxygen

Species

and Cell Death

The generation of oxygen free radicals following traumatic or ischemic injury followed by reperfusion has been described in the nervous system and is held to be a biologically significant phenomenon (HalliweU and Gutteridge, 1985; McCord, 1987; Braughler and Hall, 1989; Hall and Braughler, 1989). An increasing amount of attention has been devoted to the role of oxygen free radicals in neuronal cell death following injury in a number of pathophysiologic conditions, including seizures, ischemia-reperfusion, and inflammation (Halliwell and Gutteridge, 1985; McCord, 1987; Braughler and Hall, 1989). Experimental investigations in the field of free radicals are fraught with technical difficulties, not the least of which are the instability and transient nature of many species of interest. Consequently, many studies seeking to link oxygen radicals to pathophysiology have employed indirect means, including analysis of end products of free radical-produced reactions, such as malondialdehyde (Dahle et al., 1962; Gutteridge, 1981). An alternative indirect approach has been to demonstrate inhibition of these reactions by known antioxidants (Simon et al., 1981; Hall and Braughler, 1989; Topinka et al., 1989). Neither kind of indirect approach, despite its utility in assessment of radical-induced injury, provides conclusive evidence linking free radical effects with pathology. Activated oxygen species, both endogenous and exogenous to the nervous system, may arise in a number of physiologic and pathologic states besides ischemia-reperfusion. The superoxide anion can be produced in mitochondria in several ways, including quinone autoxida-

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PARADIGMS OF NEURAL INJURY

tion (Misra and Fridovich, 1972; Boveris et al., 1976; Patole et al., 1986). Peroxisomes generate H202 directly (Masters and Holmes, 1977). The superoxide ion may be formed during arachidonate metabolism catalyzed by cyclooxygenase and lipoxygenase (Kukreja et al., 1986). Oxidation of catecholamines by monoamine oxidase may be an important endogenous source of H202 in the nervous system (Marker et al., 1981). Exogenous sources of reactive oxygen species may become increasingly important as the response to injury progresses. The generation of reactive oxygen metabolites by phagocytic enzyme systems such as NADPH oxidase and myeloperoxidase, as well as the microbicidal function of these mechanisms, have been documented (Klebanoff, 1968; Babior et al., 1976; Gabig and Lefker, 1984). The role of oxygen radicals in tissue destruction during inflammation has also been demonstrated (McCord, 1987); whether analogous processes occur in the late stages of injury to the nervous system, when secondary waves of neuronal cell death may emanate from primary traumatic or ischemic foci, is not known. Also, the generation of substantial amounts of reactive oxygen species, such as H202, by activated phagocytes may be a major source of such species following injury (Means and Anderson, 1983; Hallenbeck et al., 1986). Reactive oxygen metabolites considered to be of biologic importance include H202, the superoxide anion (O~), and the hydroxyl radical (. OH), and nitric oxide (NO). Lipid peroxidation in unsaturated fatty acids of membrane phospholipids may be initiated by 9OH (Raleigh et al., 1977; Gutteridge, 1982). Once initiated, membrane lipid peroxidation may be propagated, or terminated through the action of a number of cellular antioxidants, including glutathione and c~-tocopherol (Tappel, 1954; Lucy, 1972; Tsan et al., 1985). Hydroxyl radicals are one of the most reactive metabolites of oxygen. By interacting with ferrous iron, H202 is capable of forming 9OH in a Fentontype reaction [Eq. (1)]: F e 2+ + H202---> F e 3+ + O H - + . O H O~ + H202 -'--> 0 2 -}- O H - + 9O H

(1) (2)

Hemoglobin released from erythrocyte lysis following trauma may result in 9OH formation by the Fenton reaction (Gutteridge, 1986). The generation o f . OH can also occur by the Haber-Weiss reaction (2), in which iron catalyzes a one-electron transfer from O~ to H202 (Haber and Weiss, 1934). The ferrous iron then reacts with H202 to generate ferric iron, OH-, and 9OH. During ischemia, degradation of ATP may result in accumulation of hypoxanthine (Kleihues et al., 1974). Xanthine dehydrogenase may be converted proteolytically to xanthine oxidase during ischemia (Batelli, 1980).

[1] NEUROTROPHIN EFFECTS IN OXIDANT INJURY

H20 O2+H20 .= (R) l~k GSSG NADPH OH <--... CAT~ \ # X~// ~f Hexose F~~ /GSH-~[ GSH-R~ C ~ P ~ Monophosphate -~> _ s ~1 /~. jz/X~ / \ Shunt 02 O~i > H202 / GSH ~ NADP R

-

--glutamy~ (~ t r ~ t i d a ~ ~'~-> Cys~Gly cycle

_

~'-Glu-Cys

~l;lutamylcysteine

3'-Glu-AA Cys ~ Glu ~, ~ ~+ f -glutarnyl~ I protein I / s-ox~o,,~=~ cyclotransferase ) ~.~ / AA ~-" 5-Oxoproline

FIG. 1 Overview of the metabolism of reactive oxygen species. For details, see text. From Jackson, G. R. Werrbach-Perez, K., Pan, Z., Sampath, D., and PerezPolo, J. R., Dev. Neurosci. (1995), with permission.

The action of this enzyme on hypoxanthine results in the formation of O~ (McCord and Fridovich, 1968). Under biologic conditions, the Haber-Weiss reaction occurs very slowly, but substantial 9OH formation by the Fenton reaction takes place (Freeman and Crapo, 1982). Nitric oxide is capable of interacting with superoxide to form the toxic peroxynitrile ion. On the other hand, nitric oxide may under some conditions exert a neuroprotective effect via interaction with a redox modulatory site of the NMDA receptor (Lipton et al., 1993). Figure 1 shows a simplified scheme of reactive oxygen metabolites and antioxidant defenses. Superoxide dismutase catalyzes the conversion of the superoxide anion to H202 (McCord and Fridovich, 1969). Catalase and glutathione peroxidase catalyze the degradation of H202 (Mills, 1957, 1960; Deisseroth and Dounce, 1970; Chance et al., 1979). The kinetic properties of the latter enzyme suggest that it may be of more importance under relatively low H202 concentrations (Cohen and Hochstein, 1963; Flohe and Brand, 1969). The importance of the glutathione peroxidase system as compared to catalase in oxidative injury has been emphasized by some investigators but disputed by others (Sadrzadeh et al., 1984; Suttorp et al., 1986; Gaetani et al., 1989; Giblin et al., 1990). On the other hand, the high capacity of catalase for H202, based on its rapid turnover number, would suggest that catalase activity is diffusion limited (Chance et al., 1979). Because the activity

6

PARADIGMS OF NEURAL INJURY

of the peroxidase system is closely linked to regeneration of GSH by GSH reductase and NADPH (Arrick et al., 1982; Harlan et al., 1984; Schrauffstatter et al., 1985), the availability of reducing equivalents for regeneration of GSH may become a critical factor regulating this pathway. The activity of enzymes capable of generating NADPH, such as malic enzyme and the hexose monophosphate shunt enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, may also play a critical role in cellular responses to oxidative stress by supplying reducing equivalents (Harlan et al., 1984; Schrauffstatter et al., 1985; Suttorp et al., 1986; Meister et al., 1988; White et al., 1988). The coordinated operation of a number of cellular defense mechanisms is required for an integrated response to oxidative stress, the relative contribution of each element to the overall response depending on the severity of the injury sustained. Cellular effects of reactive oxygen species may be grouped into five categories: membrane damage, protein modifications, ion deregulation, DNA strand breakage, and effects on energy homeostasis. Interactions between effects in each category make these simplistic classifications artificial but do not limit their usefulness. The effects of lipid peroxidation on membrane integrity and the formation of cytotoxic by-products, such as aldehydes, have been documented (Benedetti et al., 1984; Demopoulos et al., 1979; Masaki et al., 1989). Despite a large body of evidence implicating lipid peroxidation in pathology, using indirect indices such as lactate dehydrogenase release or malondialdehyde formation, in most cases the demonstration that such changes are necessary and sufficient to elicit cytotoxic effects is lacking. Protein inactivation by thiol oxidation, cross-linking, and other modifications may rapidly begin to disrupt the normal functions of other systems, such as glycolysis due to effects on glyceraldehyde-3-phosphate dehydrogenase, or ion regulation due to effects on Na§247 (Fligiel et al., 1984; Kim et al., 1985; Girotti et al., 1986; Wolff and Dean, 1986; Hyslop et al., 1988; Richards et al., 1988; Kyle et al., 1989). Protein-phospholipid cross-linking may also impair cell function as a consequence of oxidative damage (Nielsen, 1981). Cellular ATP losses during oxidative stress may further compromise Na§247 activity and ion regulation, resulting in cellular swelling (Maridonneau et al., 1983). Elevation of intracellular free calcium, derived both extracellularly and from intracellular sources such as mitochondria, occurs as a consequence of membrane disruption and ion deregulation and activates enzymes such as phospholipases and endonucleases (Mallis and Bonventre, 1986; Hyslop et al., 1986; Cantoni et al., 1989a,b). Hydroxyl radicals formed from H202 in the Fenton reaction efficiently generate singlestrand breaks in DNA (Imlay and Linn, 1988; Olson, 1988). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase, which catalyzes the ADP-ribosylation of chromatin proteins at the expense ofNAD § (Berger,

[1]

N E U R O T R O P H I N EFFECTS IN OXIDANT INJURY

1985; Berger et al., 1986; Schrauffstatter et al., 1986a,b; Olson, 1988). ADPribosylation of chromatin proteins such as histone H1 counteracts histone inhibition of DNA repair processes, such as DNA ligase activity (Durkacz et al., 1980; James and Lehmann, 1982; Morgan and Cleaver, 1983). One hypothesis of cell death following oxidative stress is that irreversible depletion of NAD + and hence ATP generation via oxidative phosphorylation occurs as a consequence of ADP-ribosylation in response to DNA damage (Spragg et al., 1985; Cantoni et al., 1986; Carson et al., 1986; Gille et al., 1989; Junod et al., 1989; Varani et al., 1990). Depletion of NAD + may result in decreased glycolytic flux, further perturbing energy homeostasis (Berger et al., 1986). Thus, the ability of cells to recover from depletion of pyridine nucleotides may be crucial to the final outcome of oxidative injury. Inhibition of poly(ADP-ribose) polymerase has been demonstrated to confer protection against chemically induced oxidative stress and against traumatic injury in hippocampal slices (Wallis et al., 1993). Another link between oxidative stress and the activation of poly(ADP-ribose) polymerase is the observation of nitric oxide-induced activation of the enzyme (Zhang et al., 1994). I n V i t r o P a r a d i g m s for S t u d y o f O x i d a n t I n j u r y

Various systems are available for study of neurotoxicity in vitro. Primary dispersed cultures of neurons, astrocytes, or oligodendrocytes provide a means of assessing effects of agents on a defined cell type. The use of transformed cells lines that exhibit neural properties has the advantage that large quantities of cells may be grown, permitting detailed biochemical analysis. Organotypic explants (Hauser and Stiene-Martin, 1991; Lyman et al., 1991, 1992; Newell et al., 1993), reaggregate cultures (Jones et al., 1993), synaptosomes (Pifl et al., 1993), and slice preparations (Schurr et al., 1993; Wallis et al., 1993) all provide ways of assessing neurotoxicity. Each of these techniques possesses its own inherent advantages and disadvantages (for review see Atterwill et al., 1992). Cell culture approaches to the study of injury have the advantage that the responses of isolated cell types can be analyzed in detail. The cell culture milieu may be precisely controlled and manipulated to study the effects of individual elements. The caveat that information gleaned from in vitro experiments with single cell types provides only a partial explanation for complex physiological processes does not negate the utility of such approaches. As indicated in Table I, a number of methods are available for generating oxidant injury experimentally. These include hypoxia, either chemically induced or generated by incubation in a low-oxygen atmosphere followed by return to normal oxygen content (Cazevieille et al., 1992; Zhang and

8

PARADIGMS OF NEURAL INJURY TABLE I

Methods for Inducing Oxidant Stress a

Hypoxia Rose bengal Glucose/glucose oxidase Xanthine/xanthine oxidase H202 Sodium nitroprusside/nitroglycerin/S-nitrosocysteine 6-Hydroxydopamine MPTP Amyloid fl-protein a For references, see text.

Piantadosi, 1992; Akaneya et al., 1994). Both the glucose/glucose oxidase and the xanthine/xanthine oxidase systems are capable of generating the superoxide anion, although singlet oxygen may also be a source of oxidative stress when these systems are employed (Fridovich and Handler, 1962; Griot et al., 1990; Pan and Perez-Polo, 1993; Hiraishi et al., 1994; Naveilhan et al., 1994). Rose bengal is another source of singlet oxygen (Fridovich, 1989; Van Reempts et al., 1993). Hydrogen peroxide can be added directly to culture medium, resulting in the production of the hydroxyl radical via the Fenton reaction (Jackson et al., 1990, 1992, 1994; Naveilhan et al., 1994). NO can be added to cultures directly or generated using nitroprusside or nitroglycerin (Chen et al., 1991; Lustig et al., 1992). Reactive oxygen species may also be generated by 6-hydroxydopamine, the toxicity of which depends in part on the generation of H 2 0 2 (Heikkila and Cohen, 1972; Cohen and Heikkila, 1974; Tiffany-Castiglioni and Perez-Polo, 1980, 1981; Spina et al., 1992; Cerutti et al., 1993). Another source of reactive oxygen species is 1methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). The toxicity of MPP +, the biotoxic metabolite of MPTP, has been suggested to depend in part on the hydroxyl radical (Johannessen, 1991; Chiueh et al., 1992; Cleeter et al., 1992; Hartikka et al., 1992; Cerutti et al., 1993; Pifl et al., 1993; Hartley et al., 1994). Amyloid fl-protein toxicity has been suggested to depend on the generation of H 2 0 2 (Behl et al., 1994~. Studies of cell survival require reliable, reproducible techniques to assess cell viability. Table II lists a number of methods available for quantitating cell survival. A number of approaches have been taken to this end, including techniques measuring properties such as membrane integrity, protein or nucleic acid synthesis, or colony-forming ability. Each of these measures is perturbed by cell injury, but no single one is an entirely satisfactory index. Tests of membrane integrity, such as trypan blue exclusion, have long been

[1]

NEUROTROPHIN EFFECTS IN OXIDANT INJURY TABLE II

M e t h o d s of Assessing Cell Viability a

Radiolabeled precursor incorporation MTT reduction Trypan blue exclusion Rhodamine 123 fluorescence Propidium iodide fluorescence Morphology Acridine orange fluorescence 5~Cr release Cytosolic enzyme release Clonogenic survival a For references, see text.

accepted as measures of viability (Pappenheimer, 1917; Eaton et al., 1959; Tennant, 1964). Leakage of intracellular enzymes has been widely exploited both in vivo and in vitro (Smith et al., 1987; Loeb et al., 1988; Martin et al., 1988; Olson, 1988). Measurement of the release of cytoplasmic enzymes, such as lactate dehydrogenase (LDH) or adenylate kinase, is an effective index at late stages of cell injury but a poor measure of early homeostatic changes (Martin et al., 1988; Jackson et al., 1992; Cazevieille et al., 1993; Behl et al., 1994). Release of 51Cr may also be used as an index of cell viability (Sarafian and Verity, 1990). Dye-exclusion measurements fail to provide a sensitive index of cell viability because each cell must be classified as either alive or dead with no intermediate stages. Indices that rely on metabolic activities, such as tetrazolium salt reduction and incorporation of radioactive precursors, provide more sensitive measures of early perturbations in cell viability. Reduction of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was originally employed in mitogen studies of lymphoid cells, but has found application elsewhere (Mosmman, 1983; Hansen et al., 1989). This assay depends on mitochondrial activity, specifically succinate dehydrogenase, to reduce a tetrazolium salt to a colored product (Slater et al., 1963). Although the assay is highly sensitive, it may be inappropriate for measuring responses to injury when cells display impaired metabolic activity but nevertheless remain viable. Measurement of radioactive amino acid, uridine, or thymidine incorporation affords a sensitive index of activity in some cell types, but is useless in phases of the cell cycle when viable cells fail to incorporate precursors. Clonogenic survival may be particularly problematic in some cell types, such as neurons, that normally do not proliferate once they have reached maturity (Puck and Marcus, 1955; Tiffany-Castiglioni and Perez-Polo, 1980; Anderson, 1994;

10

PARADIGMS OF NEURAL INJURY

Van der Maazen et al., 1992). Fluorescence assays using agents such as rhodamine, propidium iodide, and acridine orange are convenient and may be applied to flow cytometry (Detta and Hitchcock, 1990; Gagliardinini et al., 1994; Deckwerth and Johnson, 1993; Ziv et al., 1994; Favit et al., 1992; Detta and Hitchcock, 1990; Darzynkiewicz et al., 1992; Sauer et al., 1992). Metabolic indices such as energy charge, which measures high-energy phosphate content (Atkinson, 1968), have been considered among the most definitive measures of metabolic capability. Nevertheless, even this technique may not be appropriate in all circumstances.

E f f e c t s o f N G F o n O x i d a n t I n j u r y in P C 1 2 Cells Our laboratory has used the PC12 rat pheochromocytoma cell line as a model for elucidating the effects of NGF on oxidant injury. Figure 2 shows the dose-response to HzO2 in control and NGF-treated cultures. The minimal concentration of NGF tested that was capable of enhancing survival above the control level was 1 ng/ml, a level effective only against 0.1 mM H202. Higher levels of NGF were effective against a wider range of H202 concentrations. The effects of NGF on cell viability under serum-free conditions were measured using protein synthesis and MTT reduction (Fig. 3). Under these conditions, the minimal dose of NGF tested that was capable of enhancing PC12 cell viability above the basal level was 1 ng/ml. Higher doses (10-100 ng/ml) had a more pronounced cell-sparing effect. The dose-response relationship between NGF concentration and survival in serum-free medium resembled that for survival following H202 treatment (Fig. 2). The time course for cell death following 0.5 mM H202 treatment is shown in Fig. 4, using dye exclusion, [35S]methionine incorporation, MTT reduction, and LDH release as viability assays. Decreases in all indices were observed following 0.5 mM H202 injury at the earliest time points measured, i.e., 2 hours postinjury for dye exclusion and LDH release and 4 hours for [35S]methionine incorporation and MTT reduction. NGF enhancement of survival was evident at the earliest time points obtained for dye exclusion and amino acid incorporation, but not for the other two indices. Enhanced survival of NGF-treated cells was not evident until 8 hours postinjury for MTT reduction and 4 hours for LDH release. No evidence for substantial recovery from initial viability losses was apparent except in the MTT reduction assay. This apparent recuperation from initial damage was most pronounced in NGFtreated samples. LDH release, in contrast, continued to increase such that the maximal value attained was at 24 hours. NGF-treated samples continued

[1]

NEUROTROPHIN EFFECTS IN OXIDANT INJURY

11

to release LDH after 4 hours, although these continued losses were less marked than those observed in control cultures. Previous studies of NGF effects on HzO 2 toxicity in PC12 cells and the SY5Y human neuroblastoma cell line used chronic NGF treatment regimens (6-11 days) before performing cytotoxicity tests (Tiffany-Castiglioni and Perez-Polo, 1981; Perez-Polo et al., 1986; Jackson et al., 1990). The data presented here indicate that NGF induction of a H202 refractory state does not require extended treatment. NGF treatment for 24 hours elicited a significant cytoprotective effect against oxidant injury. This effect was apparent prior to withdrawal of PC12 cells from the mitotic cycle, an effect requiring more prolonged NGF treatment (Greene and Tischler, 1976; Greene, 1978). Thus, cell cycle dependency of H202 injury, an important determinant of cellular ability to repair single-strand DNA breaks (Frankenberg-Schwager, 1990; Kleiman et al., 1990), does not appear to bear on enhanced survival of cells treated with NGF for 24 hours prior to injury. The minimal NGF concentration tested was capable of increasing PC12 cell survival above the control level was 1 ng/ml, although this NGF level was less effective than higher concentrations against high H202 levels. Similar doses of NGF were required for enhancement of survival following oxidant injury and serum deprivation. The ECs0 for NGF (i.e., the concentration necessary for a half-maximal effect at any given HzO 2 concentration) was estimated to be between 1 and 10 ng/ml. This dose-response relationship is similar to that previously described (Greene, 1978) and bears physiologic relevance in that the minimal effective dose tested (1 ng/ml or 3.7 nM) was similar to the Kd of the low-affinity NGF receptor (Sutter et al., 1979). Thus it appears from the dose-response characteristics that NGF induction of an HzOz-refractory state is a physiological rather than a pharmacological effect. Losses of viability following 0.5 mM HzO 2 treatment occurred at the earliest time points measured, regardless of the assay employed. Trypan blue exclusion decreased more rapidly in these studies than in those reported following a higher HzO 2 concentration (2.5 mM) in macrophage-like cells (Schrauffstatter et al., 1986b). The more rapid decreases in viability seen in PC12 cells were likely to reflect an enhanced susceptibility of neurally derived cell types to oxidant injury as compared to other cell types (Tiffany-Castiglioni et al., 1982). 6-Hydroxydopamine, a neurotoxin that generates H202 (Heikkila and Cohen 1972; Cohen and Heikkila, 1974), studied in the SY5Y human neuroblastoma line, revealed that dye exclusion was among the last viability index tested to be impaired (Tiffany-Castiglioni and Perez-Polo, 1980; TiffanyCastiglioni et al., 1982). The delayed changes in trypan blue exclusion reported in early studies may be a consequence of the prolonged generation of oxygen radicals by 6-hydroxydopamine as compared to delivery of a preformed HzO 2 concentration described here.

12

P A R A D I G M S OF N E U R A L I N J U R Y

110

o~ "-" z O_

~ 0 ~. nO o z__ bw "-~ U3

%.

100 90

' •

80

70

\"'-i

60

"*--*

%

50

.....

.....

~_ -e..

40 30

•

20 10 Illl

II, OH

,

i

~,. .

"i

-eZ~

-.

------_v.~" "e-

i i illl

i

i

i

i

0.10

i ilia

"k~

i

1.0

i

i

i i|

10.0

H20 2 (mM)

FIG. 2

Thus, the effect of NGF o n H 2 0 2 resistance was rapid and dose dependent. A direct comparison of four different viability assays demonstrated NGF protection from H 2 0 2 cytotoxicity. These findings refute the contention that NGF enhancement of cell viability as assessed by radiolabeled amino acid incorporation is an artifact secondary to NGF enhancement of protein synthesis. Homeostatic perturbation by oxidant injury has widespread repercussions on cellular function. The outcome of oxidant injury depends on a balance between the severity of the insult sustained and the capacity of cells to withstand and recover from such injury. Mechanisms of cellular recovery from the toxicity of reactive oxygen species may be as critical to the outcome of injury as are mechanisms for preventing such injury, such as antioxidant capabilities. The generation of single-strand DNA breaks in cultured cells during oxidant injury sets in motion a number of processes that may culminate in cell death (Olson, 1988; Schrauffstatter et al., 1986a,b). The activation of nuclear poly(ADP-ribose) polymerase (PADPRP) following DNA damage results in rapid depletion of NAD + (Sims et al., 1983; Berger, 1985; Schrauffstatter et al., 1985, 1986a; Berger et al., 1986; Junod et al., 1989). If extensive DNA damage causes irreversible NAD + depletion, cell death may ensue from inhibition of energy metabolism via effects on glycolysis and oxidative phosphorylation (Spragg et al., 1985; Berger et al., 1986; Carson et al., 1986; Schrauffstatter et al., 1986b). Inhibition of PADPRP using inhibitors such as 3-aminobenzamide (AB) has been reported to prevent NAD + depletion and cell death following oxidant exposure (Sims et al., 1983; Schrauffstatter et

13

[1] NEUROTROPHIN EFFECTS IN OXIDANT INJURY

FIG. 2 D o s e - r e s p o n s e effect of H20 2 on viability of PC12 cells treated with varying doses of N G F , using [35S]methionine incorporation as an index. Studies of N G F effects on H20 2 toxicity were performed as described previously (Jackson et al., 1990, 1992). PC12 cells (7.5 • 104) were plated in poly(D-lysine)-coated 6-mm-diameter wells and allowed to attach overnight in complete m e d i u m before addition of N G F . After 24 hours, these media were r e m o v e d and replaced with varying dilutions of H20 2 in RPMI 1640 medium. After 30 minutes o f H 2 0 2 t r e a t m e n t at room t e m p e r a t u r e , these solutions were r e m o v e d and replaced with complete medium. Following a 24hour r e c o v e r y period, viability was assessed. Incorporation of 35S]methionine was performed as previously described (Jackson et al., 1990, 1992). Each point represents the mean _+ S E M for quadruplicate samples. T , 0; O, 1; , , 10. II, 100 ng N G F / m l . Statistical analysis was carried out using A N O V A followed by F i s h e r ' s L S D test, comparing the values b e t w e e n N G F treatment groups at each H20 2 concentration. df, 29; F, 66.52. Statistics are summarized in the tabulations below, which give the most significant a level obtained for each comparison (NS, not significant, i.e., p -> 0.05) (note: at 5 m M H202, all values are NS):

NGF (ng/ml) NGF (ng/ml)

0

0 0.1 1.0 10

--

0.1

1.0

mM H202 NS NS -NS --

10

100

0.001 0.001 0.005

0.001 0.001 0.001 0.001

0.5

--

100 0.10

0 0.1 1.0 10

--

NS --

mM H20 z 0.01 NS ~

0.001 0.001 0.05 --

0.001 0.001 0.005 NS

100

mM HzO2 NS NS -NS

0.5

0 0.1

--

1.0

--

10

0.001 0.001

0.001 0.001

0.001

0.001

--

NS

100

0 0.1 1.0 10 100

--

1.0 mM HzOz NS NS -NS --

0.001 0.001 0.005 --

0.001 0.001 0.001 NS

14

PARADIGMS OF N E U R A L I N J U R Y 0.70 E tO

tO

Z

0.60

/

0.50

LU tO Z < rn n" O

0.20

<

0.10

nn

60

0.40 0.30 -

T

//~,~~

48

I ~

-36

o.. n" 0 0 Z

-

-F-

7/ 24

LU

-

0.00

/'/" 0

i

i

i

0.01

0.10

1.0

NGF

=

10

12

tt)

co

0

100

(ng/ml)

FIG. 3 Correlation between [35S]methionine (T) incorporation and MTT (O) reduction as indices of viability on the fourth day of serum deprivation. Serum deficiency experiments were performed by plating PC12 cells in the presence of serum, washing, and culturing 72 hours under serum-free conditions. PC12 cells were plated in complete medium at a density of 5 • 104 cells/well in 16-mm-diameter wells. After allowing the cells to attach overnight, they were washed three times in RPMI 1640 medium with 15 mM HEPES and then treated with various dilutions of NGF (0.01-100 ng/ ml). After 48 hours under serum-free conditions, these media were replaced with fresh media (containing the same growth factor concentrations). Cells were cultured another 24 hours before viability determination. Each point represents the mean ___ SEM for quadruplicate wells. For MTT reduction, wells treated with 1, 10, and 100 ng NGF/ml differed from the untreated value (df, 5; F, 518.69; p < 0.001 vs. 0 NGF value); for [35S]methionine incorporation, 10 and 100 ng/ml differed from the control (df, 5; F, 49.25; p < 0.001 vs. 0 NGF value; ANOVA with Fisher's LSD test).

al., 1986b; Stubberfield and Cohen, 1988). Other studies using this inhibitor, however, have reported no change or even enhancement of cytotoxicity following oxidative stress (Cantoni et al., 1989a; Gille et al., 1989). A further complication in the experimental use of AB is the reported effect of the inhibitor on processes other than ADP-ribosylation (Milam and Cleaver, 1984; Hunting et al., 1985; Milam et al., 1986). The connection between depletion of NAD § ATP, and cell death has been challenged by studies that have demonstrated a dissociation of ATP maintenance from viability (Stubberfield and Cohen, 1988; Varani et al., 1990). The experiment presented in Fig. 4 revealed a cytoprotective effect in NGF-pretreated PC12 cells as early as 2 hours following H202 injury. Our studies of N G F effects on antioxidant systems suggested that, although significant enhancement in the activity of defenses such as catalase and glutathione peroxidase occurred following N G F treatment, many such effects

A

B

""mt.u 125 [

125

._1

< uJ ._1 uJ

O

100

z O o

rr"

g

75

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--b

100

75

50

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> 0 0

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I 16

I

20

0

24

TIME FOLLOWING H 2 0 2 (HR)

C

g x w D z_

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I

I

I

8

12

16

20

24

TIME FOLLOWING H 2 0 2 (HR)

D

125

._1

O tr Iz O

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125

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,~

100

100

-

,..q

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75 X

UJ

121

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25

~

s~-

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9176

_.1

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25

i

4

-_~---'F

8

12

l

16

i

20

TIME FOLLOWING H20 2 (HR)

24

0

0

a.

4

l

8

=

12

l

I6

20

24

TIME FOLLOWING H20 2 (HFI)

FIG. 4 Time course for cell death following 0.5 mM H20 2 treatment in control and NGF-pretreated cells as assessed by dye exclusion (A), [35S]methionine incorporation (B), MTT reduction (C), and LDH release (D). Experimental conditions were as described in the legend to Fig. 2. Viability assays were performed as described (Jackson et al., 1992). All points are mean _+ SEM for quadruplicate wells. For trypan blue exclusion, data were expressed as percentage of cells excluding dye. Values for NGF-treated samples (T) differed significantly from controls (0) at 2, 4, 8, and 24 hours postinjury (p < 0.05, 0.05, 0.005, and 0.05, respectively). For [35S]methionine incorporation, values were normalized to the initial value (100% for either control or NGF-treated wells). NGF-treated samples differed significantly from controls at 4, 8, and 24 hours postinjury (p < 0.001, 0.001, and 0.01, respectively). For MTT reduction, values were normalized to the initial value (100% for either control or NGF-treated wells). NGF-treated values differed from controls at 8 and 24 hours (p < 0.001). Values for LDH were expressed as a percentage of the total activity released into the medium at each time point. NGF-treated samples differed from controls at 0, 4, and 24 hours (p < 0.01, 0.005, and 0.001, respectively; Student's t-test). From Jackson, G. R., Werrbach-Perez, K., Ezell, E. L., Post, J. F. M., and Perez-Polo, J. R., Brain Res. 592, 239 (1992), with permission. 15

16

PARADIGMS OF NEURAL INJURY

required culture for longer times and with higher concentrations than those necessary to decrease HzO2 sensitivity (Jackson et al., 1990, 1994; Pan and Perez-Polo, 1993; Sampath et al., 1994. These efforts to elucidate effects of NGF on the degradation of activated oxygen species suggested that investigation of an aspect of oxidant injury other than the defense against initial injury might prove worthwhile, i.e., the recovery phase. NGF treatment rapidly decreases the content of PADPRP and its mRNA in PC12 cells (Taniguchi et al., 1988). Rapid (within 15 minutes) depletion of NAD + was observed following 0.5 mM H202 treatment in both control and NGF-pretreated cells (Fig. 5). By 1 hour following HzO2 treatment, intracellular NAD + concentrations had decreased to less than 20% of initial values. No evidence for NGF effects on initial depletion of NAD + was obtained; on the contrary, the mean NAD + concentration for NGF-treated cells (as a percentage of the NGF-treated control) was actually lower than the corresponding control value. At later time points, however, NGF-treated cells began to evidence a more extensive recovery from this initial depletion. Although there was an apparent enhancement of recovery from NAD + depletion in NGF-treated cells as compared to controls by 2 hours following injury, this increase did not attain statistical significance until 4 hours following H202 treatment. In control cells there was some evidence for enhanced recovery of NAD + from the minimum levels observed at 4 hours following injury, but the recovery was delayed and less complete than that observed in NGF-pretreated cells. By 24 hours following oxidant treatment, the NAD + concentration in NGF-treated cells had recovered to 80% of the initial value; in control cells, by contrast, the NAD + concentration was less than 50% of the initial value by this time. Analysis of LDH release confirmed that the more complete recovery of NAD + noted in NGF-treated as compared to control cells was matched by an enhanced viability, as indexed by greater enzyme retention (Jackson et al., 1992). These data indicated that NGF pretreatment did not prevent NAD + depletion occurring as a consequence of PADPRP activation; rather, the effect of the growth factor was to enhance recovery from initial NAD + depletion. Further experiments used a competitive inhibitor of PADPRP, AB, to examine further the role of this enzyme in H202 cytotoxicity. AB pretreatment results in a dose-dependent increase in cell survival following injury with H202 (Jackson et al., 1992). The effect of a single concentration of AB in control and NGF-pretreated cells, as assessed 24 hours following H202, is illustrated in Fig. 6. The protective effect of NGF in the absence of AB was qualitatively similar to that observed in experiments already described. In this particular experiment, incubation with AB had a protective effect on control PC12 cells only at 0.1 mM H202. The combination of AB with NGF

[1] NEUROTROPHIN EFFECTS IN OXIDANT INJURY

17

12o[

~. ~_ 0

110~ 100~ 90 80

60

//.L

50

,"

.~

40

z

30

" ~'

20 ~-~y,,~ 0

0

o..... - ~ ' ~ i 4

i 8

i 12

I 16

i 20

24

TIME FOLLOWING H20 2 (HR)

FIG. 5 Effect of treatment with 0.5 mM H202 on intracellular NAD + in control (0) and NGF-treated (!t) PC12 cells. Analysis of NAD + depletion was performed as described previously (Jackson et al., 1992). Briefly, cells (106) were plated on poly(Dlysine)-coated 60-mm-diameter dishes and pretreated 24 hours with NGF. NAD + concentrations were determined in perchloric acid extracts of cells harvested at various times following H202 treatment. Points are the mean + SEM for triplicate determinations. Values for NGF-treated cultures were significantly different from those for controls at 4, 8, and 24 hours following H202 treatment (p < 0.001, 0.001, and 0.05, respectively); the value for NGF-treated cells was significantly different from the control at 1 hour (p < 0.05, Student' s t-test). From Jackson, G. R., WerrbachPerez, K., Ezell, E. L., Post, J. F. M., and Perez-Polo, J. R., Brain Res. 592, 239 (1992), with permission.

pretreatment had a dramatic effect on survival. NGF-pretreated cells incubated with AB displayed an enhanced survival as compared to cells without AB at H202 concentrations between 0.1 and 1.0 mM. N G F treatment alone was insufficient to enhance survival above the control level at H202 concentrations above 0.1 mM. In cells treated with both N G F and AB, by contrast, robust tetrazolium salt reduction was observed at H202 levels as high as 1 mM. Thus, inhibition of PADPRP had an effect in NGF-treated cells that was more pronounced than that observed in undifferentiated cells. Consistent with studies of oxidant injury in other cell lines (Cantoni et al., 1989a; Gille et al., 1989; Junod et al., 1989; Schrauffstatter et al., 1986a,b), rapid depletion of N A D + was observed following 0.5 mM H202 treatment. Because N G F treatment diminished the PADPRP content of PC12 cells within the same time frame used in these experiments (Taniguchi et al.,

18

PARADIGMS OF N E U R A L INJURY 0.55 T 0.50 - ~I~, ~,

0.45

i,,.

0.40

t.u

0.30

z<

0.25

n"

0.20 -

co O m ,a:

0.15 -

or

o m

" ,,~,..

-I"~

""" ~ . . ' ~

0.35 "

"

0.05

"

-

, ,tr ,,,

0

0.01

........

\

: .

•,, '~',, M

O.lO o.oo

""I\

"

9

9 \~1~

-.

~~1% , %" '

0.1

,

~- ',~-

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"N

, ,"-_ ....

10

H 2 0 2 (mM)

FIG. 6 Effect of aminobenzamide (AB) on H202 toxicity in control (0) and NGFpretreated ( . ) cells, as assessed 24 hours following injury. Culture conditions were similar to those described in the legend to Fig. 2. Pretreatment with AB was performed as described (Jackson et al., 1992). The value for AB-treated (Ir) cells differed significantly from the control at 0.1 mM H202 (p < 0.05). The value for AB- and NGF-treated (11) cultures differed significantly from the NGF-treated control at 0.1, 0.5, and 1.0 mM H202 (p < 0.05, 0.001, and 0.001, respectively; df, 27; F, 208.6; ANOVA with Fisher's LSD test). From Jackson, G. R., Werrbach-Perez, K., Ezell, E. L., and Post, J. F. M., and Perez-Polo, J. R.,Brain Res. 592, 239 (1992), with permission.

1988), NAD + depletion as a consequence of PADPRP activity was expected to be attenuated in NGF-treated cells. Contrary to this expectation, however, NAD § depletion was observed to a similar degree in NGF-treated cells and controls, suggesting that the previously reported effect of the growth factor on PADPRP is irrelevant to enhancement of responses to oxidative stress. A competitive inhibitor of PADPRP was used to probe further the role of this enzyme and pyridine nucleotide metabolism in cell death following oxidative stress (Jackson et al., 1992). The inhibitor minimized cell death following H202 treatment in a time- and dose-responsive fashion, implying that NAD § depletion was a significant component of the metabolic consequences of oxidative stress culminating in cell death. Two different observations suggest that the reported reduction of PADPRP content of PC12 cells by N G F treatment (Taniguchi et al., 1988) is not related to the cytoprotective effect of the factor in oxidant injury. First, N G F pretreatment did not prevent NAD § depletion following H202 injury. Second, incubation with AB had an effect in NGF-treated cells that was more pronounced than that observed in controls. If the premise is accepted

[1] NEUROTROPHINEFFECTS IN OXIDANT INJURY

19

that irreversible NAD + depletion is a critical determinant of cell death as a consequence of oxidant injury, a scenario may be envisioned in which the capacity of NGF to enhance cell viability by stimulating recovery from NAD + depletion may be limited by the number of cells that remain at or above the threshold for reversibility of injury. By inhibition of PADPRP, the number of cells that remain above this critical threshold at a given level of injury is increased, increasing the effectiveness of NGF enhancement of recuperation via stimulation of NAD synthesis. Details of the relationship between NAD + depletion and cell death following oxidative stress have not been fully elucidated. Some investigators have reported reductions of ATP following NAD + depletion (Carson et al., 1986; Schrauffstatter et al., 1986a; Spragg et al., 1985), whereas others have demonstrated a dissociation between reductions of NAD +, ATP, and survival (Stubberfield and Cohen, 1988; Gille et al., 1989; Varani et al., 1990). The mechanism(s) responsible for losses of ATP following NAD + depletion have been attributed to increased utilization of ATP for NAD + cycling, to activation of the hexose monophosphate shunt, or to diminished oxidative phosphorylation secondary to decreased activity of NAD-dependent enzymes and reduced glycolytic flux (Carson et al., 1986; Berger et al., 1986; Spragg et al., 1985).

Acknowledgments Supported in part by NS Grant 18708 and by a grant from the American Paralysis Association.

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[2]

Nitric Oxide Toxicity in Central Nervous System Cultures Valina L. Dawson* and Ted M. Dawson

Introduction Primary neuronal cell cultures are ideal as a model system for investigating isolated cellular mechanisms in that they retain most of the physiological and biochemical characteristics of in situ neurons (1) and are free from the influences of the microvasculature. Furthermore, manipulation of neuronal circuitry and composition of supportive cells and extracellular buffering allow for a variety of diverse processes to be investigated. Culture conditions can be optimized to observe the mechanisms of interest. However, several considerations must be made when using cultured neurons. Individual neuronal cell types, in different brain regions, mature at different rates throughout the development of the central nervous system. Therefore, the developmental age of the animal is important in generating optimal cell cultures from different brain regions. For instance, mesencephalic cultures should be obtained at embryonic day 12 or 13, cortical cultures at embryonic day 14 or 15, hippocampal cultures at embryonic day 16 or 17, and cerebellar granule cell cultures at postnatal day 1. Additionally, neurons in culture will continue to mature after the initial plating, altering the physiology and biochemistry of the culture system. Thus, it is particularly important to determine when the proteins or pathways of interest are present in a particular culture system. For instance, in cortical neurons, to study optimally oxidative stress that is unrelated to glutamate-mediated events, cultures should be used at 72 hours after plating (2). To study glutamate responses in cortical neurons without a nitric oxide (NO) component, the cultures should be examined before day 17 in culture. However, to study nitric-oxide synthase (NOS; EC1.14.13.39) activation and NO production, cultures must be examined after day 20 in culture, when NOS is fully expressed at in vivo levels (3). The ability to manipulate neuronal cultures allows for maximal optimization of the pathway of interest. However, the data derived from neuronal cultures must be regarded as potential components of a larger system and must ultimately be judged relative to the results obtained in vivo. Primary

* To whom correspondence should be addressed.

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Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

[2]

NITRIC OXIDE TOXICITY IN CNS CULTURES NArg, NMA NIO, LNAME NMMA

JCaMJ

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DPI

+

I FMN FAD INADP"I

Calmidazolium W-7 GTlb GM1

FIG. 1 Arginine analogs compete with arginine (LARG) for the catalytic site on NOS and decrease the amount of NO produced. NO production can be decreased by the removal of L-arginine from the media. Calmodulin (CAM) is an essential cofactor and calmodulin antagonists or agents that bind calmodulin can reduce N0S catalytic activity and decrease NO production. The shuttling of electrons by the flavoprotein moieties is critical for the conversion of L-arginine to citrulline and NO. NOS catalytic activity can be inhibited by flavoprotein inhibitors. NOS is phosphorylated by four different kinases on four different amino acids. In the phosphorylated state NOS is inactive. NOS is dephosphorylated and activated by calcineurin. Calcineurin antagonists keep NOS in the phosphorylated, inactive state and thus decrease NO production.

neuronal cultures are useful for modeling isolated mechanisms, but because they lack many in situ components, they may not accurately reflect neuronal function in an intact nervous system. Nitric oxide mediates many biological processes, including vasodilation, cytotoxicity of activated macrophages, and glutamate-stimulated formation of cGMP in the nervous system. Nitric-oxide synthase converts L-arginine to NO and citrulline. Endothelial and neuronal NOS is activated by an increase in intracellular calcium. In neurons entry of Ca 2+ through glutamate receptor channels can activate NOS. In primary neuronal cultures, glutamate and N-methyl-D-aspartate (NMDA) produce neuronal cell death, which can be prevented by several classes of NOS inhibitors or by removing arginine from the incubation medium. Additionally, scavenging NO with reduced hemoglobin attenuates glutamate or NMDA neurotoxicity. Molecular and biochemical studies indicate that NOS utilizes several cofactors and NO formation is regulated at multiple levels (Fig. 1). Thus, a variety of pharmacologic interventions can modulate enzyme activity (Fig. 1) and provide novel agents for neuroprotection. In primary cell cultures, NO donors can directly induce neurotoxicity that is not attenuated by NOS inhibitors, but is blocked

28

PARADIGMS OF NEURAL INJURY

~ra L-.<-.-..,-,-.-.-.~ w-~ Calci~urin4-Ca~aM

I

~,,

Taraet Neuron 1 r (--GTP /~.Cyclase ~,~ cGMP

L-Arg citrullineJ r Hgb

DNA~DamagepARS NAD ~ Poly Ribose "~.._J NAm 4 ATP

FIG. 2 From our studies, a model of NO-initiated glutamate neurotoxicity is proposed. Activation of the NMDA receptor subtype of glutamate receptors causes an increase in intracellular calcium. Calcium binds to calmodulin and the calciumcalmodulin complex binds to NOS, activating it, and also binds to calcineurin, which dephosphorylates NOS, resulting in further activation of the enzyme. NOS converts L-arginine to citrulline and NO. NO reacts with O2 to form ONOO-. DNA is damaged and PARS is activated. NAD levels drop, followed by a drop in ATP levels. NO or ONOO- could inhibit enzymes in the mitochondrial respiration chain, which would impair restoration of cellular ATP and NAD levels. Energy-dependent processes are subsequently inhibited and the cell dies.

by reduced hemoglobin (3). Both N M D A and NO donor-mediated neurotoxicity is attenuated by superoxide dismutase (SOD), which scavenges the superoxide anion (3). Therefore, the formation of peroxynitrite from the reaction of superoxide anion and NO may be important in the genesis of neurotoxicity. One pathway toward neuronal cell death may involve NOinduced DNA damage overactivating the nuclear enzyme, poly(ADP-ribose) synthase (PARS; NAD + ADP-ribosyltransferase), depleting the cell of NAD followed by depletion of ATP (4) (Fig. 2).

Isolation, Culture, and Characterization of Primary Neurons from Rodent Forebrain Preparation of Tissue Culture Plates Forebrain neurons grow best in 16-mm wells of 4- or 24-well plates and the preference for a specific manufacturer's plastic appears to be N U N C (Naperville, IL) > Costar (Cambridge, MA) > Corning (Corning, NY) > Falcon (Lincoln Park, NJ). Primary cultures can also be grown in 35-mm dishes.

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N I T R I C O X I D E T O X I C I T Y IN CNS C U L T U R E S

TABLE I

Stock Solutions Reagent

Eagle's minimal essential medium (MEM) with Earle's salts, without glutamine Fetal bovine serum ( F B S ) d heat inactivated at 56~ for 30 minutes then sterile filtered 5- Fluoro- 2'-deoxyuridine (5F2DU) b is highly toxic; wear gloves when handling Glucose b Glutamine a HEPES b Horse serum (HS), a heat inactivated at 56~ for 30 minutes then sterile filtered Polyornithine b Trypsin a

Source

Concentration

Storage

G I B C O - B R L (Gaithersburg, MD)

1x

500 ml/4~

GIBCO-BRL

1x

27 m l / - 2 0 ~

10 m M

10 m l / - 2 0 ~

Sigma (St. Louis, MO)

J. T. Baker (Phillipsburg, NJ) GIBCO-BRL Sigma GIBCO-BRL

Sigma GIBCO-BRL

2 M 200 m M 1M 1•

0.3 mg/ml 10•

100 ml/4~ 5.5 or 6.5 m l / - 2 0 ~ 100 ml/4~ 27 m l / - 2 0 ~

10 m l / - 2 0 ~ 1 ml/-20~

a Avoid repeated freeze/thaw cycles with these reagents. For best performance, aliquot once into sterile test tubes the volume needed for each preparation and thaw only the necessary aliquots. b Stock solutions made with autoclaved Milli-Q water and sterile filtered (0.2 txm) prior to aliquoting and freezing.

When neuronal cultures are grown in 96-well plates there is a nonuniform distribution of neuronal cell types in each well due to the small number of neurons plated in each well. For instance, the number of NOS neurons varies greatly in each well and some wells will have no NOS neurons. Thus, if the experiment is designed to examine activation of NOS there will be considerable variability among wells. Prior to plating the culture well with neurons, the well must be precoated with an attachment matrix. Stock polyornithine (0.3 mg/ml) (Table I) is diluted into autoclaved Milli-Q water at a l : 1 0 0 dilution (1 ml polyornithine/99 ml H 2 0 ). This solution is filtered through a 0.2-ixm filter to remove any crystals that may have formed, because the crystals are toxic to neurons. Using sterile technique, add 1 ml of polyornithine solution to each well, cover, and incubate in an oven for 60-90 minutes at 37-42~ Cultured neurons will lie flat and will not aggregate (clump) on a well-coated plate. Our optimal conditions are 42~ for 60 min-

30

PARADIGMS OF N E U R A L INJURY

TABLE II Culture Media Formulations a Reagents 10: 10:1 M E M MEM FBS HS Glutamine 5 : 1/5F2DU M E M MEM HS Glutamine 5F2DU 5:1 MEM MEM HS Glutamine M E M + 21 m M glucose MEM 2 M Glucose

V o l u m e (ml)

500 63.3 63.3 6.3 500 26.6 5.3 1.45 500 26.6 5.3 500 5.25

a Media containing glutamine should be used within 4 weeks of being made. Glutamine converts to glutamate with time in aqueous solution and may alter the cell culture conditions. If media cannot be used within 4 weeks, make a smaller volume.

utes. After incubation, aspirate off the polyornithine solution with a sterile Pasteur pipette. Gently rinse the well one time with autoclaved Milli-Q water and aspirate off the water. Apply water by placing the pipette on the side of the well and slowly releasing the water so as not to disturb the layer of polyornithine. Let the plates dry overnight in a clean hood. Store in a dustfree environment. For optimal results use the plates within the first week.

Culture Procedure At the beginning of the dissection, warm 10" 10" 1 Eagle's minimal essential medium (MEM) solution and the 1 x trypsin solution (Table II) in a 37~ H20 bath. Add 10-12 ml sterile Brooks-Logan solution (Table III) into a 15-ml test tube (one for each brain region to be dissected) and 15-25 ml of Brooks-Logan solution into several sterile 100-mm round tissue culture plates. Place dissecting tools into 70% ethanol. For cortex or caudate-puta-

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N I T R I C O X I D E T O X I C I T Y IN CNS C U L T U R E S TABLE III Reagent Sucrose o-Glucose NaCl KC1 Na2HPO 4 KHzPO 4 1 M HEPES

Brooks-Logan

Solution a Concentration (mM) 44 25 137 2.7 10 1.8 10

a Dissolve all reagents in autoclaved Milli-Q water. Adjust pH to 7.4. Sterile filter through a 0.2-~m filter. This solution can be kept for several weeks at 4~ but should be sterile filtered before each use to prevent contamination of cultures by fungus or mold. The solution should be discarded if there are any visible particles or growth.

men cultures, decapitate a 14-day-old pregnant rat, or a 16-day-old pregnant rat for hippocampal or thalamic cultures. Place the rat supine, spray the abdomen with 70% ethanol, and expose the uterus by making an incision through the abdomen with a pair of scissors. Using forceps and a pair of scissors, remove the uterus and place in a sterile 100-mm culture plate. Make a longitudinal incision along the uterus, remove the fetuses, followed by removal of the heads to a sterile 100-mm culture plate containing 15-25 ml of sterile Brooks-Logan solution (enough to cover the heads completely). One at a time, remove a head to a different sterile 100-mm culture plate cantaining 10-15 ml Brooks-Logan solution. Utilizing a dissection microscope to assist with visualization, remove the brain from the cranial cavity by making a continuous longitudinal incision along the dorsal and ventral surface of the cranium. Take care to avoid cutting the underlying brain tissue. Remove the brain from the cranial cavity and remove the olfactory bulbs, cerebellum, midbrain, and spinal cord. Remove as much of the meninges as possible. Dissect out the brain region of interest (i.e., cortex, hippocampus, caudate-putamen, or thalamus) and put the dissected pieces of brain tissue into a 15-ml sterile conical test tube containing 10-12 ml of Brooks-Logan solution. Pool all the dissected tissue from one brain region from one pregnant rat in one test tube. Repeat for each fetal brain. After all the tissue has been dissected, draw off the Brooks-Logan solution from the 15-ml conical test tube with a sterile pipette. Be careful to avoid aspirating off the dissected tissue. Replace with 1.5-3 ml of a 1 x trypsin solution and place in a 37~ incubator for 20-25 min. Trypsin facilitates the dissociation of cells by digesting the protein contacts between cells; 37~ is the optimal temperature for

32

PARADIGMS OF NEURAL INJURY

trypsin activity. Do not overtreat the tissue, because trypsin will digest cell walls with time. The 1 x trypsin solution is made by adding 9 ml of Brooks-Logan solution to 1 ml of a 10x trypsin stock. After the trypsin digest, aspirate off as much of the trypsin solution as possible with a sterile pipette. Because trypsin-treated tissue is sticky, care must be used not to remove any tissue. This is followed by adding 5-7 ml of warm 10" 10" 1 MEM. (The serum in the MEM solution will inactivate the remaining trypsin.) Using sterile 9-inch Pasteur pipettes, with mouth openings of decreasing area (made by flaming the tips), triturate the cells and disperse them by gently moving the cells in and out of the pipette. The tissue should immediately form a cloudy suspension. Do not triturate more than 10 times. Let the tissue settle for a few minutes. Undissociated tissue will settle to the bottom of the test tube. Pipette 10/zl of cell suspension with a sterile pipette tip and eject between the coverslip and hemocytometer for cell counting and observation. There should not be any cell clumps, only individual cells. Count the number of cells in several squares and calculate the mean number of cells per square. Calculate the volume of media necessary to plate the cells at a particular density by the following equation: (mean number of cells per square x 16 • 10,000 x milliliters of dissociated cells) divided by desired density equals final volume (in milliliters). To dilute the dissociated cells to the desired density for plating, remove by pipette the suspended dissociated cells, leaving the bulk tissue in the test tube. Dilute the suspended cells in an appropriate volume of 10" 10" 1 MEM. Plate 1 ml of the diluted cells per well of a 24-well plate. Place in a 37~ 7% (v/v) CO2 humidified incubator. After 4-5 days change the media to 5 : 1/ 5F2DU MEM (Table II) to inhibit nonneuronal cell growth. The culture media are changed to 5"1 MEM 3-4 days later. The media are changed twice weekly by aspirating off one-half of the volume and replacing it with fresh 5:1 MEM (Table II). The cell cultures are maintained for 21 days.

NADPH-Diaphorase Stain for NOS in Primary Neuronal Cultures NOS is fully expressed by day 20 in culture and it is important to determine that the cultures are mature and express a full complement of NOS neurons. To determine the percentage of NOS neurons in the total cell population, stain the cultures for NADPH-diaphorase. In the CNS, NOS has been shown to account for the NADPH-diaphorase stain under conditions of paraformaldehyde fixation (3). To stain, fix the cell cultures in 4% freshly depolymerized paraformaldehyde/0.1 M phosphate-buffered solution (w/v) for 30 minutes at room temperature. Wash fixative off cells with control salt solution (CSS,

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N I T R I C O X I D E T O X I C I T Y IN CNS C U L T U R E S

TABLE IV Control Salt Solution a Reagent

Concentration (mM)

NaC1 KC1 CaC12 D-Glucose Tris-HC1 MgCI2 b

120 5.4 1.8 15 25 0.8

a Dissolve all reagents in Milli-Q water and adjust pH to 7.4 at the temperature the experiment is to be performed (the pH of Tris is temperature sensitive). Sterile filter the solution through a 0.2-/~m filter and store at room temperature until use. All solutions should be sterile filtered before use and discarded if there is visible growth. b Except for NMDA experiments.

Table IV). Apply NADPH-diaphorase stain mix (see Appendix at end of chapter) to the cell cultures, approximately 500/xl per well. Float the dish in a 37~ water bath for 30 minutes. Check the development of the stain. The stain is complete when the neurons and projections are a deep purple/ black. If the glia begin to stain blue the cultures have been overstained. If the stain is not complete, return the plate to the water bath. Typically it takes between 30 and 90 minutes for the stain to develop in tissue culture. To stop the reaction wash the stain off with CSS. The cultures can be kept for a few days at 4~ if 0.05% sodium azide is added to the CSS. Greater than 100 NOS neurons are required per well of a 24-well plate to observe an NO component of glutamate neurotoxicity.

Toxicity Experiments

Exposure'Day 1 We routinely use cultures that are 21 days old because NOS is not fully expressed until day 20 of culture. Prepare exposure solutions that contain the pharmacologic agent of interest in sterile CSS (Table IV). Prior to exposing the neurons to excitatory amino acids (EAA) (see Appendix at end of chapter), the MEM must be removed because it contains salts and metals that may alter the outcome of toxicity experiments. Aspirate off growth media and replace with 1-1.5 ml CSS. Wash the wells with 1-1.5 ml CSS

34

PARADIGMS OF N E U R A L INJURY

2x to completely remove the MEM. Aspirate off the last rinse of CSS and replace with the exposure solution (0.5-1 ml). NO seems to play a major role in delayed neurotoxicity--a 5-minute exposure to glutamate or NMDA elicits cell death 24 hours later. Thus, exposure solutions should be applied to the neurons for exactly 5 minutes. Monitor intervals with a timer to deliver exposure solutions to each well. We have found that 15 seconds is the maximal time interval between wells that can safely be achieved without dislodging cells or losing time during the washes. Terminate the exposure by aspirating off the exposure solution and replacing with CSS (1-2 ml) and washing 2x. Replace the CSS with 1 ml MEM containing 21 mM glucose (Table II). Return the tissue culture plate to the incubator for 18-24 hours. Other forms of neurotoxicity require exposure times longer than 5 minutes. For exposure times greater than 30 minutes we use an exposure solution containing MEM + 21 mM glucose and perform the exposures in the incubator. Note: the buffering salt in CSS is Tris-HCl, which is temperature sensitive. Therefore, it is critical that the pH be adjusted to 7.4 at the same temperature at which the experiment will be performed.

Assay for Cell Death: Day 2 There are a variety of methods available to assay for cell death. We have found that the "gold standard" is cell counting and that other methods are not as sensitive for small but statistically significant changes. We routinely stain dead cells with 500 ~1 of 0.4% trypan blue for 20 minutes. Trypan blue is a vital stain that stains dead cells dark blue. Live cells exclude the die and remain phase bright. Very gently remove the trypan blue and wash with CSS until the cells can be visualized under a microscope. Photomicrographs or permanent images of several random fields of cells should be obtained for counting by an observer blinded to study design and treatment protocol. We routinely obtain between three and five images per cell well, representing approximately 20% of the total neuronal population. Viable versus nonviable cells should be counted. We usually perform at least two separate experiments utilizing four separate wells and routinely count a minimum of 4000-12,000 neurons for each data point. To determine whether cells are being "washed" away or are destroyed by either the treatment or staining protocol, photomicrographs should be made before and after treatment using a transparent grid etched with a diamond tip pen on the bottom of each culture plate. In our laboratory no appreciable loss of neurons has been identified when comparing "before" and "after" photomicrographs from cortical cultures. Other assays for cytotoxicity are based on biochemical parameters that

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NITRIC OXIDE TOXICITY IN CNS C U L T U R E S

35

change in response to cell death. These assays work very well in homogeneous cell cultures. However, in mixed primary neuronal cell cultures that contain glia as well as neurons, the specificity of these methods can be clouded by background noise. The lactate dehydrogenase (LDH) assay is widely used for many cytotoxicity paradigms and has been found to be reliable in some cortical culture and retinal culture models (2, 6, 7). However, LDH activity is affected by the oxidative environment and appears to be a molecular target for transition metal-mediated radical attack in some cultured neuronal systems (8; R. Ratan, personal communication), limiting its use in these experimental paradigms. Additionally, a relatively large proportion of LDH activity resides in the small astrocytic population present in primary neuronal cultures. These astrocytes can become "leaky" under ischemic conditions and contribute nonspecifically and disproportionately to the measurement of LDH release (9). Therefore, LDH activity must be carefully evaluated before use as a measure of cell viability when the experimental paradigm is designed to examine certain models of oxidative stress or ischemia. Although the glia comprise less than 20% of the total cell population in primary neuronal culture, they contain more than 60% of the mitochondria. Cytotoxicity assays that measure mitochondrial function, such as rhodamine 123 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye assays, measure the response of the glia disproportionately to the response of neurons (9). The vital dye, neutral red, accumulates faster in glia than in neurons in primary neuronal cultures, again generating a larger glial than neuronal response. Therefore, these dye assays should be used in homogeneous neuronal cultures that contain minimal numbers of glia, such as cerebellar granule cell cultures or sympathetic ganglion cultures. An additional failing of these assays is the lack of assessment of live versus dead cells. In cultures that have survived for 3 weeks, the cell number can vary among wells. This is not a problem encountered in cultures that survive for only a few days before the experimental paradigm. Another method of cell counting that assesses both live and dead cells relies on fluorescent dyes. These are usually based on ethidium homodimer or fluorescein diacetate staining of dead cells (red) and calcienAM staining live cells (green) (Cytoprobe, Millipore, Bedford, MA; Live/Dead, Molecular Probes, Eugene, OR). These fluorescent vital dyes have the advantage that the incubation media do not need to be changed and they give graphic colorful photomicrographs. Additionally, the stained cultures can be analyzed by a fluorescent plate reader. The disadvantage to these dyes is that color photomicrographs must be made for cell counting, which is potentially cost prohibitive. If a plate reader is used to assess cell death by quantitating the amount of red versus green fluorescence, the center of each well must be uniform because the reader takes measurements from the center of each well.

36

PARADIGMS OF NEURAL INJURY

Criteria f o r N O S Activation A short (5 minutes) application of NMDA or glutamate to primary neuronal cultures clearly elicits neuronal cell death when determined 24 hours later. We and others have shown that NO mediates a component of neuronal cell death in these experimental paradigms (3). A number of criteria must be satisfied to establish that NO mediates cell death. A number ofNOS inhibitors with different mechanisms of action should be used (see Appendix). Potency of inhibition of toxicity should parallel their potencies as NOS inhibitors. The neuroprotection afforded by arginine analog inhibitors should be reversed by excess substrate L-arginine, and o-arginine should have no effect. Additionally, stereoisomers of the NOS inhibitors should be inactive. Depletion of arginine from the culture media should be neuroprotective, and hemoglobin, which complexes NO, should attenuate neurotoxicity. If the toxic insult occurs in part through NO, then increased levels of NO should be present during the exposure of the toxic agent. We routinely use cGMP as an indirect measure of NO formation in cultures (Fig. 2). Additional methods exist for measurement of NO formation, including the Greiss reaction and the NO electrode. The Greiss reaction is based on a colorimetric assay of nitrite (NO2) and nitrate (NO~-), reaction by-products of NO. Although the method is specific, it is usually not sensitive enough in primary neuronal cultures to access NO formation accurately. Commercially available NO electrodes allow the direct measurement of NO. Whether these electrodes are sensitive enough to reflect accurately changes in NO content in cultures is not known. Questions remain as to the specificity of the measurements as well as the calibration of the electrodes. This is an emerging technology and it may become the method of choice once the technical problems are resolved. Regardless of the choice of NO measurement, experiments should be performed to establish the specificity of NO formation. These experiments would be identical to those used in the toxicity experiments and include using a variety ofNOS inhibitors, stereoisomers, arginine-free media, and hemoglobin.

NO Chemistry NO can exist in three valence states: NO-, NO +, and NO .. The predominant species of NO formed in vivo is not known, but is probably dependent on the redox milieu of the local environment around NOS. NO- reacts with molecular oxygen to form NO2 and NO~- in the absence of metal ions. Several NO releasers are available, which preferentially release different valence states of NO. For instance, NO + is released by nitrosocysteine and organic

[2] NITRIC OXIDE TOXICITY IN CNS CULTURES

37

nitrates and reacts with tissue sulfhydryl groups such as those on the NMDA receptor, thus inactivating the NMDA receptor. In this way NO + is neuroprotective by decreasing NMDA receptor activity (10). Sodium nitroprusside (SNP) releases NO + or NO. depending on the redox environment of the experimental system, and 3-morpholinosydnonimine (SIN-l) preferentially releases NO .. The toxic valence state of NO appears to be N O . , which reacts with superoxide anion (O~) at a rate of 6.7 • 109 m -~ sec -~ to form the powerful oxidant ONOO- (11). There are several isoforms of superoxide dismutase (SOD) that convert O~ to hydrogen peroxide ( H 2 0 2 ) , which is then converted to H 2 0 by catalase or glutathione peroxidase. SOD is highly and ubiquitously expressed in all tissues as a first-line defense against oxidative stress. Of all identified enzymes, the copper-zinc isoform of SOD has the fastest rate constant for its substrate, O~ (2 • 109 M -~ sec-~), but the reaction between O~ and N O . is faster. Thus, NO. is the one biological molecule known that can outcompete SOD for O~. NO. is freely diffusible across lipid membranes; with a tl/2 < 1 second, it is a relatively "stable" free radical with a diffusion radius of approximately 40 tzm in tissue. In contrast, O~ has a diffusion radius of 1-3/zm intracellularly and can pass through cell membranes only at ion channels. Extracellularly, O~ has a diffusion radius of 20 /zm. Thus, O~ and N O . are ideally suited to react and form ONOO-. ONOOis somewhat membrane permeable and can diffuse several cell widths. It is estimated that approximately 10% of ONOO- exists in the trans form, which demonstrates hydroxyl-like chemistry and decomposes primarily to nitrate. The remainder of ONOO- exists in the cis form, which reacts with metals to nitrosylate amino acids such as tyrosine (12). Additionally, ONOO- oxidizes sulfhydryl groups, or ONOO- is protonated to peroxynitrous acid (ONOOH) (13), which decomposes to nitrogen dioxide and hydroxyl free radicals. The chemical state and the preferential reactions of NO and ONOO- are directly related to both the oxidative state and the pH of the tissue. Many of these conditions can be regulated in cell culture but clearly become quite complex in in vivo models of stroke and neurodegeneration.

Targets of NO The widespread role for NO in mammalian physiology and pathophysiology has just recently been recognized, and target molecules for NO that may play an active role in the genesis of neurotoxicity are still being discovered. In addition to reacting with O~ to form ONOO-, NO can activate hemecontaining enzymes, such as guanylate cyclase, through displacement of the heine moiety. NO can also induce the ADP-ribosylation and modification of several intracellular proteins, such as glyceraldehyde-3-phosphate dehydro-

38

PARADIGMS OF NEURAL INJURY

genase. Enzymes with iron-sulfur centers in both the respiratory cycle and in the pathway for DNA synthesis are also targets for NO. Target enzymes in this class so far identified include aconitase (aconitate hydratase; part of the Krebs cycle), reduced nicotinamide adenine dinucleotide phosphate" ubiquinone oxidoreductase [mitochondrial complex, NADH dehydrogenase (ubiquinone)], succinate:ubiquinone oxidoreductase [mitochondrial complex II, succinate dehydrogenase (ubiquinone)], and ribonucleotide reductase (a rate-limiting enzyme in DNA synthesis) (14, 15). Modulation of the function of any of these enzymes could obviously have a dramatic consequence to the target cells and possibly result in neurotoxicity. NO can cause deamination of DNA (16), which could result in sufficient DNA damage to be directly cytotoxic to the neurons. DNA damage resulting from deamination is different from DNA damage characterizing apoptotic, or "programmed," cell death in which DNA is degraded by endonucleases into specific fragments, resulting in a DNA ladder. One potential pathway leading toward neurotoxicity could be activation of poly(ADP-ribose) synthase in response to DNA damage. PARS has been extensively studied in eukaryotic cells such as thymocytes and in many secondary cell lines. PARS is a chromatin-bound enzyme that cleaves NAD and then transfers the ADP ribose moieties to various nuclear proteins, including histones, topoisomerase, DNA ligase II, and PARS itself. PARS can form lengthy ribose polymers of more than 80 residues to target proteins. Under normal physiologic conditions the biologic half-life of these polymers is less than 1 minute; therefore, nuclear enzymes can utilize NAD to create rapidly high molecular weight polyanions, which can have a drastic but transient effect on chromatin structure and enzyme function (for review, see Ref. 17). However, for every mole of NAD used as substrate by PARS, 4 moles of ATP are required to regenerate NAD. Therefore, when DNA is excessively damaged and PARS activation is sustained, both NAD and ATP intracellular levels drop precipitously. This depletion of NAD and ATP results in drastic reductions in energy-dependent processes, including synthesis of DNA, RNA, and protein. Application of PARS inhibitors such as benzamide blocks the depletion of NAD and ATP, resulting in restoration of energydependent processes and preservation of cellular integrity and neuroprotection against NMDA and NO donor-mediated neurotoxicity (4). It is possible that under certain conditions NO and other free radicals trigger massive DNA damage, overactivating PARS, resulting in depletion of the energy sources, NAD, and ATP, culminating in neuronal cell death. If this is the case, PARS inhibitors might have widespread therapeutic effects in diseases and neurodegenerative disorders that may involve glutamate neurotoxicity, such as stroke, Huntington's disease, and Alzheimer's disease.

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NITRIC OXIDE TOXICITY IN CNS CULTURES

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Appendix

NADPH-Diaphorase

Stain

FIXative: 4% Paraformaldehyde/O.1 M PB Use 8% paraformaldehyde (8 g/100 ml Milli-Q water); heat to 80~ for 30 minutes (do not boil). Clear with 1-2 drops of 10 N NaOH and replace volume of evaporated water. Add an equal volume of 0.2 M PB (100 ml 0.2 M NaHzPO4:400 ml 0.2 M NazHPO 4, pH 7.4) and pass through a 0.45tzm filter.

NADPH-Diaphorase Stain Solution Buffer 0.1 M Tris-HC1 (pH 7.2) 0.2% Triton X- 100 7.5 mg Sodium azide/100 ml buffer Mix 1 mM NADPH, reduced form (Sigma); 8.33 mg/5 ml 0.2 mM Nitroblue tetrazolium (NBT) (Sigma); 1.64 rag/5 ml Final volume is 10 ml; solubilize NADPH and NBT in separate test tubes to prevent precipitation. NBT may need to be sonicated to solubilize. Mix soluble NADPH and NBT to form stain mix.

E x p o s u r e Solutions Excitatory Amino Acids NMDA is readily soluble in water or buffer solutions up to 10 mM. Glycine is necessary for full activation of the NMDA receptor. Glycine is present in most water supplies, but to ensure consistent activation, a final concentration of 10/~M glycine should be added. Quisqualate is not readily soluble in water or buffer solutions. Make a 1 mM stock solution and heat to 52~ in a water bath. Vortex vigorously and heat until the fine crystals go into solution. Kainate is readily soluble in MEM. Add kainate to MEM and expose the cultures for 24 hours in the incubator to the kainate solution. Glutamate is readily soluble in water or buffer solutions.

40

PARADIGMS OF NEURAL INJURY

NOS Inhibitors NG-Nitro-L-arginine (N-Arg) competes with arginine for the catalytic site and is moderately soluble in water or buffer solutions up to a 1 mM concentration with vigorous vortexing and heating up to 42~ N-Methylarginine (NMA), N-iminoethyl-L-ornithine (NIO), NG-nitro-L arginine methyl ester (L-NAME), and NG-methyl-L-arginine acetate salt (L-NMMA) compete with arginine for the catalytic site and are readily soluble in water or buffer solutions. 7-Nitroindazole competes with arginine for the catalytic site and is more potent in inhibiting neuronal than endothelial NOS. Note: All of the arginine analog inhibitors of NOS inhibit all NOS isoforms. If more than one isoform is present in the preparation, care must be taken to assure that inhibition of all isoforms present does not confound the results. Diphenyleneiodonium (DPI) inhibits electron shuttling by flavoproteins. Dissolve in dimethyl sulfoxide (DMSO) and dilute in buffer solution. DMSO (Kodak, Rochester, NY) concentration cannot exceed 0.1%. GM1 ganglioside inhibits calmodulin, an essential cofactor for NOS activation. It is barely soluble in buffer solutions and requires a 2-hour preincubation before initiation of experiment. GTlb ganglioside inhibits calmodulin, an essential cofactor for NOS activation. It is barely soluble in buffer solutions and requires a 2hour preincubation before initiation of experiment. FK506, or immunophilin (Fujisawa Pharmaceuticals, Japan), binds to FKBP-12, and the FK506FKBP12 complex binds to calcineurin, inactivating the phosphatase. NOS is activated by calcineurin-mediated dephosphorylation.

Arginine Depletion Arginine is the precursor to NO formation by NOS. Cell cultures can readily be depleted of arginine by two methods. 1. Culture cells for 24 hours in arginine-free MEM (GIBCO, Grand Island, NY, special order) in the presence of 2 mM glutamine to inhibit argininosuccinate synthase. Arginine is a semi-essential amino acid and is synthesized by the cells unless the synthesis pathway is blocked by glutamine. After exposure of the cultures to experimental conditions the MEM + 21 mM glucose must also be arginine flee. 2. Treat both the media and the cell cultures with arginase, 10 units for 2 hours. Following exposure to experimental conditions place cells in arginase-treated MEM + 21 mM glucose.

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NO Donor Reagents Sodium nitroprusside (SNP) is readily soluble in water or buffer solutions and releases NO and Fe(CNs) 2-. S-Nitroso-N-acetylpenicillamine (SNAP) (RBI, Natick, MA or Molecular Probes, Eugene, OR) is soluble in DMSO up to 100 mM and then can be diluted carefully into buffer solutions. Final DMSO concentrations cannot exceed 0.1% because DMSO is a free radical scavenger and is neuroprotective at concentrations greater than 0.1%. Additionally, penicillamine is a peroxynitrite (ONOO-) scavenger. In culture, a balance must be obtained between release of NO and scavenging of ONOO-. 3-Morpholinosydnonimine (SIN-l) (Molecular Probes) is soluble in water or buffer solutions. SIN-1 releases both NO and superoxide anion. Note: The NO donors should be weighed, kept in a foil-wrapped test tube, and solubilized immediately before use. In pure deoxygenated water, NO is stable for up to 30 minutes, but in buffer solutions the stability of NO is related to the degree of oxygenation and metal ions. For example, the stability of NO in Krebs buffer is less than 5 minutes. The NO rapidly converts to nitrite in the presence of metal ions. Choice of buffer solutions is therefore very important. Superoxide Anion Scavenger Superoxide dismutase is soluble in water and is a very stable protein. Hemoglobin Reduction Dissolve 2 mM hemoglobin (Sigma) in water. Dissolve 20 mM dithionite (Sigma) in water. When both reagents are in solution, mix them together in a 1:1 (v/v) ratio for a final concentration of 1 mM hemoglobin and 10 mM dithionite. The hemoglobin should turn from a brown color to a deep red color. Put the reduced hemoglobin in dialysis tubing with a pore size less than 60 kDa. Dialyze overnight in 1000-fold excess water at 4~ The dialysis vessel should be wrapped in foil. Replace the water with fresh water and store at 4~ wrapped in foil until use. Hemoglobin oxidizes very rapidly so prepare the cultures for the experiment and have all the solutions ready prior to diluting the hemoglobin to 500 tzM for the experiments. Long exposures to hemoglobin (in hours) should be avoided because hemoglobin can be toxic to neurons. Note: Hemoglobin does not exclusively scavenge NO, but other oxidants as well, including superoxide anion. PARS Inhibitors Benzamide (Aldrich) is soluble in DMSO; dilute (w/v) to less than 0.1% DMSO in final exposure solution. 3-Aminobenzamide (Aldrich) is soluble in DMSO; dilute (w/v) to less than 0.1% DMSO in final exposure solution. 1,5-

42

PARADIGMS OF NEURAL INJURY Dihydroxyisoquinoline (DHIQ), or 1,5-isoquinolinediol (Aldrich) is soluble in DMSO; dilute (w/v) to less than 0.1% DMSO in final exposure solution. Peroxynitrite

Peroxynitrite is not commercially available. O N O O - can be synthesized by the methods of Reed et al. (1974) and Beckman et al. (1994). O N O O - is relatively stable in alkaline solutions but rapidly decomposes in buffer solutions at physiologic pH. Both the volume of stock O N O O - added and the volume of exposure solution should be kept to a minimum to try to decrease the variability in concentration to which the cells will be exposed. O N O O decomposes and the concentration drops constantly while the reagent diffuses through the media to the cells. N o t e : Bicarbonate scavenges O N O O - ; therefore, bicarbonate-based buffers should be avoided when examining this chemical species in culture.

Acknowledgments VLD is supported by grants from National Alliance for Research on Schizophrenia and Depression, American Foundation for AIDS Research and the American Heart Association. TMD is supported by grants from the PHS, CIDA NSO 1578, the International Life Science Institute, and American Health Assistance Foundation. The authors own stock in and are entitled to royalty from Guilford Pharmaceuticals, Inc., which is developing technology related to the research described in this chapter. The stock has been placed in escrow and cannot be sold until a date that has been predetermined by the Johns Hopkins University.

References 1. M. A. Dichter, Brain Res. 149, 279 (1978). 2. R. R. Ratan, T. H. Murphy, and J. M. Baraban, J. Neurochem. 62, 376 (1994). 3. V. L. Dawson, T. M. Dawson, D. A. Bartley, G. R. Uhl, and S. H. Snyder, J. Neurosci. 13, 2651 (1993). 4. J. Zhang, V. L. Dawson, T. M. Dawson, and S. H. Snyder, Science 263, 687 (1994). 5. T. M. Dawson, D. S. Bredt, M. Fotuhi, P. M. Hwang, and S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 88, 7797 (1991). 6. D. W. Choi, J. Koh, and S. Peters, J. Neurosci. 8, 185 (1988). 7. S.A. Lipton, N. J. Sucher, P. K. Kaiser, and E. B. Dreyer, Neuron 7, 111 (1991). 8. E. R. Stadman, Science 257, 1220 (1992). 9. B. H. J. Juurlink and L. Hetrz, Dev. Brain Res. 70, 239 (1993).

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10. S. Z. Lei, Z.-H. Pan, S. K. Aggarwal, H.-S. V. Chen, J. Hartman, N. J. Sucher, and S. A. Lipton, Neuron 8, 1087 (1992). 11. R. E. Huie and S. Padmaja, Free Rad. Res. Commun. 18, 195 (1993). 12. J. S. Beckman, H. Ischiropoulos, L. Zhu, M. van der Woerd, C. Smith, J. Chen, J. Harrison, J. C. Martin, and M. Tsai, Arch. Biochem. Biophys. 298, 438 (1992). 13. J. P. Crow, C. Spruell, J. Chen, C. Gunn, H. Ischiropoulos, M. Tsai, C. Smith, R. Radi, W. Koppenol, and J. S. Beckman, Free Rad. Biol. Med. 16, 331 (1994). 14. T. M. Dawson, V. L. Dawson, and S. H. Snyder, Ann. Neurol. 32, 297 (1992). 15. T. M. Dawson, V. L. Dawson, and S. H. Snyder, in "Neurobiology of NO. and 9OH." Annals of the New York Academy of Science. In press (1994). 16. D. A. Wink, K. S. Kasprzak, C. M. Maragos, R. K. Elespuru, M. Misra, T. M. Dunams, T. A. Cebula, W. H. Koch, A. W. Andrews, J. S. Allen, and L. K. Keefer, Science 254, 1001 (1991). 17. N. A. Berger, Rad. Res. 101, 4 (1985). 18. J. W. Reed, H. H. Ho, and W. L. Holly, J. Am. Chem. Soc. 96, 1248 (1974). 19. J. S. Beckman, J. Chen, H. Ischiropoulus, and J. P. Crow, in "Methods in Enzymology" (L. Packer, ed.), Vol. 233, p. 229. Academic Press, San Diego, 1994.

[3]

Development of in Vitro Injury Models for Oligodendroglia A. Espinosa, P. Zhao, and J. de Vellis

Introduction One of the major tasks of neural cultures has been to provide an understanding of the mechanism(s) involved in the development and function of the central nervous system. The role of environmental agents and naturally occurring substances that affect CNS activity has been elucidated to a large extent by using brain culture systems. Oligodendrocyte/astrocyte primary glial cultures are usually prepared in our laboratory from newborn rat brain (McCarthy and de Vellis, 1980). These cultures are the source of oligodendrocytes and their progenitors, which can be mechanically isolated from the astroglial monolayer (Cole and de Vellis, 1989). Both cell populations can be replated separately for further studies. These cultures have been extremely important for the characterization of oligodendroglial cell development (de Vellis and Espinosa, 1992). Recently we have focused our efforts to the establishment of various in vitro injury models for oligodendroglia. Such models facilitate the study of the serial changes suffered by these cells following injury. They allow evaluation of the either permanent or transient dysfunction and the potential of oligodendrocytes to recover. Here we describe a "freeze-thaw" method applied to two kinds of cultures: (a) primary 2-week-old glial cultures and (b) 3-day-old oligodendrocyte cultures prepared as described (Cole and de Vellis, 1989). The experimental design consists in the exposure of the cell cultures to severe hypothermia in an attempt to stop virtually all their metabolic functions. In order to preserve the cell structure and cytoplasmic membrane, the regular culture medium is substituted by a special freezing medium. Cultures are kept at -20~ and cells are reanimated 1 or 2 weeks later. Reanimation of the cells consists of placing them under regular culture conditions. We will discuss the phenotypic changes displayed by the cells as assessed by double-fluorescent immunostaining and the applications of this method for further studies on oligodendroglial cell injury.

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Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Experimental Procedures

Preparation of Glial Cell Cultures Primary glial cultures are prepared from newborn rat brains (Cole and de Vellis, 1989). Cultures are maintained in the standard culture medium, Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12). The DMEM/ F12 medium is supplemented with 10% calf serum. After 1 week in culture the flasks or multiwell plates are rinsed twice with phosphate balance salt (PBS) solution (NaC1, 120 mmol/liter, KC1, 2.7 mmol/ liter in 10 mM phosphate buffer at pH 7.4) at room temperature. The liquid is removed from the flasks by aspiration, leaving the cell layer virtually dry, and the freezing medium is slowly added to the cell layer. Observation of these cultures under phase-contrast microscopy reveals a confluent flat layer of astrocytes overlaid with oligodendrocytes. Secondary cultures of pure oligodendrocytes are similarly treated. For immunocytochemistry, glass coverslips are prepared in 24-well plates, coated with poly(D-lysine), and rinsed prior to cell plating.

Freezing Medium Preparation A mixture of dimethyl sulfoxide (DMSO) or glycerol, calf serum, and "glial development" culture medium is freshly prepared as follows: DMSO, 10%; calf serum, 89%; and GDM, 1% (v/v). This mixture is applied to the culture layer at room temperature (12 ml of freezing medium (FM) per 75 r The freezing medium should cover the total surface of the flask(s). For immunocytochemistry, 700 ~l to 1 ml per well of freezing medium is recommended.

Preparation of Cultures for Freezing Prior to freezing, the flasks or well plates are individually wrapped with a thick layer of cotton. A second layer should be prepared with dippers (to isolate the surfaces of the plate) followed by a third layer of thin foil. This step will allow a gradual decrease in temperature necessary to preserve plasma membrane integrity. Cultures should be placed in a -20~ freezer. The flasks are placed at -70~ 24 hours later. They can be kept indefinitely under these conditions. The minimum freezing period recommended is 48 hours.

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Basal Medium DMEM/F12

Additives

GDM

Insulin Putrescine Sodium selenite a o(+)-Galactose Penicillin-streptomycin Transferrin Sodium bicarbonate L-Glutamine

5.0 mg/liter 16.1 mg/liter 10.0/A/liter 4.6 g/liter 1.0 ml/liter 50.0 mg/liter 2.2 g/liter

a Stock solution for sodium selenite is 0.8 mg/ml. Note: DF mix powder contains a large (4.5 g/liter) amount of glucose.

Culture Media Composition Our studies for the nutrient requirements of oligodendroglial cells indicate that, in a chemically defined medium, cells from the oligodendrocytelineage display an ability to proliferate and mature in a manner similar to the in vivo situation. The culture medium used is the glial developmental medium (GDM). The composition of the culture GDM is shown in Table I. Upon or after reanimation, oligodendroglial cells are fed with either GDM or with DMEM/F12 (1 : 1, v/v) mixture supplemented with 10% calf serum (DF-10).

Reanimation of Samples Regular culture medium is warmed at 37~ The frozen samples are totally unwrapped and placed for 10 to 12 minutes in a 37~ incubator. As soon as the freezing medium is liquid it is aspirated, leaving the cell layer as dry as possible. Then the flasks are rinsed twice with fresh culture medium, leaving the second rinse at least 3 minutes in each flask. After the last rinse add 10 ml of the same medium or 1 ml/well for the twenty-four or four well plates and keep the cultures in the incubator, at least 24 hours. Cultures can be studied any time (beyond the initial 24 hours) and it is recommended that the medium be changed 24 hours after thawing. These cultures can be used to address a variety of questions. For the present study we used mixed glial cultures kept frozen for 2 weeks, followed by a 2-week incubation at 37~ or we used pure oligodendrocyte cultures reanimated in either DF-10 or GDM. Samples were fixed 3 days after reanimation.

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Immunocytochemical

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Characterization

Characterization of the phenotype of the cells present in the cultures after the freeze-thaw treatment is essential. Double immunofluorescence has been very useful to assess the expression of specific markers by a given cell population (Espinosa and de Vellis, 1988, 1990). It is well known that the morphological features of a cell together with the expression of specific cell markers define not only the cell type but in many cases also indicate its developmental or functional stage (Espinosa and de Vellis, 1995). Many techniques have been described for the characterization of cells in culture. All these techniques offer advantages and disadvantages that need to be considered in order to select the most adequate method for the examination of the changes produced by the freeze-thaw injury model. The most current technique for visualization of a single antigen is the avidin-biotin complex (ABC) method. This procedure allows a very sharp detection of even very slight changes of antigen expression and/or its distribution within the cell. Such small changes would be undetectable by using the peroxidase-antiperoxide method or fluorescent immunocytochemistry. Another advantage of the ABC technique is that it often requires the use of much less antibody than do other methods. At the present time, many products are commercially available for the ABC method. To study a small number of samples, a kit is convenient and cost effective. If the technique is to be used on a regular basis for a large number of samples, however, it is more cost effective to purchase the reagents separately. For the study presented here, we selected double and triple immunofluorescence for the characterization of the cell populations present in reanimated cultures. We used the following markers: sulfatides detected with the monoclonal antibody 04, galactocerebroside (GC) and myelin basic protein (MBP) for oligodendrocyte/myelin markers, and the glial fibrillary acidic protein (GFAP) for identification of astrocytes. Transferrin (Tf) has been described as an early marker for oligodendroglia (Espinosa et al., 1988) and appears in these cells prior to any myelin component, including GC. We also studied Tf expression in our samples. For visualization of these markers, a secondary goat antimouse immunoglobulin G (IgG) tagged with fluorescein, a goat antirabbit IgG tagged with Texas Red, and a goat antimouse IgM are combined. All the antibodies are purchased from Boehringer Mannheim (Indianapolis, IN). The dilutions for primary and secondary antibodies are predetermined by titration. The method for immunostaining used in the present study has been described in detail for freshly dissociated cells (Espinosa et al., 1989). It has been successfully used for the characterization of well-established cell cultures such as glial cells from normal rat brains (Espinosa and

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de Vellis, 1988) or from myelin-deficient (md) rat mutant (Espinosa et al., 1989) cell lines and other cell cultures. Briefly, the method consists of permeabilization of the cells with Triton X-100 treatment and blockage of nonspecific binding sites with normal goat serum. Preparation of the cocktail of primary antibodies and their incubation overnight are carried at 4~ The next day, primary antibodies are rinsed and secondary antibodies are prepared. Samples are incubated at room temperature with a mixture of secondary antibodies for 1 hour, rinsed, and mounted. Samples are observed under a micro Fx fluorescent Nikon microscope equipped with the appropriate filters and barriers.

Results

Observation of Samples Primary glial cultures are characterized by the presence of a layer of astrocytes and oligodendroglial progenitors that appear within the first week postplating. Oligodendrocyte progenitors display a dark and flat appearance, and very short (or no) processes. Initially, the majority are found as single cells (Espinosa et al., 1985). During the second week in culture dark cells change to round bright cells with thin long cell processes either in clusters or as single cells surrounding such clusters (Espinosa et al., 1985; Cole and de Vellis, 1989). When primary glial cultures that were frozen are reanimated at 37~ in the incubator, the freezing medium melts within 10 to 12 minutes; at this time cells are observed under the microscope. The layer of astrocytes and small clusters of oligodendrocytes present in these cultures appear normal when compared to nonfrozen cultures. However, large oligodendrocyte clusters, as well as some single oligodendrocytes, detach and float in the culture medium. At this time, the freezing medium is substituted for fresh culture medium at 37~ Cultures are placed in the incubator and subsequently observed daily. Observation of the cultures reveals no morphological differences in frozen versus nonfrozen cells. Cultures are fed every fourth day. When oligodendroglial cultures are reanimated with the same procedure used for primary glial cultures, a large number of cells detach from the coverslip, leaving approximately 40% of viable cells on the coverslips. Floating cells are eliminated by the replacement of freezing medium for either GDM or DF-10 fresh culture medium. Cultures reanimated and fed with either one of the media look very similar under phase-contrast microscopy. No difference is detected morphologically in reanimated cultures fed with

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FIG. 1 Phase-contrast views of oligodendroglial cells cultured for 10 days after their isolation from primary glial cultures. (A) The oligodendroglial cells display multiple cell processes; some of the cells have formed clusters and some single cells interconnect with neighboring cells, forming a network with their processes. All cells have a small cell body with similar morphology. (B) View of a reanimated culture. Cells were grown for 1 week, as was their sister culture (A), then were frozen for 1 week. Cells were reanimated as described in the text and cultured for 3 more days. These cultures differ from their sister culture counterparts in cell numbers, the almost total absence of cell clusters, the large amount of debris, and vacuolated cells. However, the most remarkable finding is the morphological diversity of the cell populations. None of these oligodendrocytes have long cell processes; many cells lack cell processes, their cell bodies appear larger, and some are vacuolated. A subpopulation of astrocyte-like cells is present. These GFAP § cells have multiple long cell processes and are not found in normal glial cultures. Magnification: x200.

DF-10 (Fig. 1) or G M D (now shown). Despite the c o n s i d e r a b l e loss of cells during r e a n i m a t i o n , a diversity of cell types is still p r e s e n t in these cultures (Fig. 1B). This h e t e r o g e n e i t y in r e a n i m a t e d cultures s h o w s that the t r e a t m e n t affects to a different e x t e n t cells from the same lineage t r e a t e d identically

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through the duration of the experiment. All the different cell types survive and remain healthy in these cultures until fixation time (in this case 3 days after reanimation). In contrast, oligodendrocyte cultures are composed of a homogeneous cell population with similar morphology. These cells present a healthy appearance with their characteristic small round and bright cell body and multiple thin cell processes. These cells are either single or are in small clusters homogeneously distributed on the surface of the coverslip (Fig. 1A).

Fixation and Characterization of Samples After 3 days or 2 weeks after reanimation, primary glial cells or oligodendroglial cultures are fixed. Nonfrozen sister samples are fixed either 3 days or 2 weeks after their counterparts are frozen and stored at 4~ until reanimated samples would be fixed. Previous studies have led us to use formaldehyde (3.7% v/v) as a routine fixative for most of our studies instead of other methods, such as acetone-ethanol, paraformaldehyde, or cold air flow, which have also been used to study mature oligodendroglia in culture (Espinosa et al., 1986a,b; Espinosa, 1987). Every fixative offers advantages and disadvantages with respect to others; for instance, paraformaldehyde offers an excellent preservation of cell morphology, whereas cold air flow or acetoneethanol enhances the visualization of membrane components, but in some cases soluble antigens are poorly detected or even undetectable due to their partial or total extraction during fixation procedure. Formaldehyde allows the detection of a large panel of cell markers independently of whether they are in a soluble form in the cytoplasm, membrane associated, membrane components, or cytoskeleton proteins. Formaldehyde fixation provides a good preservation of cell morphology and of the antigens we studied. The appearance of reanimated primary glial cultures under phase-contrast after fixation is very similar to that of nonfixed cells. At the time of fixation, cells do not show any changes and remain attached to the culture dish. However, when fixative is added to secondary cultures of oligodendrocytes, 10-12% of cells lift from the coverslip; these cells are eliminated from the coverslip. The rest of the cells remain attached throughout the procedure.

Immunocytochemical Analysis of the Cultures After fixation, double or triple immunofluorescence is performed as described in the methods section. Reanimated primary glial cultures are examined by double immunofluorescence for GFAP and GC or MBP. Figure 2 (color

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plate) shows the comparison of reanimated samples (Fig. 2A and B) to nonfrozen sister age-matched cultures (Fig. 2C and D). Reanimated cultures are characterized by the presence of GC + cells in clusters or as single cells. However, they do not have any cell processes. GFAP + cells with round cell body, eccentric nucleus, and multiple long cell processes are frequently observed. These cells are also GC +, giving an orange-yellow color by the colocalization of rhodamine and fluorescein-tagged antibodies in the same cell (Fig. 2A). In some cases, these cells have extremely long GFAP + cell processes (in green), whereas GC expression is restricted to the cell body (in orange-yellow, Fig. 2B). In sister cultures that were never frozen, all the cells either in clusters or single are GC +, with branches forming a GC + network (Fig. 2C). All the cells in these cultures (100%) express the myelin marker MBP, of the cells in the cell bodies, their processes, and extensive layers of membrane, as seen in Fig. 2D. These cultures are virtually devoid of GFAP immunoreactivity (data not shown). The most remarkable finding is the presence of MBP + membrane sheaths in control cultures and the absence of both membrane sheath and cell processes in reanimated cultures. Reanimated oligodendrocytes fed with either one of the culture media (DF-10 or GDM) are analyzed by triple immunofluorescence. The reason for this is the diversity of cell types found in treated samples. Phase contrast does not reveal a major difference between DF-10 and GDM reanimated cultures, because only the cell bodies are visible. The primary antibodies are prepared in combination, Tf/O4/GFAP. Both oligodendrocyte markers Tf and 04 appear early in the lineage. Tf is expressed only by 5% of mature oligodendrocytes, but 100% by young oligodendrocytes. Sulfatides (detected by 04) appear later than Tf but persist in the mature cell and its membrane(s). The pattern of expression of 04 and GC is very similar (04 is used here as an alternative to GC because it is a monoclonal IgM, allowing its combination with mGFAP, IgG, and polyclonal TfIgG). Immunocytochemistry reveals more information about the effects of the treatment. Besides the expression of the antigens studied, the cell morphology is more distinct when viewed with epifluorescence, instead of phase contrast. The appearance of cultures fed with DF-10 after reanimation is shown in Fig. 3 (color plate); the cells are flat and have either no processes or irregular processes. These cells have a weak expression for GFAP (Fig. 3A) as well as for 04, they have retracted all their processes, and their morphology differs from what is expected for a normal 04 + cell (Fig. 3C). These cells are extremely immunoreactive for Tf (Fig. 3B), which appears soluble or in vesicles within the cell cytoplasm. Some spheres of Tf + are also frequently outside the cells (on the substrate). In some cases these vesicles are in the inner side of the plasma membrane. Tf + vesicles are

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negative for 04 and GFAP. Occasionally a short display of cytoplasmic extension is found but no cell processes or myelin-like sheaths are visible. When sister reanimated samples are cultured in GDM, the findings are very different. Most of the cells have cytoplasmic extensions in a perinuclear symmetric manner. Various intensities of 04 are observed, at the level of the cell body, with a smooth appearance as a soluble product. In a few cells 04 is within intracellular vesicles (Fig. 3F). However, the most remarkable finding is the preservation of extensive myelin-like sheaths that are also O4+, as shown in Fig. 3D. All of the cells have very strong expression of Tf (Fig. 3F) and more than 50% of them display a weak GFAP immunoreactivity (Fig. 3D). It is important to note that the cell bodies are smaller than those from cells maintained in DF-10.

Interpretation of Data and Concluding Remarks The elucidation of the mechanisms elicited in oligodendrocytes by injury is necessary in order to be able to provide support for recovery of function in these cells. Several in vitro approaches have been described to study chemical damage focusing mainly on a nutrient deficit or cytotoxicity. The experimental injury model for oligodendrocytes that we have described here demonstrates that injury is not necessarily accompanied by cell death as a result of injury. However, cells suffer major phenotypic changes as a result of severe hypothermia. This model allows direct observation of the changes induced by the freeze-thaw treatment. The model offers a simple yet effective way of evaluating the extent of injury. Furthermore, this system can be used as a working model to test potential factors that may promote recovery of the cells. Examination of reanimated cultures can be done not only by immunocytochemistry but samples can also be prepared in culture dishes or flasks for Northern blot analysis of mRNA expression of the mRNA markers of interest. Western blots can also be prepared, yielding not only qualitative results, but also quantitative information. The application of this method can be very extensive, going from time course studies to different cell types or culture conditions. Furthermore, this method can be used to study interspecies response to injury and plasticity going from fish to mammals. Using the model described in this review, we have learned that the freeze and thaw treatment appears to affect mature oligodendrocytes differently when cultured as mixed glia versus purified oligodendroglia. We observed that in mixed glial cultures cell loss was relatively low, but oligodendrocytes had lost their cell processes and myelin sheaths. All of these cells were GC + both in clusters or single cells. The expression of GFAP by a subpopulation of cells in these cultures was frequent, approximately 30% of the total number

[3]

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of cells. GFAP + cells displayed multiple long cell processes; some of them were also GC +, indicating their oligodendroglial origin and suggesting that oligodendrocytes, just like astrocytes, can be reactive under certain conditions. A large number of studies of oligodendrocytes in culture have led to the finding of GFAP expression by some or most of these cells, depending on the culture conditions (Raft et al., 1983). When these GFAP + cells were first found in cultures prepared from optic nerve or brains of newborn rat pups, they were considered an alternative phenotype to oligodendrocytes, derived from the same progenitor, termed a "bipotential progenitor," thus the GFAP + cell was named a type II astrocyte. The culture conditions wherein type II astrocytes were present have been considered as the optimal conditions for the appearance and maintenance of this type II phenotype. To our knowledge, the present study is the first of its kind to show the changes resulting from injury to mature oligodendroglia. Extensive studies have shown that reactive gliosis occurs as a result of trauma or pathology. Initially, this phenomenon was assessed in the mature CNS and the term "gliosis" was and is still used for astroglial cells. Among the features characteristic of a reactive astrocyte are cell proliferation, hypertrophy of the cell body and cell processes, as well as overexpression of the intermediate filament protein GFAP (Duffy, 1983). Our preliminary findings using the freeze-thaw model indicate that injury induces the expression of the GFAP gene in oligodendroglia to a different extent. The colocalization of 04 and GFAP confirms that GFAP + cells had synthesized 04 before freezing and that they are oligodendrocytes. On this basis, GFAP expression by these cells indicates a "reactive" stage(s) of oligodendrocytes. The overexpression of Tf experienced by reanimated cells indicates that injury up-regulates this gene as well. The colocalization of GFAP, 04, and Tf in oligodendrocytes is not an unusual finding. In other experiments performed in our laboratory whereby cultured oligodendrocytes were exposed to abnormal conditions, such as hyperoxia, colocalization of oligodendroglial markers with GFAP was observed (Espinosa and de Vellis, 1995). On the basis of these observations it is clear that plasticity of oligodendrocytes may also include a reactive stage under nonphysiological conditions, e.g., the so-called type II astrocyte arising from oligodendroglial progenitors in culture exposed to fetal calf serum. It remains to be determined whether the reactive oligodendrocyte described here is a transient or permanent phenotype. The use of different culture media resulted in both protection of performed myelin sheaths and their maintenance after reanimation. This last finding strongly suggests that oligodendroglia can support their myelin sheath after injury if they are provided with adequate conditions for their recovery.

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Acknowledgments We thank Dennis Espejo, Danny Vu, and Nancy Wainwright for help in the preparation of this manuscript. Research was supported by NIH Grant HD-06576.

References Cole, R., and de Vellis, J., in "A Dissection and Tissue Culture Manual of the Nervous System," pp. 121-133. Alan R. Liss, New York, 1989. de Vellis, J., and Espinosa de los Monteros, A., Neuromethods 23, 323-352 (1992). Duffy, P. E., "Astrocytes; Normal, Reactive and Neoplastic." Raven Press, New York, 1983. Espinosa de los Monteros, A., Ph.D. Thesis. Universit Louis Pasteur de Strasbourg (1987). Espinosa de los Monteros, A., and de Vellis, J.,J. Neurosci. Res. 21, 181-187 (1988). Espinosa de los Monteros, A., and de Vellis, J., in "Cellular and Molecular Biology of Myelination (H. Althaus, ed.). Springer-Verlag, Berlin and New York, 1990. Espinosa de los Monteros, A., and de Vellis, J., "Vulnerability of Oligodendrocytes to Environmental Insults: Potential for Recovery (M. Aschner, ed.). CRC Press, Boca Raton, Florida, 1995. Espinosa de los Monteros, A., Roussel, and Nussbaum, J. L., and Labourdette, G., Dev. Biol. 108, 474-480 (1985). Espinosa de los Monteros, A., Roussel, G., and Nussbaum, J. L., Biochem. Soc. Trans. 14, 648-650 (1986a). Espinosa de los Monteros, A., Roussel, G., and Nussbaum, J. L., Dev. Brain Res. 24, 117-125 (1986b). Espinosa de los Monteros, A., Chiappelli, F., Fisher, R. S., and de Vellis, J., Int. J. Dev. Neurosci. 6, 167-175 (1988). Espinosa de los Monteros, A., Pena, L. A., and de Vellis, J., J. Neurosci. Res. 24, 125-136 (1989). Levison, S., and McCarthy K., pp. 103-104. Alan R. Liss, New York, 1990. Raft, M. C., Miller, R. H., and Noble, M., Nature (London) 303, 390-396 (1983).

[4]

Glia Models to Study Glial Cell Cytotoxicity Antonia Vernadakis and M. Susan Kentroti

Introduction The neuron-glia functional partnership first proposed by Hyden in 1961 is now generally accepted, and several reviews have been written describing the cellular events in neuron-glia interactions [see three volumes, "Astrocytes," edited by Fedoroff and Vernadakis (1986a-c); see also the reviews by Vernadakis (1988) and by Abbott (1991)]. The role of cell-to-cell interactions involved in neurogenesis and neuronal growth and differentiation has been a major theme of developmental neurobiology in the past two decades. The coexistence of neurons and glial cells during early neuroembryogenesis places these cells in a strategic position to interact with each other and thus to influence their individual growth and differentiation. In vivo and in vitro studies described in the above-mentioned reviews demonstrate (1) the influence of glial cells on neuronal growth and differentiation and (2) the influence of neurons on glial growth and differentiation. Such interactions appear to be mediated through cell surface components and cell-secreted factors in the microenvironment. The second volume of "Astrocytes" edited by Fedoroff and Vernadakis is dedicated to the physiological and pharmacological aspects of astrocytes. Specific attention is given to intracellular metabolic activity; to membrane components and functions, including receptors, uptake, and transport; to responses to neurotransmitters and other intrinsic factors; and to neuron-glia interactions. Hertz and Schousboe (1984) eloquently describe the role of astrocytes in compartmentation of amino acid energy and metabolism. They position the astrocyte functionally between the y-aminobutyric acid (GABA)ergic and glutamatergic neuron, thus regulating the amount of both glutamate and GABA in the extracellular environment (Fig. 1). One can, therefore, deduct from this interrelationship that any changes occurring in the fucntion of astrocytes would be reflected in both the glutamatergic and GABAergic neuronal function. Such changes can be produced in astrocytes both by exogenous administration of cytotoxins and by cytotoxic substances produced endogenously; the latter are implicated in aging and in neuropathological conditions. A re vie w of Aschner and LoPachin ( ! 993) describes astroc ytes as targets and mediators of chemically induced injury. Several glial cell models have been used to study glia cell cytotoxicity and include both glioma Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

55

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PARADIGMS OF NEURAL INJURY

Astrocyte

GABAergic Neuron

GABA

GIu

Glutamatergic Neuron

FIG. 1 Schematic drawing of evoked release and uptake of glutamate and GABA in GABAergic or glutamatergic neurons and in astrocytes. The sizes of the arrows give an estimate of the relative magnitudes of the respective fluxes. It can be seen that neuronally released glutamate, to a major extent, is accumulated in astrocytes, whereas most of the released GABA is reaccumulated in neurons. Reproduced with permission from Hertz, L. and Schousboe, A., in "Model Systems of Development and Aging of the Nervous System" (A. Vernadakis, A. Privat, J. M. Lander, P. S. Timiras, E. Giacobina, eds.), pp. 19-32. Martinus Nijhoff, Boston, 1984. cell lines and primary glial cells. In this chapter method of studying cytotoxicity using C6 glial cells and primary astrocytes derived from chick embryos will be described. Methodology for isolation of astrocytes from rodents is described in other chapters in this book and thus will be omitted here.

Glial Cell Models

Primary Glial Cell Cultures Glial-enriched cultures can be prepared from various animal species, but the most commonly used animals are rodents (e.g., mouse or rat) or birds (e.g., chicken). In this chapter we will describe glial-enriched cultures obtained from chick brain.

Glial-Enriched Cultures Derived from Chick Brain: Mixed Astrocyte-Oligodendrocyte Cultures Glial-enriched cultures can be prepared from cerebral hemispheres (or other brain areas) of 15-day-old chick embryos or from the telencephalons of 3-day-old chick embryos. In cultures derived from 15-day-old chick embryo cerebral hemispheres, neuronal cells do not survive after 5 days in culture, and by 15 days the cultures consist predominantly of glial cells and about 10% fibroblasts (after removal of meninges). In cultures derived from the

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MODELS TO STUDY G L I A L C E L L CYTOTOXICITY

57

3-day-old chick embryonic telencephalon, neuronal elements disappear within 1 week. The uniqueness of these early embryonic cultures is that they remain glioblastic until they are subcultured at least once. Thus these cultures can be used to study the influence of cytotoxins on early gliogenesis and glial phenotypic differentiation.

Preparation of Glial-Enriched Cultures Glia Cultures Derived from 15-Day-Old Chick Embryo (E15CC) The cerebral cortices from 15-day-old chick embryos are dissected under sterile conditions and collected on a piece of 73-~m mesh placed on a 100mm petri dish containing 2 ml Dulbecco's modified Eagle's medium (DMEM) + 20% fetal bovine serum (FBS). Tissue is mechanically dissociated through the mesh and the cell suspension counted. Each paired cerebral cortex yields approximately 1.0 x | 0 6 cells. Cells are plated directly on 60-mm plastic dishes at a concentration of 2.0 x 10 6 cells/dish in 3 ml DMEM + 20% FBS. Cells are grown at 37~ in 7.5% (v/v) CO2 in air. After 24 hours, the medium is changed to remove floating cells. Cultures are further grown with media changes every 4-6 days until neuronal elements disappear and cells form confluent cultures. After approximately 3 weeks, cultures are ready to be subcultured. Cultures grown in 20% FBS exhibit a high percentage of astrocytes. If one desires a higher population of oligodendrocytes, cultures are first plated in DMEM + 20% FBS and after 24 hours medium is changed to medium containing 2.5-5% FBS. Glia Cultures Derived from 3-Day-Old Chick Embryo (E3H) The telencephalic region from 3-day-old chick embryos is dissected under sterile conditions and collected on a piece of 48-~m mesh placed on a 100mm petri dish in 2 ml DMEM + 20% FBS. Tissue is mechanically dissociated through the mesh and the cell suspension counted. Each telencephalic region yields approximately 0.385 x 10 6 cells. Cells are plated directly on 60-mm plastic dishes at a concentration of 1.0 x l 0 6 cells/dish in 3 ml DMEM + 20% FBS. After 24 hours, the medium is changed to remove floating cells. Cultures are further grown with media that have been changed every 4-6 days until neuronal elements disappear and cells form confluent cultures. After approximately 3 weeks, cultures are ready to be subcultured.

Biochemical Profiles of Glial Marker Enzymes in Culture For biochemical studies, culture medium is aspirated and a 2-ml solution containing 0.1% trypsin is added to each 100-mm dish, or ! ml for a 60-mm dish, and immediately aspirated. This is to neutralize any peptidases left

58

PARADIGMS OF NEURAL INJURY

from the serum in the medium and to minimize the effectiveness of trypsin in detaching the cells from the dish surface. In order to detach the cells from the dish, cultures are incubated at 37~ with 0.1% trypsin (5 ml for a 100mm dish or 3 ml for a 60-mm dish) for 3-5 minutes. The cell suspension from each dish is transferred with a Pasteur pipette to a conical tube containing an equal volume of DMEM + 10% FBS, centrifuged at 1000 rpm, 4~ for 10 minutes. Medium is aspirated and cell pellets are resuspended in 2-5 ml Earle's balanced salt solution (EBSS) and centrifuged as above. This procedure is repeated twice in order to remove all traces of FBS, which will interfere with protein analysis. The final pellet is frozen at -20~ until enzyme assays are performed. Pilot studies have shown that the enzyme activity does not decrease during this time. Two biochemical markers are used to identify astrocytes and oligodendrocytes. The activity of glutamine synthetase (GS; glutamate-ammonia ligase) is used for astrocytes (Norenberg and Martinez-Hernandez, 1979) and 3',5'cyclic-nucleotide phosphohydrolase (CNP) is used for oligodendrocytes (Podulso, 1975; Podulso and Norton, 1972). The biochemical assays used to determine the activities of GS and CNP are described in detail in Sakellaridis et al. (1983). Figures 2 and 3 illustrate the enzyme profiles of glial-enriched cultures prepared from 15-day-old chick embryo cerebral hemisphers (Sakellaridis et al., 1983). The decrease in CNP with days in culture is attributed to the lack of neurons in the cultures. To test this hypothesis, conditioned medium prepared from neuronal cultures can be added to the medium. For such a study the reader is referred to the report by Sakellaridis et al. (1984). Figure 4 illustrates the GS activity profile in E3H cultures, passage 7. I m m u n o c y t o c h e m i c a l Characterization o f E 3 H and E15CC Cultures Glial-enriched cultures are prepared from telecephalons of 3-day-old chick embryos or 15-day-old chick embryo cerebral hemispheres, as described above. An aliquot of 4000 cells is plated in four chamber slides. At various days in culture, slides are immunostained for glial markers. Markers used are galactocerebroside (GalC) for labeling oligodendrocytes (Raft et al., 1978); A2B5 (Eisenbarth et al., 1979) for labeling neurons, bipotential progenitors, type 2 astrocytes, and immature oligodendrocytes (Abney et al., 1983; Raft et al., 1978; 1984); glial fibrillary acidic protein (GFAP) for labeling type 1 and type 2 astrocytes (Bignami and Dahl, 1973; Raft et al., 1984); and vimentin, a major cytoskeletal component of immature glia (Dahl et al., 1981). These cell markers are identified using specific monoclonal or polyclonal antibodies directed against the appropriate antigen. Cells to be stained for GFAP or double-stained for GFAP and A2B5 are first fixed in 100 acetone at -20~ for 5 minutes. Cells to be stained for GalC or double-stained for GalC and A2B5 are first fixed in 3.7% formaldehyde in PBS for 30 minutes at room temperature. Cells are incubated in the primary antibody for 30

[4]

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Days in Culture FIG. 2 Changes with days in culture in glutamine synthetase activity in glial-enriched cultures dissociated from cerebral hemispheres of 15-day-old chick embryos. Activity is expressed in mircomoles of y-glutamylhydroxamic acid formed in 15 minutes/mg protein and plotted vs. days in culture. Points represent means +- SE of 3-4 separate culture dishes. From Sakellaridis, N., Bau, D., Mangoura, D., and Vernadakis, A., Neurochem. Int. 5, 685-689 (1983), with permission. minutes at room temperature, washed with PBS, and incubated an additional 30 minutes with secondary antibody conjugated to either fluorescein (GFAP; GalC) or rhodamine (A2B5). After a single passage, glia from E3H exhibit a glioblastic phenotype (Vim +, GFAP-) early in culture (C1) and progress to mature astrocytes (Vim +, GFAP +) later. Cells follow this same transition with each successive passage. In E15CC cultures, glial cells exhibit multiple phenotypes, with cells representing astroblasts (Vim+, GFAP-), intermediate astrocytes (Vim +, GFAP+), mature astrocytes (GFAP+), and oligodendrocytes (GalC+), and some cells express the extracellular matrix protein, fibronectin. Data in Figs. 5 and 6 illustrate the glia phenotypes exhibited in E 15C and E3H cultures.

C6 Glial Cells as M o d e l s C6 glial cells from a rat glioma cell line have been used to study glial cell properties and function. This cell line has generally been designated as an astrocytoma (Benda et al., 1968). C6 glioma cells can be obtained from the

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FIG. 3 Changes with days in culture in 2',3'-cyclic-nucleotide 3'-phosphohydrolase activity in glial-enriched cultures dissociated from cerebral hemispheres of 15-day-old chick embryos. Activity is expressed in micromoles of 2'-adenosine monophosphate formed in 20 minutes/mg protein and plotted vs. days in culture. Points represent means -+SE of 3-4 separate culture dishes. From Sakellaridis, N., Bau, D., Mangoura, D, and Vernadakis, A., Neurochem. Int. 5, 685-689 (1983), with permission. American Tissue Culture Collection (Rockville, MD). We use C6 glioma cells, 2B clone obtained courtesy of Dr. Jean deVellis from the University of California at Los Angeles. This cell line was given to us at passage 11 and we currently have passages ranging from 11 to 80 frozen in liquid nitrogen. Cell Passage Procedure Cells are plated at a density of 0.5 x 106 for a 100-ram petri dish or 0.1 x 10 6 for a 60-mm dish (Falcon, Lux, respectively). The growth medium is Dulbecoo's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO). For a 100-mm dish, 6 ml of medium is used and for a 60-mm dish, 3 ml of medium is used. Cells are grown at 37~ in 7.5% (v/v) CO2 in air for 3 days, at which point medium is aspirated and replaced with new medium. At culture day 7 (C7), medium is aspirated and a solution containing 2 ml 0.1% trypsin is added to the 100mm dishes or 1 ml is added to the 60-mm dishes and again aspirated. In order to detach the cells, cultures are incubated at 37~ with 0.1% trypsin (5 ml for a 100-mm dish and 3 ml for a 60-mm dish) for 3-5 minutes. The cell suspension is transferred with a sterile pipette to a beaker containing 10-20 ml DMEM + 10% FBS. The cell suspension is mixed using a syringe with a large needle (14-gauge) or a pipette attached to a pipettor moved "up

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and down" about l0 times. An aliquot is counted using a hemocytometer, which gives the amount of cells in 1 ml. This number is then multiplied by the total volume in the beaker to give a total number of cells. From this cell suspension new cultures can be plated. Cell passages up to passage 30 are characterized as early passage, whereas passages 70 and above are considered late passage. Figure 7 shows the morphology of early and late passage C6 glial cells (Lee et al., 1992).

Freezing Cells The procedure for freezing cells is similar to passing cells, up to the point when cells are counted. Instead of plating, the cell suspension is divided into sterile, screw-cap centrifuge tubes (15-ml volume) and centrifuged at room temperature for 5 minutes at low speed. The supernatant is decanted and the cell pellet resuspended in a solution containing 90% FBS and 10% glycerol. The volume to be used should be adjusted to contain 4-3 million cells/ml (labeled with cell passage, number, and date). An aliquot of 1 ml is transferred to sterile cryotubes, which are then capped. The above procedure, except for the centrifugation, is performed under a sterile laminar

62

PARADIGMS OF NEURAL INJURY

FIG. 5 Double-staining immunofluorescence labeling of glial cells derived from 15day-old chick embryo cerebral cortex. Cultures were double stained for GFAP and vimentin at various passages (P) and days in culture (C). (A and B) Cells from P1 at C11. At this early passage, the presence of intermediate astrocytes (GFAP § Vim§ large arrowhead) and of mature astrocytes (GFAP+; small arrowhead) is observed. (C and D) Cells from P4 at C 1; (E-H) cells from P4 at C 13. At C 1 cells are predominantly immature glioblasts exhibiting Vim +, GFAP- immunostaining (occasional cells are also GFAP +, Vim +, as shown by the arrowheads in (C and D). By C13 of the same passage 9P4), many more cells exhibit the intermediate astrocyte phenotype [GFAP § Vim§ small arrowheads (F)] as well as the phenotype of immature glioblasts [Vim§ GFAP-; large arrowheads (E)]. (G) Cells immunoreactive for the oligodendrocyte marker, GalC (outlined arrowhead); (H) same frame with a large, epitholioid cell exhibiting positive staining for the basement membrane protein, fibronectin.

[4]

MODELS TO STUDY GLIAL CELL CYTOTOXICITY

FIG. 5

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flow hood. The cryotubes are transferred to a bucket containing ice and refrigerated for 30 minutes. The cryotubes are then transferred to a bucket containing dry ice for 4 hour at -20~ Last, the cryotubes are placed in labeled racks and immersed in liquid nitrogen. Liquid nitrogen is replenished weekly. This method has resulted in our successfully keeping frozen cells for over 10 years.

Thawing Cells To retrieve cells for use from liquid nitrogen, cryotubes are removed and rapidly thawed in a 37~ water bath. Under a sterile hood, cryotubes are wiped with 70% (v/v) ethanol and the caps carefully removed. Cells are aspirated into a sterile, cotton-plugged Pasteur pipette and transferred to a tissue culture flask (75 cm) containing 30 ml DMEM + 10% FBS. This volume

64

PARADIGMS OF NEURAL INJURY

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Double-staining immunofluorescence labeling of glial cells derived from 3-day-old chick embryo telencephalon (head) at various passages and days in culture. (A-D) Cells from passage 0 (primary cultures) at C 17. Cultures consist predominantly of immature glioblasts [Vim+, GFAP-; large arrowheads (B)] with occasional intermediate astrocytes [Vim+, GFAP+; small arrowheads (A)]. (C) Cluster of oligodendrocytes, which stain positive for GalC (outlined arrowhead). A light micrographic representation of cultures is shown (D). (E-J) Cells at P1, C1, and C13. Whereas at C1 most cells are Vim § glioblasts (large arrows) with few cells exhibiting GFAP § Vim § immunostaining, by C13, cultures continue to contain Vim § glioglasts as well as cells that exhibit exclusively GFAP § immunostaining, considered mature astrocytes (G; small arrowhead). (I) Numberous GalC § oligodendrocytes. It should be noted here that this morphological pattern also agrees with biochemical expression of glutamine synthetase in Fig. 4. FIG. 6

[4]

MODELS TO STUDY GLIAL CELL CYTOTOXICITY

FIG. 6

(continued)

65

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PARADIGMS OF NEURAL INJURY

FIG. 7 Photomicrographs of early (P23-P24) and late (P73) passage glial cells grown in DMEM + 10% FBS (A, early passage; B, late passage) or CDM + TIPS (C, early passage; D, late passage). (A) Cells are primarily glioblastic (large arrows), with some stellate cells representing mature-like glial cells (small arrows). (C) In contrast, cells are differentiated, mature stellate-typema mixture of astrocyte (large arrow) and oligodendrocyte-like (small arrow) cells. Morphology of late passage cells in B and D is not strikingly different except for a decrease in proliferation and apparent increase in cell size. Also, in D, the appearance of oligodendrocytic cells is evident (small arrows) Magnification: x590. From Lee, K., Kentroti, S., Billie, H., Bruce, C., and Vernadakis, A., Glia 6, 245-257 (1992), with permission.

is necessary to remove the glycerol from the cells. After 24 hours the medium is replaced with 10 ml fresh D M E M + 10% FBS. Culture medium is again changed (C3). Cells can be passed at C5 for stocks.

Characterization of Glial Phenotypes in C6 Glial Cell Cultures Biochemical Markers Using CNP and GS as biochemical markers in an early study, Parker et al. (1980) found that early passage (20-26) C6 glial cells (2B clone) exhibit high activity levels of CNP with low levels of GS and that late passage (80-88)

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MODELS TO STUDY GLIAL C E L L CYTOTOXICITY

67

cells exhibit high activity levels of GS and low levels of CNP. From these studies it appears that early passage cells exhibit oligodendrocytic properties, whereas late passage cells exhibit astrocytic properties. Immunocytochemical Characterization of C6 Glial Cells C6 glial cells from passage 23 (early) or 111 (late) are plated on four-chamber plastic slides at a concentration of 20,000 cells/chamber in a volume of 0.3 ml DMEM + 10% FBS. Figure 8 shows early passage C6 glial cells grown in the presence (Fig. 8A-D) or absence (Fig. 8 E - H ) of serum (Lee et al., 1992). As noted in Fig. 8, we only considered cells that exhibited intense immunoreactivity, because faintly stained cells may express nonspecific fluorescein staining. In the presence of serum, many cells are A2B5 + and are assumed to be glioblastic progenitor cells. A few cells were A2B5 + GFAP +, indicating type 2 astrocytes (Fig. 8A and B), and, as expected, several cells were A2B5 + GalC + (Fig. 8C and D), indicating oligodendrocytes. In the absence of serum, most cells were A2B5 + GFAP + (Fig. 8E and F), indicating the differentiation to type 2 astrocyte. A2B5 + GalC + oligodendrocytic cells were very rare (Fig. 8G and H). In late passage C6 glial cells grown in either the presence or absence of serum, the predominant cells are A2B5 + GFAP +, indicating type 2 astrocytes (Fig. 9A, B, E, and F). Of importance was the presence of several A2B5 + GalC + in cultures grown in the absence of serum (Fig. 9G and H), suggesting that possible progenitor cells still present in the late passage cells differentiate into oligodendrocytes in the absence of serum, as has been reported by others (Raft et al., 1984).

Usefulness o f Early and Late Passage C6 Glial Cells Based on both biochemical and immunocytochemical characterizations, early passage C6 glial cells can serve as a model to study various conditions that shift phenotypic expression, oligodendrocytic versus astrocytic. The late passage cells can be used to study effects on a predominantly astrocytic population. As an example, a study by Mangoura et al. (1989) has shown similarities of early (20-22) and late (78-82) passage C6 glial cells with primary chick brain glial cells in culture. The conditions tested were culture substrata [collagen, poly(e-lysine), plastic] or supplements for the culture medium, i.e., DMEM (fetal bovine serum, heat-inactivated fetal bovine serum, or media conditioned from mouse neuroblastoma cells or primary chick embryo cultured neurons). GS and CNP were the biochemical glial markers used. The

68

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findings showed that C6 glial cells are pluripotential and have the plasticity to express both astrocytic and oligodendrocytic properties, whereas the late passage cells are more committed to astrocytic expression.

Paradigm of Astrogliosis Using C6 Glial Cells as Models The potential role of glial cells in neuroinflammation and reactive gliosis was examined by testing the response of C6 glial cells to platelet-activating factor (PAF) (Kentroti et al., 1991). P A F (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent biologically active phospholipid found in various cells, including endothelial, mast, and kidney cells, as well as platelets and macrophages. It is normally released during inflammation and immune reactions to modulate cellular functions. In the central nervous system, PAF exerts modulatory effects on neuronal differentiation and calcium fluxes and its synthesis is enhanced by certain neurotransmitters. In this study, cells from passages 18-25 were designated early passage whereas cells from passages 152-167 were designated late passage. Cells were plated in D M E M + 10% fetal bovine serum on uncoated 100-mm polystyrene petri dishes (Lux) at a density of 0.5 x 106 cells/dish. Cultures were incubated at 37~ in an atmosphere of 7.5% CO2 in air, saturated with H20. Because PAF is rapidly metabolized in the presence of serum, and in order to assess the direct effect of test substances without the interaction of serum factors, media were replaced with chemically defined media (CDM) supplemented with transfer-

FIG. 8 Double-staining immunofluorescence labeling of C6 glial cells, 2B clone, early passage (P23), grown in chamber slides in DMEM + 10% FBS (A-D) or CDM + TIPS without serum (E-H), 2 days in culture. (A and B) Cells were stained with anti-GFAP (fluorescein optics in A) and anti-A2B5 (rhodamine optics in B). Note two A2B5 + cells (B), which also express GFAP (A) and are considered type 2 astrocytes. The remaining cells in A cannot be considered positive for GFAP when compared to the two cells that express both A2B5 and GFAP intense immunoreactivity. All other cells in B are exclusively A2B5 +. (C and D) Cells were stained with anti-GalC (fluorescein optics in C) and anti-A2B5 (rhodamine optics in D). Note three A2B5 + cells (D) that are also GalC + (C, arrows) and are considered oligodendrocytes; most other cells in D are exclusively A2B5+. Again, the faint stain in the remaining cells of C is considered nonspecific fluorescein staining. (E and F) Cells were stained with anti-GFAP (fluorescein optics in E) and anti-A2B5 (rhodamine optics in F). Note that marked difference, when compared to A and B, that most, if not all, A2B5 + cells also express GFAP and are considered type 2 astrocytes. (G and H) Cells were stained with anti-GalC (fluorescein optics in G) and anti A2B5 (rhodamine optics in H). Note three A2B5 + cells (H), which are also GalC + (G, arrows) and are considered oligodendrocytes. All other cells are exclusively A2B5 +. From Lee, K., Kentroti, S., Billie, H., Bruce, C., and Vernadakis, A., Glia 6,245-257 (1992), with permission.

70

PARADIGMS OF N E U R A L INJURY

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rin, insulin, and penicillin/streptomycin (TIPS) after 24 hours in culture (C 1). At that time, cells were gently washed twice with D M E M and the media replaced with 6 ml CDM + TIPS. Cells were initially administered test substances at this time in culture. Groups of cultures (four to six/group) from early or late passages were treated at C1 with PAF (2-200 nM), lyso-PAF (3-300 nM), dBcAMP (1.0 mM), or RO20-1724 (0.1 mM). Cells were harvested at C4 with 0.2% trypsin, centrifuged for 5 minutes at 250 g and 4~ washed twice with Earle's balanced salt solution (EBS), and the pellet frozen at - 2 0 ~ for assay of GS activity. PAF increases GS activity in early passage (glioblastic) cells and, more importantly, it increases GS activity in late passage cells already committed to the astrocytic phenotype. Furthermore, cells from both passages fail to respond to addition of lyso-PAF, the nonbiologically active analog of PAF, to the medium. We compared PAF effects with that of dibutyryl cyclic AMP (dBcAMP) and RO20-1724, a phosphodiesterase inhibitor. Cells from the early passage responded to both dBcAMP and RO20-1724 treatments with a significant increase in GS activity, whereas late passage cells showed no significant change, confirming earlier reports from this laboratory. These findings indicate that the response of C6 glioma cells to PAF (at least in the late passage) is not mediated via cyclic AMP. We suggest that in early passage cells PAF promotes expression of the astrocytic phenotype, and in the late passage cells PAF mediates a gliosis-type response.

FIG. 9 Double-staining immunofluorescence labeling of C6 glial cells, 2B clone, late passage (P73), grown in chamber slides in DMEM + 10% FBS (A-D) or CDM + TIPS without serum (E-H), 2 days in culture. (A and B) Cells were stained with anti-GFAP (fluorescein optics in A) and anti-A2B5 (rhodamine optics in B). All A2B5 + cells (B) also express GFAP (A) and are considered type 2 astrocytes. (C and D) Cells were stained with anti-GalC (fluorescein optics in C) and anti-A2B5 (rhodamine optics in D). Note three A2B5 + cells (D) that are also GalC + (C, arrows) and are considered oligodendrocytes. One cell (D) appears to be A2B5 + and GalC + and is considered to be a precusor (p?). The remaining cells in panel C are faintly stained, probably nonspecific fluorescein staining. (E and F) Cells are stained with anti-GFAP (fluorescein optics in E) and anti-A2B5 (rhodamine optics in F). Note that all A2B5 + cells are also GFAP + and are considered type 2 astrocytes. Cells grown in CDM + TIPS (E, F) have a more differentiated stellate appearance than those grown in DMEM + 10% FBS (A, B). (G and H) Cells were stained with anti-GalC (fluorescein optics in G) and anti A2B5 (rhodamine optics in H). Note three A2B5 + cells (H) that also express GalC + (G, arrows) and are considered oligodendrocytes. From Lee, K., Kentroti, S., Billie, H., Bruce, C., and Vernadakis, A., Glia 6, 245-257 (1992), with permission.

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Glial C y t o t o x i c i t y P a r a d i g m s

Ethanol Gliotoxicity and Relation to Glutamate Neurotoxicity There is now abundant evidence of the neuropathologic effects of ethanol both in the brains of chronic alcoholics and in children afflicted with fetal alcohol syndrome, the preponderance of these studies center on the structure, physiology, and biochemistry in neurons. The extent to which glial cells are vulnerable to ethanol has been less thoroughly examined [see review by Davies (1992)]. Studies from our laboratory using the chick embryo as an animal model have established that the critical period for ethanol neuroembryotoxicity is between embryonic days 1 and 3 (Brodie and Vernadakis, 1990; Kentroti and Vernadakis, 1992). Neuronotoxic effects of ethanol include decline in neuronal survival, shifts in neuronal phenotypic expression, neuronal migration, and neuronal maturation. In view of the role that astrocytes play in neuronal homeostasis from development to aging (Vernadakis, 1988; Muller, 1992; Abbott, 1991), the cytotoxicity of ethanol to glial cells may be an important factor on ethanol neuronotoxicity. One can investigate ethanol gliotoxicity using various models, including C6 glial cell cultures. We have reported ethanol gliotoxicity (Davies and Vernadakis, 1986) in late passage C6 glial cells because, as discussed previously, these late passage cells consist predominantly of an astrocytic phenotype. C6 glial cells at passage 76 (late passage) were plated at 1 x 10 6 cells per 100-mm dish in DMEM + 10% FBS. On culture day 3 (logarithmic growth) or on culture day 10 (postconfluency) the cultures were divided into control and ethanol-treated groups (five to six cultures per group). Medium is aspirated and replaced with DMEM + 5% FBS containing either 0.2, 0.5, or 1% ethanol. Ethanol-treated cultures are placed over a pan containing water plus ethanol up to 2% and the entire pan is enclosed in a thick plastic bag and sealed. We have found that this procedure maintains constant levels of ethanol in the culture medium for several days (Kentroti and Vernadakis, 1990). Control and ethanol-treated cultures can be removed after 24-72 hours and cultures are harvested with 0.1% trypsin for cell counts using a hemocytometer and also for biochemical analysis for GS activity. In this study, cultures treated with ethanol at C3 and C4 (logarithmic growth phase) were harvested at C5 whereas cultures treated with ethanol at C10 and C11 (confluent growth phase) were harvested at C12. The results obtained have shown that glial cells exposed to ethanol at postconfluency growth phase are more vulnerable to ethanol and exhibit a marked decrease in GS activity.

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We have also studied ethanol gliotoxicity using glial cultures prepared from 15-day-old chick embryo cerebral hemispheres as described above (Davies and Vernadakis, 1984). Cultures are prepared as described above. On day 6, in vitro cultures are assigned to one of five groups: experimental cultures treated with DMEM + 10% FBS containing ethanol at one of four dosages [0.1, 0.5, 1.0, or 2.05 (w/v) (i.e., 21.7, 108.5, 217, or 434 mM)]. Control cultures receive the same culture medium without ethanol. Ethanoltreated cultures are placed over a pan containing water with 500 mM ethanol and the pan with cultures is placed in a plastic bag and sealed. Cellular maturation of cultures is followed by phase microscopy. The proliferation of cells is assessed on culture days 5, 7, 8, and 10 in the control group and the 0.5% and 1.0% ethanol groups. At each designated time point, four or five petri dishes from each group are harvested with 0.1% trypsin as described above. Viable cells are counted in a hemocytometer; the trypan blue exclusion method is employed to evaluate cell death. Cultures are harvested on day l0 in vitro for determination of GS activity and protein content (GS is expressed per mg/protein). Although the doses of ethanol appear high, they represent neurotoxicity doses in vivo. The striking finding in this study is the marked effect of 1% ethanol on cell n u m b e r ~ a n almost 50% decrease by day 10. Also, the surviving cells exhibit immature characteristics, i.e., appearing as flat, epitheloid cells. Of interest is the finding that GS activity is also markedly decreased, which is reflected by the decline in cell number. Another significant observation is the presence of reactive astrocytes exhibiting enlarged somata extending numerous branching processes. For comparison of the ethanol response of glial cells from various species, we present here the results of a study by Bass and Volpe (1988) using glia derived from newborn rat brain. Newborn Sprague-Dawley rats, <24 hours old, were used to prepare both astrocyte-enriched and oligodendrocyteenriched cultures. They used GS and CNP activity as biochemical markers for astrocytes and oligodendrocytes, respectively. Anhydrous ethanol was added to the media in concentrations of 0.10, 0.25, and 0.50% (v/v) (respective concentrations: 17, 43, and 86 mM). The medium was changed every 2 days and 24 hours prior to each cell harvest for biochemical determination. Cultures were harvested at 4, 8, 12, 16, 20, and 24 days in culture. These authors found no changes in GS activity in the ethanol-treated astrocytic cultures. However, they observed a twofold increase in CNP activity in the ethanol-treated oligodendrocytic cultures as compared to controls. Again, for comparative reasons, we present the results of a study on the effects of prenatal exposure to ethanol on rat cortical astrocytes (Mayordoma et al., 1992). The astrocytes were obtained from 21-day-old fetuses of both control and alcohol-fed rats. The authors report that in cultures derived from

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fetuses prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultastructural alterations compared to controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum, and Golgi apparatus. These comparative models of ethanol gliotoxicity demonstrate that each model system provides different but relevant information to the complexity of glial cell cytotoxicity.

L e a d Gliotoxicity and Relation to L e a d Encephalopathy For many years, it has been known that severe lead intoxication in young children causes acute encephalopathy manifested particularly by impairment of the blood-brain barrier and as a consequence of brain edema. It is only recently that the possibility that lead toxicity may also affect glial cells, which in turn will be reflected in the well-known lead neurotoxicity, has been considered. An excellent review on lead toxicity in neuroglia has been published by Tiffany-Castiglioni et al. (1989) and thus this subject will be only briefly described here. The authors report two problems that confound the selection of a suitable dose for investigating lead neurotoxicity in vitro: the lack of reported measurements of lead concentrations in brain extracellular fluid and a poor understanding of what forms of lead are biologically available to brain cells (ionic protein-bound or colloidal suspensions). Most investigators take the approach that cells are affected by the total lead concentration in the medium, including all three possible forms. The authors also report that another consideration regarding the lead dose used in culture is its proper measurement. Very few studies have quantified the amount of lead contained in the medium that was used to treat the cultures.

Astrocytes Engle and Volpe (1990) studied lead toxicity in primary glial cultures derived from newborn rat cerebral hemispheres that were 95% enriched with differentiating astrocytes. The cultures were used for experiments on day 18. Lead was added in the assay mixture. They report a 70% inhibition of GS activity with a lead concentration of only 2.5 ~M. Prevention of the inhibition by addition of EDTA or dithiothreitol is compatible with the conclusion that the effect is mediated by binding of lead ion to sulfhydryl moieties of enzyme. Among several other cations tested, only mercury, which has a similarly high binding affinity for sulfhydryl moieties, inhibited the enzyme. The inhibitory effect of lead was relatively specific, because no inhibition of another astro-

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cytic marker enzyme (lactate dehydrogenase), of the oligodendroglial marker enzyme (2',3'-cyclic-nucleotide 3'-phosphohydrolase), or of the plasma membrane marker (Na+, K+-ATPase), was observed with concentrations of lead that produced a 70% decrease of GS. Due to the critical role of GS in regulation of extracellular glutamate (as discussed earlier in this chapter), the findings raise the possibility that glutamate-induced neuronal injury is involved in the genesis of the cognitive defects associated with chronic lowlevel lead exposure in young children.

Oligodendrocytes Tiffany-Castiglioni et al. (1989) described two studies on the effects of lead on oligodendroglia in culture, comparing their responses with those of other cell types. In one study, four types of cells in culture were exposed acutely to lead acetate: astroglia-enriched, oligodendrocyte-enriched, and meningeal fibroblast cultures prepared from neonatal rat cerebral hemispheres, and human neuroblastoma cultures prepared from the SK-N-SH-SY5Y cell line. The lead level in the medium was 0.1-1000/xM before filter sterilization and about one-third this value afterward. Of the four cell types, only oligodendroglia showed marked sensitivity to lead treatment as assessed by cell number. In another study, sensitivity to lead was compared among astroglia, oligodendroglia, and meningeal fibroblasts cultured from neonatal rat cerebra and exposed to a single dose of lead (0.3, 3.0, or 30/xM) for 3 days. Again, oligodendroglia were most sensitive. However, astroglia were shown to take up lead from the culture medium and concentrate it to at least 55 times the extracellular level. An interesting proposal put forward by Holtzman and co-workers (1984) is that mature astroglia have a specific mechanism for sequestering lead and thus act as lead depots in the brain.

Role o f Astrocytes in Aluminum Chloride Neurotoxicity Aluminum is a potent neurotoxin and its excessive accumulation in the CNS has been implicated in a variety of neurological disorders, including dialysis encephalopathy, Alzheimer's disease, and amyotrophic lateral sclerosis dementia complex of Guam [see the review by Sturman and Wisniewski (1988)]. The mechanisms involved in aluminum neurotoxicity are not understood but some evidence supports the hypothesis of disturbances in the glutamic acid-GABA system (see Fig. 1). In addition, evidence suggests that astrocytes are the cellular compartment most affected by aluminum. Again, in vitro studies may provide some clues to this astrocyte cytotoxicity and its reflection to neuronal toxicity.

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A study by Albrecht et al. (1991) uses neonatal rat cortical astrocytes in culture to study the effects of aluminum on the release of endogenous glutamate, taurine, and adenosine. Cells are dissociated according to this group's methodology (but astrocytic cultures can be prepared using a variety of methodologies described in this book). These authors use 5-week-old cultures of neonatal cortical astrocytes grown in the presence of dibutyryl-cAMP (not less than 95% of the cells are glial fibrillary acidic protein positive). Cells maintained in 35-mm dishes are washed twice with buffered Krebs-Ringer medium (in mM: NaC1, 140; KC1, 3; CaC12, 1; MgC12, 0.6; glucose, 10; and HEPES, 50; pH 7.4) and then incubated in 1 ml of the same medium for 15 minutes at 37~ The cells are then incubated for 5 minutes at 37~ either in Krebs-Ringer medium alone (control cells) or in Krebs-Ringer medium containing 0.5 or 5.0 mM aluminum chloride. Following incubation, 0.9-ml samples of the medium are collected. Afterward, 1 ml of Krebs-Ringer medium is added, the 5-minute incubation is repeated, and 0.9-ml samples are again collected. The second incubation step is included to account for possible delay in the maximal release response, known from previous studies to occur with taurine and adenosine. Mean values of these two samples are used in determining the stimulated levels of release in five separate experiments. Samples are freeze-dried and subjected to reversed-phase HPLC analysis. Incubation of cerebral cortical astrocyte cultures for 5 minutes in the Krebs-Ringer buffer leads to measurable basal release levels of taurine, adenosine, and gluatamate. GABA is detected, confirming the extremely low content of this amino acid in cultured astrocytes. Basal release levels of the neuro-inert amino acid serine are highest in the medium after incubation. The 5-minute treatment with 0.5 mM AIC13 increases the taurine release to approximately 170% of basal levels, but has no effect on glutamate, adenosine, or serine basal levels. Treatment with 5.0 mM A1CI3 increases the basal release of taurine to more than 800%, that of glutamate to more than 10-fold, and the release of adenosine to 250% of control levels. By contrast, 5.0 mM A1CI3 causes an approximately 30% inhibition of serine basal release. The authors conclude from these in vitro findings that the enhanced release of these neuroactive compounds from astrocytes to the extracellular environment will contribute to the neurotoxic effect of aluminum.

Role o f Astrocytes in Parkinsonism-lnducing Effects of Neurotoxin MPTP Recent evidence has renewed interest in the puzzling pathogenesis of idiopathic Parkinson's disease. This is due to the demonstration that the drug 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Aldrich, Milwaukee,

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WI) causes a similar clinical syndrome and neuropathological changes. Indirect evidence suggests that neuroglia cells may play a role in MPTP neurotoxicity. Ransom et al. (1987) has tested the hypothesis that astrocytes convert MPTP to its active metabolite MPP + using primary cultures of astrocytes prepared from 1-day-old mouse forebrains. MPTP is applied at concentrations ranging from 0.1 to 100 ~g/ml. In some experiments, the MAO inhibitor, pargyline, is applied at a concentration of 50-100/~M. Cultures are maintained in exactly 1.5 ml of growth medium consisting of minimum essential media supplemented with 10% horse serum. Studies are carried out on 2- to 4-week-old astrocyte cultures. Standard aliquots (150 tzl) of growth medium are removed after various periods of incubation in control or drug-containing medium, and are assayed for MPP + using gas chromatography-mass spectrometry. Concentrations of MPP + are expressed as/~g/mg of total cellular protein (determined using standard methods) in experimental or control sister cultures. The 2-week-old astrocyte cultures rapidly convert MPTP to MPP +. Measurable concentrations of MPP + are present within 1 hour of incubation with MPTP and rise steeply during the first 24 hours. MPP + concentrations increase at a less rapid rate over the next 4 days of incubation. Control experiments are carried out in which MPTP is added to culture dishes containing normal culture medium but no cells; no measurable MPP + is produced in this situation. Given the large volume of culture medium overlying the astrocyte monolayer, the significant amount of MPP + present after only a few hours of incubation suggests that the conversion from MPTP to MPP + is happening at a very rapid rate. The conversion of MPTP to MPP + is partially blocked by coincubation with parglyine. MPP + production is inhibited by approximately 85% at the 1- and 2-day time points. Moreover, MPTP at concentrations beyond 10 ~g/ml produce a marked decline in astrocyte survival. Thus astrocytes are involved not only in the production of the active metabolite, MPP +, but also exposure to MPTP markedly affects astrocyte growth. Therefore, in this case astrocytes are both the mediators and targets of MPTP neurotoxicity.

M e t h y l m e r c u r i c Chloride Gliotoxicity Neurological disorders in humans caused by methylmercuric chloride ( C H 3HgC1) are referred to as Minamata disease. CH3HgC1 accumulates in the central nervous in rather large proportions after ingestion or after intravenous or intraperitoneal administration. Prasad et al. (1979) have compared the effects of CH3HgCI on neuroblastoma (NB) and C6 glial cells (passage 30-40).

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C6 glia and NB cells are plated at different densities due to differences in their growth rates. CH3HgC1 (0.1-1 /xM for NB; 0.05-0.3/zM for C6 glia) is added 24 hours after plating and immediately after the addition of cyclic AMP-stimulating agents RO20-1724 (200/zg/ml), papaverine (10 tzg/ml), isobutylxanthine (0.25 mM), prostaglandin E1 (PGE1) (10/zg/ml). Because cyclic AMP-stimulating agents are soluble in 50% ethanol, an equivalent volume of ethanol (final concentration 0.5%) is added to a set of control cultures prior to addition of CH3HgCI. Another set of control cultures receive only CH3HgC1. Untreated cultures are also maintained during the experiment. The drug and medium are not changed during the period of experimentation in glioma cells. However, in NB cells, it becomes necessary to change the medium 2 days after treatment in the following culture: (a) untreated control, (b) alcohol-treated control, and (c) culture treated with -<-0.5/xM CH3HgC1. The medium becomes acidic in these groups because of the increased numbers of cells. At 3 days after treatment, the number of viable cells is determined by trypan blue (0.2% in saline) uptake among attached cell populations. The stained cells are considered dead. Cells are removed from the dish by incubating NB cells in pancreatin solution (30 minutes for PGE~- or RO20-1724-treated cells; 15 minutes for other treatments), and glioma cells in trypsin solution (40 minutes). After incubation, a single cell suspension is made, and an equal volume of medium is added to inhibit the protease action. The cells are counted with a Coulter counter. The number of dead cells is subtracted from the total count to obtain the number of viable cells. To evaluate the effect of CH3HgCI in the presence of cyclic AMP-stimulated agents, the cell number obtained after the treatment of cells with the individual cyclic AMP-stimulating agents alone is considered 100%. The inhibition of growth in cultures treated with CH3HgC1 and one of the cyclic AMP-stimulating agents is expressed as the percentage of cell number in cultures treated with a cyclic AMP-stimulating agent alone. The effect on cultures treated with the individual cyclic AMP-stimulating agent is expressed as a percentage of cell number in untreated cultures as controls. The effects of CH3HgC1 in the presence of cyclic AMP-stimulating agents on C6 glial cells are in part different from those on NB cells. CH3HgC1 increases prostaglandin El-induced growth inhibition (due to reduction in cell division and cell death) in C6 glia and NB cells. CH3HgC1 does not reduce PGE~-induced formation of cytoplasmic processes in C6 glia cells except at a higher PGE~ concentration, but it does decrease PGE~induced neurite formation in NB cells. The inhibition of cyclic nucleotide phosphodiesterase completely prevents the cytotoxic effect of CH3HgC1 on C6 glia, but markedly enhances the growth inhibitory effect of CH3HgC1 on NB cells.

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Conclusions and Suggestions We have presented several approaches for studying glial cell cytotoxicity. Each model has advantages and disadvantages and the researcher should choose the model system appropriate for the question under study. Primary cultures mimic most closely the in vivo situation. On the other hand, the C6 glia model has the advantage of obtaining both glioblastic and committed astrocyte populations. In order to evaluate glial cell sensitivity, both biochemical parameters and immunocytochemical characterizations should be determined. Biochemical analysis of glial enzymes reveals changes in cellular activity whereas immunocytochemistry offers specific phenotypes. We have presented a few selected studies wherein glial cell cytotoxicity has been implicated in neuronal dysfunction as assessed neurologically. We suggest that careful interpretation of the in vitro findings may provide fundamental clues to the role of glial cells in neuronal injury, neuronal cell death, and neuronal regeneration.

References Abbott, N. J. (ed.)., Ann. N . Y . Acad. Sci. 633 (1991). Abney, E. R., Williams, B. P., and Raft, M. C., Dev. Biol. 100, 166-171 (1983). Albrecht, J., Simmons, M., Dutton, G. R., and Norenberg, M. D., Neurosci. Lett. 127, 105-107 (1991). Aschner, M., and LoPachin, Jr., R. M., J. Toxir Environ. Health 38, 329-342 (1993). Bass, T., and Volpe, J. J., Dev. Neurosci. 11, 52-64 (1988). Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W., Science 161, 370371 (1968). Bignami, A., and Dahl, D., Brain Res. 49, 393-405 (1973). Brodie, C., and Vernadakis, A., Dev. Brain Res. 56, 223-228 (1990). Cookman, G. R., Hemmens, S. E., Keane, G. J., King, W. B., and Regan, C. M., Neurosci. Lett. 86, 33-37 (1988). Dahl, D., Rueger, D. C., Bignami, A., Weber, K., and Osborn, M., Eur. J. Cell Biol. 24, 191-196 (1981). Davies, D., in "Alcohol and Neurobiology: Brain Development and Hormone Regulation" (R. R. Watson, ed.), pp. 69-80. CRC Press, Boca Raton, Florida, 1992. Davies, D. L., and Vernadakis, Dev. Brain. Res. 16, 27-35 (1984). Davies, D. L., and Vernadakis, A., Dev. Brain Res. 24, 253-260 (1986). Eisenbarth, G. S., Walsh, F. S., and Nirenberg, M., Pror Natl. Acad. Sci. U.S.A. 76, 4913-4917 (1979). Engle, M. J., and Volpe, J. J., Dev. Brain Res. 55, 283-287 (1990). Fedoroff, S., and Vernadakis, A. (eds.), "Astrocytes," Vol. 1. Academic Press, New York, 1986a.

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PARADIGMS OF NEURAL INJURY Fedoroff, S., and Vernadakis, A. (eds.), "Astrocytes," Vol. 2. Academic Press, New York, 1986b. Fedoroff, S., and Vernadakis, A. (eds.), "Astrocytes," Vol. 3. Academic Press, New York, 1986c. Hertz, L., and Schousboe, A., in "Model Systems of Development and Aging of the Nervous System" (A. Vernadakis, A. Privat, J. M. Lauder, P. S. Timiras, and E. Giacobini, eds.), pp. 19-32. Martinus Nijhoff, Boston, 1984. Holtzman, D., DeVries, C., Nguyen, H., Olson, J., and Besch, K., NeuroToxicology 5, 97-124 (1984). Hyden, H. Sci. Amer. 205, 62-70 (1961). Kentroti, S., and Vernadakis, A., Dev. Brain Res. 56, 205-210 (1990). Kentroti, S., and Vernadakis, A., J. Neurosci. Res. 33, 617-625 (1992). Kentroti, S., Baker, R., Lee, K., Bruce, C., and Vernadakis, A., J. Neurosci. Res. 28, 497-506 (1991). Lee, K., Kentroti, S., Billie, H., Bruce, C., and Vernadakis, A., Glia 6, 245-257 (1992). Mangoura, D., Sakellaridis, N., Jones, J., and Vernadakis, A., Neurochem. Res. 14, 941-947 (1989). Mayordoma, F., Renau-Piqueras, J., Megias, L., Geurri, C., Iborra, F. J., Azorin, I., and Ledig, M., Int. J. Dev. Biol. 36, 311-321 (1992). Muller, C., Int. Reo. Neurobiol. 34, 215-281 (1992). Norenberg, M. D., and Martinez-Hernandez, A., Brain Res. 161, 303-310 (1979). Parker, K. K., Norenberg, M. D., and Vernadakis, A., Science 208, 179-181 (1980). Poduslo, S. E., J. Neurochem. 19, 727-736 (1975). Poduslo, S. E., and Norton, W. T., J. Neurochem. 19, 727-736 (1972). Prasad, K. N., Nobles, E., and Spuhler, K., Environ. Res. 19, 321-338 (1979). Raft, M. C., Mirsky, R., Fields, K. L., Lisak, R. P., Dorfman, S. H., Silberberg, D. H., Gregson, W. A., Leibowitz, S., and Kennedy, M. C., Nature (London) 274, 813-816 (1978). Raft, M. C., Williams, B. P., and Miller, R. H., EMBO J. 3, 1857-1864 (1984). Ransom, B. R., Kunis, D. M., Irwin, I., and Langston, J. W., Neurosci. Lett. 75, 323-328 (1987). Sakellaridis, N., Bau, D., Mangoura, D., and Vernadakis, A., Neurochem. Int. 5, 685-689 (1983). Sakellaridis, N., Mangoura, D., and Vernadakis, A., Neurochem. Res. 9, 14771491 (1984). Sturman, J. A., and Wisniewski, H. M., in "Metal Neurotoxicity" (S. C. Bondy, and K. N. Prasad, eds.), pp. 61-65. CRC Press, Boca Raton, Florida, 1988. Tiffany-Castiglioni, E., Sierra, E. M., Wu, J.-N., and Rowles, T. K., NeuroToxicology 10, 417-444 (1989). Vernadakis, A., Int. Reo. Neurobiol. 30, 149-224 (1988).

[5]

Rodent Glioma Models William W. Maggio

Introduction Most glioma models were developed for testing new treatment strategies involving radiation, chemotherapy, or immunotherapy. Of equal importance is the use of glioma models to explore mechanistic questions of carcinogenesis and invasion. Bigner (1) has stated of brain tumor models: "The theoretically ideal brain tumor model would have an autochthonous origin, be glial in composition, have solely intraparenchymal growth, be uniformly fatal within a reasonable time frame and, most importantly, have a fidelity to the therapeutic responsiveness of human brain tumors." The theoretically ideal glioma model does not exist; however, several useful glioma models have been developed in rodents. These are the nitrosourea, avian sarcoma virus (ASV), and transplantation glioma models.

Nitrosourea Models Organ specificity for the brain of the nitrosourea compounds was first demonstrated by Druckrey in 1965 (2). N-Methylnitrosourea (MNU) and N-ethylnitrosourea (ENU) have proved to be the most efficient and useful of the nitrosourea compounds for the production of brain tumors in rats. MNU is the more effective in producing brain tumors in adult rats, and ENU is more effective in fetal and neonatal rats. The precise age at which the animals change their susceptibility is unknown, but is probably within the first month of life (3). The issues of dosage, route of administration, and duration of exposure for optimal tumor production are complex because, in addition to age, the strain of rat used in the model influences the efficiency of tumor production. The nitrosourea models are complicated by the associated production of spinal, peripheral nerve, and systemic tumors. The latency between the initial exposure and tumor production tends to be very long and variable. Even with these limitations, the nitrosourea models are widely used in neurooncology because of their technical simplicity. Work with these models has provided some interesting insights into glioma progression. Several useful cell lines have been produced from nitrosourea-induced gliomas. A single subcutaneous dose of MNU produces nervous system tumors in Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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postnatal BD IX rats. At best, about 25% of the exposed rats develop gliomas with this technique. The majority of tumors are actually systemic, particularly at the site of injection (3). Limited intravenous and intraperitoneal exposure, every 2 to 4 weeks, results in brain tumor yields of 50.7% and 59.6%, respectively. A similar limited repeated oral exposure produces brain tumors in about 14.7% of rats (4); 32% of Wistar rats will develop brain tumors when exposed to l0 mg/kg intravenously every 2 weeks to a total dose of 180 mg MNU (4). More frequent exposure on a weekly or continuous schedule results in much higher yields of gliomas. Repeated intravenous administration of MNU will produce nervous system tumors in 90-100% of Sprague-Dawley rats (5). The frequency and duration of administration appears to be more important than the total cumulative dose of MNU in determining brain tumor yield (4, 6). The method of Swenberg et al. (6) uses weekly intravenous injection of 5 mg/kg MNU for 36 weeks. The types of nervous system tumors produced are gliomas and peripheral nerve tumors; 23% of the animals also develop systemic tumors such as leukemia, fibroadenomas of the mammary gland, and sarcomas of the genitourinary tract. The sex and strain of rat influence the spectrum of tumor production with intravenous exposure to MNU. The male Sprague-Dawley rat appears to be the most susceptible to gliomas. Females seem to be more prone to extraneural tumors. Using a similar dose schedule produces brain tumors in 69% of BD IX rats (7). Continuous oral administration of 100 or 200 parts per million (ppm) MNU in the drinking water for 42 weeks beginning at 7 or 11 weeks of age produces gliomas in 82.5-92.5% of adult Fischer 344 or ACI/N rats of both sexes (8, 9). There are no remarkable differences between these two strains in survival, incidence, or multiplicity of gliomas. The mean survival is 25-36 weeks, with longer survivals seen in the lower dose of 100 ppm MNU. Of the tumorbearing animals, 64% develop systemic tumors; tumors of the gastrointestinal tract, oral cavity, and genitourinary system are the most common. In Donryu rats, continuous oral administration of MNU produces primarily peripheral nerve tumors, so strain also influences the susceptibility of rats to gliomas induced by this method (10). Although ENU does not produce central nervous system tumors in adult animals (5, 7, 11), it is effective in producing brain tumors in rats exposed during the fetal or neonatal period. Transplacental MNU is less effective than ENU (12). A single intravenous dose of 20-50 mg/kg ENU given to a pregnant rat on days 15-20 of gestation will produce brain tumors in nearly 100% of the litter (13-19). Increasing the dose from 1 to 50 mg/kg increases the incidence of nervous system tumors, decreases the incidence of systemic tumors, and decreases the survival (16). The strain of rat influences the type and distribution of nervous system tumors, the latency of tumor develop-

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ment, and the survival. BD IX, CD Fischer, and Sprague-Dawley rats are suitable for transplacental ENU glioma induction (16, 18, 20). ENU is also effective when given to neonatal rats. ENU at 10-50 mg/kg injected either subcutaneously or intramuscularly into neonatal rats will result in glioma tumors in 82-88% of rats (21-24). The incidence of gliomas is relatively constant for Wistar/Furth, Long-Evans, Fischer 344, and crossbred Wistar/Furth x Long-Evans strains (22). However, the incidence of associated cranial and spinal nerve root tumors varies with the strain of rat. Long-Evans rats have an 89% relative incidence of cranial nerve tumors and a 93% incidence of spinal nerve root tumors. The Fischer 344 and Wistar/ Furth strains have much lower incidences of peripheral nerve tumors with this technique. The incidence of systemic tumors ranges from 9 to 16% depending on the strain; most of the systemic tumors are kidney tumors. Brain tumors seen with ENU and MNU are almost exclusively gliomas. Neuronal neoplasms (25) and meningiomas (16) are so rare with the nitrosourea models that, when one is reported, their relationship to the nitrosourea exposure is questionable. Granular cell brain tumors, the most common spontaneous rat brain tumor ~,11), are rarely seen after nitrosourea exposure. The histopathologies of the MNU- and ENU-induced gliomas resemble those of human gliomas. The relative frequency of the various subtypes of gliomas varies depending on the nitrosourea model studied. In the transplacental and neonatal ENU models, oligodendrogliomas and mixed oligoastrocytoma tumors predominate, whereas anaplastic astrocytomas and glioblastomas are much less common (16, 22). The continuous oral MNU model, whereby 42.5-66.2% of the tumors are glioblastomas and 19.5-50.7% are anaplastic astrocytomas, seems to more closely resemble the human condition (8). Multiple gliomas are the rule with the nitrosourea models (6, 8, 9, 16). Multiplicity seems to be directly proportional to the dose of nitrosourea. Human gliomas are usually solitary, although multifocality is seen in 4-6% of human glioblastomas (26). A significant number of gliomas induced by nitrosoureas are located in the spinal cord. In rats exposed to oral MNU, 9-22% of the gliomas are within the spinal cord. These spinal cord gliomas are usually astrocytomas, anaplastic astrocytomas, or mixed tumors (9). With repeated intravenous exposure to MNU only 3 spinal cord gliomas were encountered but 55 brain gliomas were seen (6). For transplacental or neonatal exposure to ENU, the spinal g|ioma relative incidence is 12-24%, depending on the strain of rat and dose of ENU (16). Ependymomas were the most common spinal cord tumor seen in Sprague-Dawley rats exposed to high doses of transplacental ENU. With neonatal exposure to ENU, the relative incidence of spinal gliomas was 34-72% (22). Strain of rat seems to be important in the relative incidence of spinal gliomas due to neonatal exposure to ENU; Long-Evans

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rats have the highest incidence of spinal gliomas. The majority of the spinal cord tumors seen with neonatal ENU administration are oligodendrogliomas. The occurrence of spinal gliomas in these models must be recognized because of the associated morbidity and death, which will confound survival studies, and spinal gliomas are likely to present a very different clinical picture compared to intracerebral gliomas. Although the behavior of the rat spinal cord glioma is unknown, human spinal cord gliomas differ from cerebral gliomas in progression and response to treatment. The relative incidence of peripheral nerve tumors is an important concern with nitrosourea-based rat glioma models. The incidence appears to be related to the strain of rat, the particular nitrosourea, the dose level, and timing of administration. Continuous oral administration of MNU in doses of 100-400 ppm in water to Donryu rats produces more peripheral nerve tumors than gliomas (! 0, 11). In Fischer 344 and ACI/N strains administered oral MNU, 14-23% of the nervous system tumors are in the peripheral nervous system; there was no strain difference in this study (9). The relative incidence of peripheral nerve tumors in Sprague-Dawley and CD Fischer rats exposed to repeated intravenous MNU is 12 and 24%, respectively (6). Janisch and Schreiber (4), using a variety of routes, dosages, and strains, found peripheral nerve tumors in about 18% of rats exposed to MNU as adults. Tumors of the cranial nerves and tumors of the spinal nerve roots are felt to be the cause of death in 15 and 30%, respectively, of SpragueDawley rats exposed transplacentally to 50 mg/kg ENU (16). The incidence of significant peripheral nerve tumors (i.e., felt to be the cause of death) is inversely proportional to the dose of transplacental ENU. However, the overall incidence of peripheral nerve tumors increases with increasing ENU dose. This discrepancy probably reflects a relatively greater increase in fatal gliomas with the higher doses. Peripheral nerve tumors are usually anaplastic neuromas. The trigeminal nerve is the most commonly involved cranial nerve (4, 9, 22, 25); the lumbosacral roots are the most commonly involved spinal nerve roots (22). It appears that about 25% of animals exposed to nitrosoureas will have associated peripheral nerve tumors, which may have significant impact on a particular study. Recognition of the potential for peripheral nerve tumors in nitrosourea tumor models is important if the nitrosourea model is used in a survival study. The peripheral nerve tumor may cause death or morbidity leading to e~lthanasia, which will confound survival analysis if one is attempting to study a therapy designed for gliomas. Systemic tumors are common with all of the nitrosourea models. As many as half of the animals may develop some form of systemic tumor depending on strain and the particular technique of nitrosourea exposure. The types of systemic tumors are influenced by the dose, compound, route of administration, strain, and sex. Continuous oral exposure to MNU resulted in 145

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extranervous system neoplasms in 210 rats (9). The tumors were primarily of the oral cavity, stomach, and urinary tract. In this study the sex of the animal did not influence the development of systemic tumors. The incidence of urinary tract tumors with oral MNU was strain related. Repeated intravenous MNU exposure resulted in systemic tumors in 23% of animals (6); 22% of female Sprague-Dawley and 55% of female CD Fischer rats developed fibroadenomas of the mammary gland, sarcomas of the genitourinary tract, and leukemia. No systemic tumors were seen in male animals of either strain in this study. The transplacental ENU technique also resulted in systemic tumors, most of which were seen with the lower dose of 1 mg/kg. Over half of these tumors were incidental findings at autopsy (16). Renal carcinoma, leukemia, and mammary carcinomas are the most common systemic tumors seen after transplacental ENU exposure. As for the peripheral nerve tumors, the systemic tumors may confound a therapeutic study using a nitrosoureainduced glioma model when death or impending death is used as an end point. The survival of rats after exposure to nitrosoureas is too prolonged and variable to make these models useful for therapeutic trials. The survival time for prolonged intravenous MNU exposure in adult animals is 280-421 days after the start of exposure (6). For the transplacental ENU model the survival time is 85-346 days after birth (16, 19); with continuous oral exposure to MNU, the survival time is 119-294 days (8, 9). The brain tumors are believed to be fatal in 27-69% of the animals in the studies (16). Progression of gliomas induced by transplacental ENU may involve growth from identifiable early neoplastic proliferations of oligodendroglia and astrocytes in the brain, which later develop into microtumors (13). Similar focal microscopic cellular abnormalities, felt perhaps to be intermediate between ectopias and neoplasms, have seen in human brains both in association with the phacomatoses and as rare incidental autopsy findings (27). The molecular and cytogenetic events associated with these early morphologic changes in rats and humans are unclear. The relevance of chemical carcinogenesis to human gliomas is unknown, although one study has observed an increased incidence of glioblastomas in vinyl chloride workers (28).

Avian Sarcoma Virus-Induced Glioma Model The Rous avian sarcoma virus (ASV) is a type C RNA tumor virus of the retrovirus family. It is a transducing retrovirus carrying the viral oncogene, s r c . Most strains of ASV are nondefective, carrying a complete complement of viral genes along with s r c ; therefore, most strains of ASV can both transform and replicate within susceptible host cells. Avian sarcoma virus is a very effective transformer of susceptible cells with a short latency period

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and generally a high efficiency of tumor formation (29). ASV consistently produces brain tumors when injected intracerebrally into rats, hamsters, guinea pigs, cats, dogs, and subhuman primates (1, 4, 25). In rats, glioma tumors are produced almost 100% of the time in neonatal animals (30-32). Subgroup C Bratislava-77 ASV (30, 31, 33, 34) and subgroup D SchmidtRuppin ASV (30, 32, 35) are the two ASV strains that have been used most extensively to produce glioma tumors in rats; the two viral strains appear to be equally effective. The Sprague-Dawley and Fischer 344 strains of rat appear to be very susceptible. The tumors induced in Fischer 344 rats with ASV have yielded at least two permanent, transplantable rat glioma cell lines, $635c15 (35) and RT-2 (36, 37). Bigner (30-35, 38) has developed the critical factors necessary to produce a consistent model of glioma using ASV in the rat. Large-scale viral culture with chicken fibroblasts in medium containing 1% dimethyl sulfoxide (DMSO) and concentration of cell-free virus with ultracentrifugation provide a uniform dose of ASV (30). Rats are inoculated intracerebrally with 8.7 x 104 to 4.8 x 10 6 focus forming units of ASV in a volume of 5-20 ~zl (30, 31, 33, 34). Neonatal rats, 1-10 days old, have a 91-100% incidence of brain tumors, and adult animals, 100 days old at the time of injection, have a 50% incidence of brain tumors (30, 31, 34). Neonatal Fischer 344 rats injected intracerebrally with 8.7 x 104 focus forming units on day 1 of age will form brain tumors in 100% of animals with a survival of 83.3 -+ 21.5 days; all animals die from their tumors (34). Older animals have longer survival times. A majority of brain tumors induced with ASV are gliomas. Of neonatal Fischer 344 rats inoculated intracerebrally with ASV, 100% developed gliomas, but 12.5% had meningeal sarcomas, 37.5% had spinal cord tumors, and 37.5% had hemorrhagic cysts associated with gliomas (30). In 21 rats inoculated on day 1 of life, 47 brain tumors developed. Of the 47 tumors, 68% were gliomas, 2% were gliosarcomas, and 30% were meningeal sarcomas (31). Avian sarcoma virus inoculation in 2-day-old rats produced 60% gliomas, 9% gliosarcomas, and 31% meningeal sarcomas (32). Of the gliomas produced in neonatal rats, 97% have the histologic features of anaplastic astrocytoma, gemistocytic astrocytoma, or a mixture of the two types of astrocytoma (34). The preponderance of malignant astrocytomas is similar to the situation in human gliomas. With inoculation of older rats, there is a trend toward a higher relative incidence of gliomas, although the overall tumor yield is lower. Fischer 344 rats inoculated at 100 days of age have a relative incidence of 89% gliomas and 11% meningeal sarcomas; no gliosarcomas were noted in this study (31). Older animals tended to have less multiple tumors and the astrocytomas had less anaplastic features. Pilocystic astrocytomas were 78% of all gliomas and, indeed, 65% of all brain tumors in rats inoculated at age 97-119 days (34). In neonatal dogs, the relative incidence

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of gliomas and meningeal sarcomas was influenced by the site of injection. Deep periventricular inoculation produced only gliomas in 70% of animals. Superficial inoculation in the subdural space or into the cerebellar vermis produced all sarcomas (39). The relevancy of this observation to the rat ASV glioma model is unclear, but it raises the possibility that location of inoculation could also be a factor in rats. The ASV rat glioma model has been utilized in therapeutic trials. Using Fischer 344 rats and inoculating 5-day-old animals with ASV, the response to 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) and radiation therapy has been studied. The combined treatment has a stronger positive impact on survival than either therapy alone (40). In another study using the same ASV model the dose response for fractionated external-beam radiation therapy has been examined. Higher total doses of radiation have a beneficial effect on survival (41). The model has also been used to study combined immunotherapy and chemotherapy (42). The results show improvement in survival with the combinations of therapies. Overall, the ASV glioma model parallels the human response to therapy. The model is uniformly refractive to all attempted treatments, with favorable results showing prolongation of survival but with all animals eventually succumbing to disease. The ASV rat glioma model has many attractive features. It is autochthonous, and the induced tumors are primarily glial. Peripheral nerve tumors, which are very common in the nitrosourea models, are not seen after ASV intracerebral inoculation. The tumors are predictably fatal within 3 months of ASV inoculation. In rats inoculated as adults, there is a low incidence of multiple tumors. Permanent cell lines for in vitro studies have been developed. The ASV glioma model appears to have a response to therapy about the same as that seen in human gliomas. The limitations of the ASV rat glioma model are the significant incidence of intracranial sarcomas, which must be accounted for in determining therapeutic response and cause of neurologic death in animals. With neonatal inoculation the yield of tumor is greatly increased, but at the expense of increased multiplicity and variety of brain tumor. The relevance of viral induction to human glioma pathogenesis is unclear. The techniques and precautions necessary for working with tumor viruses are cumbersome and have to be considered in using the ASV glioma model.

Transplantation Glioma Models Autochthonous glioma models have the disadvantages of multiplicity of tumor numbers and types, prolonged latency from induction procedure to death or macroscopic tumor appearance, and technical hazards of handling

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TABLE I

Rodent Glioma Models

Cell line

Recommended host

Histology

Origin

Anaplastic astrocytoma

F98

CD Fischer rats

Glioma, undifferentiated

RG2

CD Fischer rats

Differentiated glioma

BT lines (BT4C and BT4Cn) 36B10

BD IX rats

Glioma

Repeated i.v. MNU in random-bred Wistar rats Repeated i.v. MNU in CD Fischer rats Transplacental ENU or repeated i.v. MNU in CD Fischer rats Transplacental ENU or repeated i.v. MNU in CD Fischer rats Transplacental ENU in BD IX rats

65

9L

Immunosuppressed host such as nude mouse or rat CD Fischer rats

Fischer F-344 rats BD IX rats

RT2

Fischer F-344 rats

Malignant glioma

Transplacental ENU in Fischer F-344 rats Transplacental ENU in BD IX rats Avian sarcoma virus inoculation of Fischer F-344 rats

17

G-XII

Spinal malignant astrocytoma Glioma

C6

Gliosarcoma

Refs.

4 14

14, 15

97-100

51, 101 6, 7

carcinogenic chemicals or animal tumor viruses. As a result of these problems, the expense of developing and maintaining an autochthonous glioma model may be an issue for the researcher. Transplantation glioma models, in general, have much shorter latency periods, better control over location of tumor, solitary tumor production, and lower cost. The tradeoff is that more involved surgical manipulation is required, and the tumor cells are open to possible immunologic attack. Well-characterized cell lines offer the advantages of predictability and in vitro correlation. However, these cell lines are influenced to an uncertain degree by the artificial selection pressures imposed by prolonged cell culture or animal passages. Transplanted glioma cell lines have provided most of the data necessary to develop present-day standard and experimental human glioma therapy. A large number of useful cell lines have been developed from gliomas induced by the nitrosoureas (see Table I). Some critical technical aspects of the transplantation procedure are essential to the reproducibility of the model, regardless of the specific cell line utilized. Freehand injection, although rapid, appears to be associated with

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extracerebral spread of tumor. Stereotactic placement of the cells into the head of the caudate nucleus with slow delayed withdrawal of the injection cannula has minimized the risk of extracerebral spread. Kobayashi et al. (43) were the first to demonstrate the advantages of stereotactic placement over freehand injection. The freehand method produces extracranial tumors in about 17%, subdural or subarachnoid tumors in 75%, spinal cord metastasis in 20%, and lung metastasis in 13%. There is intracerebral tumor production in only 40% of rats, which is usually associated with tur,~ors elsewhere, and in 14% of animals no tumor growth occurs at all. The stereotactic technique results in a 99% incidence of intracerebral tumor take and only a 0.4% complete failure rate. The incidence of extracranial tumors is 6%, subdural or subarachnoid tumors is 2.6%, and spinal cord metastasis is 1.7%. The stereotactic injection of tumor cells into the head of the caudate is now the standard technique for intracerebral transplantation of glioma cell lines (44-49). The efficiency of tumor production is related to the amount of tumor cells inoculated intracerebrally. Most studies use from 104 to 106 cells in a volume varying from 2 to 50 txl of medium (6, 43-49, 51-56). Auer et al. (53) studied the dose response for tumor production of intracerebrally implanted C6 cells in neonatal Wistar rats. They looked at a dosage range of 5 cells to 1 x 105 cells. A 100% tumor incidence was consistently seen in doses over 1 x 10 4 cells. Less than 500 cells did not result in any tumor formation. Implantation of spheroids may be an alternative method to overcome artifactual dissemination. The method has been developed using C6 glioma cells grown in spinner culture flasks spun at 180 rpm for 2-3 weeks (57). This provides a spheroid measuring 350-500 txm in diameter and containing approximately 105 cells. A single spheroid can be isolated and implanted into the rat cortex. With this method using C6 glioma spheroids in adult Sprague-Dawley rats, tumors were produced in 93% of animals (57). Macroscopic tumors are produced by day 13 (57, 58). This model has been used to study tumor permeability (57), microvessel characteristics (57, 59, 60) and protease activity (61) in the C6 glioma implanted into the Sprague-Dawley rat. The effects of steroids and nonsteroidal antiinflammatory agents on the same model have also been studied (58, 62). In addition to the site of transplantation, the host factors that appear to be important in the success of glioma transplantation are the age and strain of the rat. The age of the host rat is important, particularly for heterotopic transplantation. Within the same species and for the same cell line, neonatal animals tend to accept intracerebral transplantation of tumor cells more readily than do adult animals (63). In syngeneic systems using highly inbred hosts and glioma cell lines derived from the same species, the success rate, even with adult animals, approaches 100%, indicating that the genetic factors

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are more important than age in tumor take. Heterotopic transplantation, particularly with human glioma lines implanted into rats, usually requires some form of immunosuppression. The sex of rat does not appear to influence the success of glioma transplantation into the brain (64), although most studies use male animals. The C6 glioma cell line was cloned from an intravenous MNU-induced glioma tumor in a random-bred Wistar rat (65). The line is well characterized, stable, and readily available; it grows very well in vitro. Because of these features, many studies have been performed using the intracerebral transplantation of C6 cells into rats. Because of the lack of a syngeneic host the reproducibility of the model and interpretation of therapeutic studies are probably influenced by immunologic factors. Studies using the C6 model demonstrate incidences of tumor production failure and prolonged survivors not seen in the models utilizing syngeneic systems, suggesting that immunologic rejection may be active in at least some animals. These issues have limited the usefulness of the C6 model in therapeutic studies. However, investigators have created useful models to study pathophysiology by intracerebrally transplanting the C6 cells into Sprague-Dawley (66), Long-Evans (50, 52), Wistar (53, 56, 67, 68), and CD Fischer (55, 69) rats. The C6 glioma intracerebral transplantation model has been used to study magnetic resonance imaging correlations (67), peritumoral brain edema (68, 70, 71), and brain invasion (56, 66). Gene therapy has been studied with the C6 line, but the studies have been performed by implantation into the nude mouse (54, 72, 73). The 9L rat glioma cell line, like the C6 line, was developed from an anaplastic astrocytoma tumor induced in a CD Fischer rat by weekly intravenous injection of MNU (74). Because the CD Fischer strain is inbred, syngeneic hosts are available for transplantation, minimizing possible immunologic rejection. The tumor model is uniformly fatal and refractory to treatment. Effective therapies generally only prolong survival, not cure the disease, in this model, which parallels the human condition. Therefore, the glioma model using transplantation of 9L glioma cells into the brain of CD Fischer rats is one of the most widely utilized systems for brain tumor therapeutic trials (45, 75). The technique of transplantation of (1-10) x 104 cells stereotactically into the caudate nucleus results in close to 100% tumor production with median survival of 20-30 days postimplantation, and the range is usually less than 10 days (45). The time of death can be accurately predicted by the weight loss pattern of the animal, which minimizes the potential suffering of moribund animals prior to actual death (76). The 9L model has been used to investigate issues of glioma tumor angiogenesis (77-79), invasion (80), and blood-to-tissue transport (81). In addition to a variety of chemotherapy and immunotherapy preclinical trials, the 9L model has also been used to

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demonstrate the potential efficacy of some novel therapeutic approaches. Boron neutron capture therapy appears to be remarkably effective against the 9L glioma in vivo without the severe toxicity of high-dose conventional external-beam radiation therapy required for similar control (82). Phenylacetate has a unique selective window of toxicity for developing glial cells, and has demonstrated promising ability to inhibit tumor growth and prolong the survival in a 9L glioma model (83, 84). The 9L model has been very useful in developing gene therapy protocols (85-87). Besides suffering from the limitations of all transplantation glioma models, the 9L glioma has unusual features that must be considered when selecting this model for study. Histologically, the 9L tumor is a gliosarcoma (45, 75). Gliosarcomas account for only about 8% of human malignant gliomas; however, the clinical behavior is the same as the glioblastoma (88). The invasion pattern for the 9L glioma is apparently different than the C6 or human gliomas. The 9L glioma invades preferentially along the perivascular Virchow-Robin spaces with no evidence of intraparenchymal invasion in the tumor periphery (80). Early attempts to study human gliomas in vivo were hampered because of immunological rejection of the xenogeneic transplants (4, 63, 88). Immunosuppression with corticosteroids or irradiation, although useful in transplanting some tumors subcutaneously, is not as effective for preventing the rejection of human glioma xenografts in the rat brain (63). The athymic, nude rat is a better host for intracerebrally xenografting human gliomas than rats immunosuppressed by other methods, including cyclosporine. Tumor production in the athymic, nude rat is 76-100% (89-91). The range in yield may likely reflect differences in the biology of the particular implanted human glioma. Using low-passage malignant human gliomas transplanted into the nude rat, in vivo human glioma metabolism has been studied using magnetic resonance imaging (MRI) spectroscopy (90). MRI also provides a means of longitudinally studying tumor growth within the same animal. Fibroblast growth factor overexpression has been demonstrated in the human glioma cell lines A172, A1207, and A1235, when implanted intracerebrally into nude rats (91). Trial therapy with intraarterial 4-hydroperoxycyclophosphamide showed a significant increase in survival in a preclinical model using the human glioma line D-54 MG intracerebrally implanted in nude rats (92). Confirmation of glioma invasion patterns that had previously only been demonstrated through extensive microscopic examination of human autopsy material has been obtained by implanting fresh human anaplastic astrocytoma cells into nonimmunosuppressed Sprague-Dawley rats (93-95). The human glioma tumor cells, harvested immediately from surgery, are first labeled with Phaseolus vulgaris leukoagglutinin and then transplanted. The label

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PARADIGMS OF NEURAL INJURY allows the individual tumor cells to be readily identified against the background of the normal invaded rat brain. In this system frank tumor growth is not seen at the site of implantation; this is possibly due to immunologic attack on most of the implanted human glioma cells. A variation of this technique using human glioma cell lines D-54 MG and GaMG, labeled first with a carbocyanine vital dye and then implanted intracerebrally into BD IX rats, demonstrated that the glioma cells migrate in a pattern similar to that of fetal glial cells (96). Rodent glioma models appear to offer the balance of a reasonable size brain for in situ study along with convenient and relatively inexpensive animal care. Rodent glioma models have undergone refinement over the past 40 years. The nitrosourea and ASV models have the advantage of producing autochthonous tumors, but tend to have very long and variable latent periods. Long-term animal care and viral facilities may make these techniques more expensive than the transplantation models. Transplantation models tend to be more predictable and less costly. Transplanted cells are the product of the artificial selection process of cell culture and many animal passages. The particular model must be chosen recognizing its limitations of efficiency and latency of tumor production, expense, treatment response, histologic features, and biologic behavior.

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11. A. Maekawa and K. Mitsumori, Crit. Rev. Toxicol. 20(4), 287-310 (1990). 12. L. Tomatis, J. Hilfrich, and V. Turusov, Int. J. Cancer 15, 385-390 (1975). 13. D. Schiffer, in "Neurobiology of Brain Tumors" (M. Saloman, ed.), pp. 85-135. Williams and Wilkins, Baltimore, Maryland, 1991. 14. L. Ko, A. Koestner, and W. Wechsler, Acta Neuropathol. (Berlin) 51, 107111 (1980). 15. L. Ko, A. Koestner, and W. Wechsler, Acta Neuropathol. (Berlin) 51, 23-31 (1980). 16. J. A. Swenberg, A. Koestner, W. Wechsler, and R. H. Denlinger, Cancer Res. 32, 2656-2660 (1972). 17. A. M. Spence and P. W. Coates, Virchows Arch. B Cell Pathol. 28, 77-85 (1978). 18. J. P. Roscoe, Br. Med. Bull. 36(1), 33-38 (1980). 19. A Koestner, J. A. Swenberg, and W. Wechsler, Am. J. Pathol. 63, 37-56 (1971). 20. S. Ivankovic and H. Druckrey, Z. Krebsforsch. 71, 320-360 (1968). 21. C. J. Vecht, M. J. Van Zwieten, B. Maat, and D. W. Van Bekkum, Eur. J. Cancer 17(6), 703-710 (1981). 22. M. Naito, Y. Naito, and A. Ito, Gann 73, 323-331 (1982). 23. M. Naito, Y. Naito, and A. Ito, Gann 72, 30-37 (1981). 24. M. Naito, Y. Naito, and A. Ito, Gann 72, 569-577 (1981). 25. D. S. Russell and L. J. Rubinstein, in "Pathology of Tumors of the Nervous System," 5th Ed., pp. 58-82. Williams and Wilkins, Baltimore, Maryland, 1989. 26. D. S. Russell and L. J. Rubinstein, in "Pathology of Tumors of the Nervous System," 5th Ed., pp. 222-223. Williams and Wilkins, Baltimore, Maryland, 1989. 27. D. S. Russell and L. J. Rubinstein, in "Pathology of Tumors of the Nervous System," 5th Ed., pp. 222-223. Williams and Wilkins, Baltimore, Maryland, 1989. 28. R. J. Waxweiler, W. Stringer, J. K. Wagoner, J. Jones, H. Falk, and C. Carter, Ann, N.Y. Acad. Sci. 271, 40-48 (1976). 29. T. Benjamin and P. K. Vogt, in "Fundamental Virology, 2nd Edition" (B. N. Fields and D. M. Knipe, eds.), pp. 291-342. Raven Press, New York, 1991. 30. D. D. Copeland and D. D. Bigner, Acta Neuropathol. 38, 1 (1977). 31. D. D. Copeland, F. S. Vogel, and D. D. Bigner, J. Neuropathol. Exp. Neurol. 34, 340 (1975). 32. R. F. Wilfong, D. D. Bigner, D. J. Self, and W. Wechsler, Acta Neuropathol. 25, 196-206 (1973). 33. D. D. Bigner, D. J. Self, J. Frey, R. Ishizaki, A. J. Langlois, and J. A. Swenberg, Recent Results Cancer Res. 51, 20-34 (1975). 34. D. D. Copeland, F. A. Talley, and D. D. Bigner, Am. J. Pathol. 83, 149-176 (1976). 35. Y. S. Lee, S. H. Bigner, L. F. Eng, P. Molnar, A. Kuruvilla, D. R. Groothuis, and D. D. Bigner, J. Neuropathol. Exp. Neurol. 45, 704 (1986). 36. F. P. Holladay, C. Rajani, T. Heitz, and G. W. Wood, J. Neurosurg. 80, 90-96 (1994). 37. M. J. Nelson, F. K. Conley, and L. F. Fajardo, Exp. Mol. Pathol. 58, 105113 (1993). 38. D. D. Bigner and C. N. Pegram, Adv. Neurol. 15, 57-83 (1976).

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PARADIGMS OF NEURAL INJURY 39. D. D. Bigner, J. P. Kvedar, T. C. Shaffer, N. A. Vick, W. K. Engel, and E. D. Day, J. Neuropathol. Exp. Neurol. 31, 583-595 (1972). 40. P. Steinbok, M. S. Mahaley, R. U., D. C. Zinn, S. Lipper, J. Mahaley, and D. D. Bigner, J. Neurosurg. 51, 581-586 (1979). 41. P. Steinbok, M. S. Mahaley, R. U., M. A. Varia, S. Lipper, J. Mahaley, and D. D. Bigner, J. Neurosurg. 53, 68-72 (1980). 42. M. S. Mahaley, R. E. Gentry, and D. D. Bigner, J. Neurosurg. 47, 25-43 (1977). 43. N. Kobayashi, N. Allen, N. R. Clendenon, and L. W. Ko, J. Neurosurg. 53, 808-815 (1980). 44. V. M. Morreale, B. H. Herman, V. Der-Minassian, M. Palkovits, P. Klubes, D. Perry, A. Csiffary, and A. P. Lee, J. Neurosurg. 78, 959-965 (1993). 45. B. F. Kimler, J. Neuro-oncol. 20, 103-109 (1994). 46. A. Rama, O. Spoerri, M. Holzgraefe, and H. D. Mennel, Acta Neurochirurg. 79, 35-41 (1986). 47. A. L. Benabid, C. Remy, and C. Chauvin, in "Biology of Brain Tumor" (M. D. Walker and D. G. T. Thomas, eds.), pp. 221-226, Martinus Nijhoff, Boston, 1986. 48. D. E. Wilkins, P. G. Raaphorst, J. K. Saunders, and I. C. P. Smith, Lab. Animal Sci. 43(5), 518-519 (1993). 49. M. Fleshner, L. R. Watkins, J. M. Redd, C. A. Kruse, and D. Bellgrau, Cell Transpl. 1, 307-312 (1992). 50. J. Mokry, S. Nemecek, J. Adler, and K. Dedic, Funct. Dev. Morphol. 3(3), 175-780 (1993). 51. H. D. Mennel and P. Groneck, Acta Neuropathol. (Berlin) 40, 145-150 (1977). 52. J. Mokry, S. Nemecek, and J. Adler, Sbor Praci LF UK Hradec Kralove 35, 293-305 (1992). 53. R. N. Auer, R. F. Del Maestro, and R. Anderson, Canad. J. Neurol. Sci. 8(4), 325-331 (1981). 54. S. H. Chen, H. D. Shine, J. C. Goodman, R. G. Grossman, and S. L. C. Woo, Proc. Natl. Acad. Sci. U.S.A. 91, 3054-3057 (1994). 55. B. D. Ross, R. J. Higgins, J. E. Boggan, J. A. Willis, B. Knittel, and S. W. Unger, Biomedicine 1(1), 20-26 (1988). 56. N. Nagamo, H. Susaki, M. Aoyagi, and K. Hirakawa, Acta Neuropathol. 86, 117-125 (1993). 57. C. L. Farrell, P. A. Stewart, and R. F. Del Maestro, J. Neuro-oncol. 4, 403415 (1987). 58. C. L. Farrell, J. Megyesi, and R. F. Del Maestro, J. Neurosurg. 68, 925-930 (1988). 59. B. L. Coomber, P. A. Stewart, E. M. Hayakawa, C. L. Farrell, and R. F. Del Maestro, J. Neuropathol. Exp. Neurol. 47(1), 29-40 (1988). 60. C. L. Farrell, C. R. Farrell, P. A. Stewart, R. F. Del Maestro, and C. G. Ellis, Int. J. Radiat. Biol. 60, 131-137 (1991). 61. I. S. Vaithilingam, E. C. Stroude, W. McDonald, and R. F. Del Maestro, J. Neuro-oncol. 10, 203-212 (1991). 62. H. R. Reichman, C. L. Farrell, and R. F. Del Maestro, J. Neurosurg. 65, 233-237 (1986).

[5] RODENT GLIOMA MODELS

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PARADIGMS OF NEURAL INJURY 86. W. W. G. Jia, M. McDermott, J. Goldie, M. Cynader, J. Tan, and F. Tufaro, J. Natl. Cancer Inst. 86(16), 1209-1215 (1994). 87. Z. Ram, S. Walbridge, T. Shawker, K. W. Culver, R. M. Blaese, and E. H. Oldfield, J. Neurosurg. 81, 256-260 (1994). 88. D. S. Russell and L. J. Rubinstein, in "Pathology of Tumors of the Nervous System," 5th Ed., pp. 222-223. Williams and Wilkins, Baltimore, Maryland, 1989. 89. C. Adams, D. E. Bullard, S. H. Bigner, and D. D. Bigner, in "Biology of Brain Tumor" (M. D. Walker and D. G. T. Thomas, eds.), pp. 97-105. Martinus Nijhoff, Boston, 1986. 90. H. J. J. A. Bernsen, A. Heerschap, A. J. van der Kogel, J. J. van Vaals, M. J. J. Prick, E. F. J. Poels, J. Meyer, and J. A. GrotenHuis, J. Neuro-oncol. 13, 119-130 (1992). 91. S. D. Finkelstein, P. Black, T. P. Nowak, C. M. Hand, S. Christensen, and P. W. Finch, Neurosurgery 34(1), 136-143 (1994). 92. J. M. Schuster, H. S. Friedman, G. E. Archer, H. E. Fuchs, R. E. McLendon, O. M. Colvin, and D. D. Bigner, Cancer Res. 53, 2338-2343 (1993). 93. E. R. Laws, W. J. Goldberg, and J. J. Bernstein, JJ, Int. J. Dev. Neurosci. 11(5), 691-697 (1993). 94. J. J. Bernstein, A. V. Anagnostopoulos, E. A. Hattwick, and E. R. Laws, J. Neurosurg. 78, 240-251 (1993). 95. J. J. Bernstein, W. J. Goldberg, and E. R. Laws, Neurosurgery 24(4), 541546 (1989). 96. P. H. Pedersen, K. Marienhagen, S. Mork, and R. Bjerkvig, Cancer Res. 53, 5158-5165 (1993). 97. K. Edvardsen, P. H. Pedersen, R. Bjerkvig, G. G. Hermann, J. Zeuthen, O. D. Laerum, F. S. Walsh, and E. Bock, Int. J. Cancer 58, 116-122 (1994). 98. O. D. Laerum, M. F. Rajewsky, M. Schachner, D. Stavrou, K. H. Haglid, and A. Haugen, Z. Krebsforsch. 89, 273-295 (1977). 99. O. Mella, R. Bjerkvig, B. C. Schem, O. Dahl, and O. K. Laerum, J. Neurooncol. 9, 93-104 (1990). 100. K. Edvardsen, N. Brunner, M. Spang-Thomsen, F. S. Walsh, and E. Bock, Int. J. Dev. Neurosci. 11(5), 681-690 (1993). 101. T. Tsukahara, M. Tamura, H. Yamazaki, H. Kurihara, and S. Matsuzaki, J. Cancer Res. Clin Oncol. 118, 171-175 (1992).

[6]

Peripheral Lesioning of Olfactory System" Expression of Neurotrophin Receptors C h r i s t o p h e r P. T u r n e r a n d J. R e g i n o P e r e z - P o l o

Introduction Nerve growth factor (NGF) and other members of the neurotrophin family are known to stimulate cell growth, division, and differentiation, and may also be involved in synaptic plasticity and neural regeneration (1). These trophic substances are thought to play key roles in the support and maintenance of specific neuronal populations throughout the nervous system (2). The low-affinity receptor for NGF, p75 NGFR, is expressed in both the peripheral and central nervous systems (PNS and CNS, respectively) during development, as well as in adult animals (2). One area where p75 NcF~ expression is particularly robust is in the rat olfactory bulb (3). The main olfactory bulb (MOB) receives peripheral input from primary sensory neurons (the olfactory receptor neurons, or ORNs) of the olfactory neuroepithelium (4). The mature ORN population continues to regenerate itself from an ever-present population of stem cells (4). This feature makes the olfactory system an appropriate area for studying the expression of neurotrophic substances and their receptors. ORNs project directly to the glomerular layer of the MOB and their regeneration in the neuroepithelium results in an unusually high degree of synaptic turnover in this region of the MOB (4). Abundant expression of p75 NGFRis localized to the glomerular layer, suggesting that p75 NGFRmay be involved in/regulated by this regenerative process (3). To test this hypothesis, we lesioned the olfactory system by chemically disrupting the peripheral input to the MOB at the level of the olfactory neuroepithelium. We chose to lesion the peripheral input by irrigating the nasal cavity with Triton X100 (TX-100), an agent that has been reported to remove mature ORNs selectively and reversibly from the neuroepithelium (5). Expression of p75 NGFR in the MOB was then monitored immunohistochemically. The reversible nature of treatment with TX-100 allowed assessment of p75 NGFR expression under degenerating and regenerating conditions. In addition, loss of one olfactory bulb has been reported to alter the regenerative capacity of both the ipsilateral and contralateral olfactory neurepithelia (6). Thus, we performed a unilateral bulbectomy to assess whether the loss of one olfactory bulb could influence p75 NGFR expression in the contralateral bulb. Again, p75 N~Fk expression was monitored immunohistochemically. Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Methods

Intranasal Irrigation The left nasal cavity of 3- to 6-month-old female Sprague-Dawley rats is irrigated with either saline (0.9% NaC1) or TX-100 (0.5% in saline) (Fig. 1). Solutions are filter sterilized prior to use. Although, initially, we anesthetized animals with Nembutal (50 mg/kg) prior to irrigation, we find that light anesthesia with halothane is sufficient for the time necessary to perform the irrigation (1-2 minutes). Three animals per treatment group are used. After 1, 2, 4, 8, and 16 weeks, animals are processed for immunohistochemistry (see below).

Olfactory Bulbectomy The 3-month-old male Sprague-Dawley rats are split into two groups. Under deep anesthesia, an opening is formed in the left, rostral cranium, exposing the left olfactory bulb. In the first, sham-lesioned (SH) group (N = 3, for each time point), no further procedures are performed. In a second, bulbectomized (OBX) group (N = 4, for each time point), the left olfactory bulb is completely removed by aspiration. Surgery is performed on request by Zivic-Miller Labs Inc. (Pittsburgh, PA). After 1, 2, 4, 8, and 16 weeks, animals are processed for immunohistochemically (see following).

Immu no his toc he mis try Animals are deeply anesthetized with Nembutal (50 mg/kg) and are nonresponsive to several reflexes (paw-pinch, tail-pinch, corneal irritation) before transcardial perfusion with phosphate-buffered saline (PBS: 0.14 M NaC1; 3 mM KC1; 1 mM NazHPO 4 ; 4.5 mM KHzPO 4) followed by phosphate-buffered 4% paraformaldehyde. The brains are then cryoprotected in 25% sucrose/ PBS, blocked, and sliced on a freezing microtome into 40-/zm sections. Freefloating sections are stained for specific p75 NGFRimmunoreactivity (p75 yCFRir), using a modified protocol of Yan and Johnson (3). All incubations are performed at room temperature and each incubation is followed by three 10minute washes with PBS. Sections are quenched for endogenous peroxidase activity by incubating for 1 hour in 100% methanol/0.003% H202, then incubated in 0.3% Triton X-100/PBS containing 3% horse serum (Vector Labs) and 3% rat serum (Sigma, St. Louis, MO) for 1 hour. The inclusion of rat serum provides a significant improvement in the reduction of nonspecific

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PIo. 1 Top: Irrigation of the nasal cavity. Appro• 700-500 #~1 of saline or 0.5% TX-IO0 was slowly injected into the left nasal cavity using a 1-ml tuberculin syringe. Animals were placed on their back with their noses pointing upward during the procedure. A few seconds were allowed to pass and the animal was turned rightside up (nose downward) and excess fluid was blotted away with a Kimwipe. The needle of the tuberculin syringe was filed to a rounded edge to prevent accidental damage to delicate tissue. ONE, Olfactory neuroepithelium; CP, cribriform plate; MOB, main olfactory bulb. Bottom: A coronal view of the olfactory system of the rat. R, Right; L, left; Med, medial; Lat, lateral; ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer. The widths of the right and left neuroepithelia were compared following irrigation with saline or TX-100. The approximate locations of the areas compared are indicated by the boxed regions.

99

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staining. Sections are incubated in avidin and biotin solutions (Vector) to block against endogenous biotin sites, followed by overnight exposure to affinity-purified monoclonal antibody (MAb)192 (0.5/~g/ml), a mouse monoclonal antibody to rat p75 NGFR(a gift from E. Johnson, Washington University, St. Louis, MO). Assay controls are performed by omitting the primary antibody (MAb 192) for negative controls, or staining sections from the basal forebrain of the same animal for positive controls. Magnocellu!ar, cholinergic neurons of the basal forebrain are known to express both the mRNA and protein encoded by the gene for p75 NGFR(3, 7). Prior to this study, we have identified these basal forebrain neurons in immunohistochemical assays with the same MAb192 preparation used here. The following day, sections are incubated with an antimouse secondary antibody for 5 hours and exposed to an avidin-biotin peroxidase (ABC) mixture for 2 hours (according to Vectastain Elite instructions; Vector). The primary-secondary ABC complex is visualized by exposing the sections to diaminobenzidine/H202 (DAB 0.5 mg/ml; 0.12% H202)for 5 minutes. This time is determined empirically and provides crisp staining with low background. The sections are left overnight in the final PBS wash at 4~ and mounted onto gelatin-chrome-alum-coated slides. After drying, the slides are passed through xylene, descending concentrations of ethanol, into distilled water, removing any debris that may have collected during the immunohistochemistry assay, and back through the alcohols to xylene. Sections are covered with Permount and coverslips. Early in our study, prominent staining of mitral cell bodies was observed in both saline- and TX-100-treated animals; this staining is apparently specific because assay control sections display no staining in this region of the MOB (8). We pursue this pattern of staining by repeating the lesions to test reliability of this mitral cell response. However, adjacent sections are now included for negative assay controls and a similar staining pattern is observed in these sections as was observed in sections exposed to the primary antibody. This observation argues against our initial conclusion that mitral cells display specific staining for p75 NGFR. It is curious why mitral cells display such robust staining while the rest of the section displays (by comparison) only background staining. Two other groups (9, 10) have reported specific staining for p75 NGFRin the mitral cell layer, which might suggest that our observations should not be dismissed as a false-positive. The use of an antibody that recognizes a different epitope from MAb 192 would shed further light on the potential of mitral cells to express p75 NGFR. To stain both olfactory neuroepithelia and olfactory bulbs in the same section, ethylenediaminetetraacetic acid (EDTA; Sigma) is employed as a decalcifying agent. The olfactory neuroepithelium is found with the bony

[6]

EXPRESSION OF N E U R O T R O P H I N RECEPTORS

101

tissue that surrounds the nasal septum. Decalcification is necessary to produce sections of uniform thickness and quality. As previously reported (11, 12) both olfactory bulb and olfactory neuroepithelium display p75NCFR-ir, suggesting that EDTA has no effect on the immunoreactivity of the antigen. In later experiments, assays occasionally failed to produce immunoreactivity in the olfactory areas (particularly in the olfactory neuroepithelium), whereas p75NGFR-ir is always observed in the basal forebrain. To determine the source of the problem, different preparations and concentrations of primary and secondary antibodies are used, all stock solutions are prepared fresh (to account for potential bacterial growth), original reagents are substituted with reagents from different sources, and, finally, blocking sera are sometimes omitted. When assays fail, these changes do not improve specific immunoreactivity in the olfactory areas of EDTA-treated tissue. In tissue that is not exposed to EDTA, olfactory bulbs always display robust staining in the glomerular layer. Other decalcifying agents (such as formic acid) destroy antigenicity in all regions, including the basal forebrain. Yan and Johnson (3) reported an absence of p75NcFR-ir in the olfactory neuroepithelium. However, Miwa et al. (13) have shown that severing the olfactory fascicles can induce expression of this low-affinity receptor in this region of the olfactory system. Perhaps the protocol used in this study was sufficiently sensitive to detect p75NGFR-ir in the neuroepithelium without having to induce such expression by lesioning. However, we were unable to obtain viable sections from the nasal septum region of nondecalcified tissue. The data we now report on are from tissue that was not exposed to EDTA.

Results

Intranasal Irrigation A detailed account of the data presented here has been reported previously (12). Table I summarizes the general effect of TX-100 on neuroepithelial width and expression of p75 NGFRin the olfactory bulb. Within the first week, TX-100 had induced a dramatic decline in neuroepithelial width on the left (lesioned) side. This reduction in width has been shown to be due to a specific loss of mature ORNs from the neuroepithelium (5, 14). A profound reduction in p75NCF~-ir was observed in the glomerular layer of the ipsilateral olfactory bulb, at a time when the neuroepithelium width was at its minimum. At the same time, an induction of p75NGFR-ir was observed in the olfactory nerve layer. As the neuroepithelium recovered in thickness, these early changes in p75NGFk-ir reversed their pattern and returned to prelesioned levels.

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PARADIGMS OF NEURAL INJURY

TABLE I Effect of Triton X-100 on Neuroepithelial Width and Expression of p 7 5 NGFR in Olfactory Bulb p75-ir in olfactory bulb b M e a n width of olfactory N E (/xm) a W e e k s postlesion Saline control 1 2 4 8 16

Left cavity 110 38 54 62 85 105

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a The mean width (+ standard deviation) of the olfactory neuroepithelium (NE) was estimated in saline control and TX100-treated (1-16 weeks postlesion) animals. Right versus left nasal cavity values are shown (see Fig. 1) and the ratio of left over right cavity (L/R) estimated. b Level of p75 NGFRimmunoreactivity (ir) in the olfactory bulbs of the same animals in the glomerular layer (GL) and olfactory nerve layer (ONL) [levels are indicated as + + + + + (intense) to + (modest); + / - (slight to none); 0 (usually below the level of detection)].

Olfactory Bulbectomy Details of the effects of unilateral bulbectomy on p75 NGFR expression in the central nervous system of rat can be found elsewhere (15). The general effects of sham-lesioning and unilateral bulbectomy on p75NGVR-ir in the olfactory bulb are summarized in Table II. In sham-lesioned animals, there was no difference in specific staining of the glomerular layer between the left and right olfactory bulbs (within the same animal, across animals within the same time group, or across time groups). In the right (remaining) bulb of OBX animals, no difference in p75NGFR-ir was observed in the 1-, 2-, and 4-week postlesion groups (when compared to sham controls) However, at 8 and 16 weeks, there was an increase in p75NGFR-ir in the glomerular layer of OBX animals above that observed for sham controls. We also observed an induction of fiberlike staining of the olfactory nerve layer, in the 8- and 16-week OBX groups.

Discussion In TX-100-treated animals, the time course for the disappearance and recovery of p75YGFR-ir from the glomerular layer of the MOB was in close agreement with the reduction and recovery of neuroepithelial width. This TX-

[6]

EXPRESSION

OF NEUROTROPHIN

103

RECEPTORS

TABLE II Staining for p75 NGFI~ in Olfactory Bulbs of Sham-Treated and Bulbectomized Animals a p75 i m m u n o r e a c t i v i t y b Left MOB Treatment a SHAM1 OBX1 SHAM2 OBX2 SHAM4 OBX4 SHAM8 OBX8 SHAM16 OBX16 a OBX,

GL

Right MOB

ONL

GL

ONL

+++

0

+++

0

-

-

+++

-

+ + +

0

+ + +

0

-

-

+ ++

-

+++

0

+++

0

-

-

+++

-

+++

0

+++

-

-

++++

+++

0

+++

0

-

-

+++++

+

Bulbectomized

animals.

Numbers

indicate

0 +/-

1, 2, 4, 8, or

16

weeks postsurgery. b Intensity of staining is the same scale as described in Table I.

100-induced change in width is reported to be due to a selective loss and recovery of mature ORNs from the neuroepithelium (5, 14). Severing the olfactory fascicles as they cross the cribriform plate (therefore depriving the olfactory bulb of its peripheral input) decreases p75NGFR-ir in the glomerular layer within 10 days, with a return of p75NCFR-ir to prelesion levels by 90 days (16). These observations are in very close agreement to the loss and recovery of p75NGFR-ir we have described (Table I). The observed increase in p75YGFR-ir within the glomerular layer of the contralateral MOB of OBX animals (Table II) might have been predicted. Regenerating ORNs project directly to the glomerular layer of the MOB (4). Changes in ORN turnover are known to take place in the contralateral neuroepithelium of unilaterally bulbectomized animals (6). Thus, the elevated levels of p75NGFR-ir we observed in the glornerular layer of the remaining (right) olfactory bulb of OBX animals may relate to the altered ORN turnover in the neuroepithelia of these animals. Unilateral occlusion of the nasal cavity (a procedure known to alter ORN neurogenesis) has been shown to increase p75NGFR-ir in the ipsilateral olfactory bulb (17). Taken together, these observations strongly suggest that regeneration events taking place in the neuroepithelium directly control expression of p75 NCFR in the MOB. The two olfactory bulbs communicate with each other through the anterior commissure (18) and, conceivably, the OBX-induced changes in p75 NCFR

104

PARADIGMS OF NEURAL INJURY expression in the contralateral bulb could be transmitted via this pathway. This explanation would require the involvement of more than one population of relay neurons (ipsilateral neurons of the anterior olfactory nucleus, and the contralateral granule and mitral cells, the latter projecting finally to the glomerular layer within the same bulb). In contrast, the projection of the ORNs to the glomerular layer is more direct. Although there was a relative absence of p75Y~FR-ir in the olfactory nerve layer of saline-treated animals, the TX- 100-induced removal of a large number of ORN axons passing through the olfactory nerve layer appears to be a sufficient stimulus for robust expression of p75 NCFRin this region of the MOB (Table I). This profound stimulation of p75NGVR-irin the olfactory nerve layer resembles the reported behavior of sciatic nerve Schwann cells following axotomy (19) and may relate to the permissive role that glia of the olfactory nerve layer are thought to play during regenerative events (20). We also observed an induction of p75NCVR-ir in the same region of the MOB, 8 and 16 weeks after bulbectomy (Table II). This induction suggests that this region of the MOB is sensitive to changes in the numbers of ORN axons traversing this layer to reach the deeper lying glomerular layer. We have previously shown that p75NaVR-ir increases in intensity during the postnatal period (11), a time during which the number of ORN axons found in the neuroepithelium and the number of ORNs projecting to the glomeruli both increase (21, 22). Thus, alterations in the input from the neuroepithelium to the olfactory bulb (either developmentally or lesioned induced) clearly influence p75 NcFR expression in the glomeruli of the olfactory bulb, suggesting that the presence (and/or turnover) of ORN axon terminals is critical to expression of glomerular p75 NCFR. It would also appear that p75 NCFR expression in the olfactory nerve layer (normally at or below the level of detection using the protocol described here) can be induced in this region of the olfactory bulb, as a result of direct (Table I) or indirect (Table II) alterations in ORN turnover. In conclusion, the remarkable capacity of the olfactory system to regenerate fully its peripheral input, despite profound, lesion-induced losses of sensory neurons, should allow critical assessment of the roles neurotrophins and their receptors play in this naturally regenerating system.

References 1. P. C. Maisonpierre, L. Belluscio, S. Squinto, N. Y. Ip, M. E. Furth, R. M. Lindsay, and G. D. Yancoupolis, Science 247, 1146-1151 (1990). 2. M. Chao, Neuron 9, 583-593 (1992). 3. Q. Yan and E. M. Johnson, J. Comp. Neurol. 290, 585-598 (1989).

[6] EXPRESSION OF NEUROTROPHIN RECEPTORS .

.

.

.

.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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P. P. C. Graziadei and G. A. Monti-Graziadei, in "Neuronal Plasticity" (C. Cotman, ed.). Raven Press, New York, 1978. F. L. Margolis, N. Roberts, D. Ferriero, and D. Feldman, Brain Res. 81, 469-83 (1974). J. E. Schwob, K. E. Mieleszko-Szumowsky, and A. A. Stasky, J. Neurosci. 12, 3896-3919 (1992). D. M. Holtzman, Y. Li, L. F. Parada, S. Kinsman, C.-K. Chert, J. S. Valleta, J. Zhou, J. B. Long, and W. C. MoNey, Neuron 9, 465-478 (1992). C. P. Turner and J. R. Perez-Polo, Trans. Am. Soc. Neurochem. 23(1), 141 (1992). Moriizumi, Personal communication, 1994. E. P. Piori and A. C. Cuello, Neuroscience 34, 57-87 (1990). C. P. Turner and J. R. Perez-Polo, Int. J. Dev. Neurosci. 10, 343-359 (1992). C. P. Turner and J. R. Perez-Polo, NeuroReport 8, 1023-1026 (1993). T. Miwa, T. Moriizumi, H. Sakashita, Y. Kimura, T. Donjo, and M. Furukawa, Neurosci. Lett. 155, 96-98 (1993). J. Verhaagen, A. B. Oestreicher, M. Grillo, Y. S. Khew-Goodall, W. H. Gispen, and F. L. Margolis, J. Neurosci. Res. 26, 31-44 (1990). C. P. Turner and J. R. Perez-Polo, Submitted for publication, 1994. H. Vickland, L. E. Westrum, and M. A. Bothwell, J. Neurosci. Abstr. 17(2) (1991). F. Gomez-Pinilla, R. M. Guthrie, M. Leon, and M. Nieto-Sampedro, Dev. Brain Res. 48, 161-165 (1989). R. C. Switzer, J. De Olmos, and L. Heimer, "The Rat Nervous System" (G. Paxinos, ed.). Academic Press, New York, 1985. M. Taniuchi, H. B. Clark, J. B. Schweitzer, and E. M. Johnson, J. Neurosci. 8, 664-681 (1988). A. R. Doucette, J. Comp. Neurol. 285, 514-527 (1989). E. Meisami, Dev. Brain Res. 46, 9-19 (1989). F. L. Margolis, J. Verhaagen, S. Biffo, F. L. Huang, and M. Grillo, "Progress in Brain Research" (W. H. Gispen and A. Routtenberg, eds.), Chap. 8, pp. 97-122. Elsevier, Amsterdam, 1991.

[7]

Basal Forebrain Cholinergic Lesions and Complete Transection of Septal-Hippocampal Pathway Lawrence R. Williams

Introduction The cholinergic neurons in the basal forebrain continue to be important foci of investigations concerning the mechanisms of neuronal plasticity and memory (1, 2). Organized as a comparatively continuous group of cells within the septum, diagonal band of Broca, and nucleus basalis (3, 4), the cholinergic neurons project primarily through the fornix/fimbria to the hippocampus, and through more diffuse pathways to the cerebral cortices (5). These neurons elicit a local sprouting response on target deafferentation, an observed phenomenon termed reactive synaptogenesis that was one of the first to indicate the plastic ability of the adult central nervous system (CNS) (6). Lesions of the basal forebrain cholinergic systems disrupt learning and memory formation in several animal models (7, 8); current hypotheses implicate the cholinergic neurons in at least an attentional requirement for memory formation (1, 9, 10). Of large clinical interest is the consistent observation in Alzheimer's disease of a profound deficit of cholinergic transmitter markers, and a coincident atrophy or death of basal forebrain cholinergic neurons (11, 12). The basal forebrain cholinergic neurons are unique in being the only neurons in the CNS that express the trk A receptor for nerve growth factor (NGF), other than two small, thalamic, noncholinergic nuclei (13). NGF treatment will reverse age-related cholinergic neuronal atrophy, stimulate the activity of cholinergic transmitter synthetic enzymes, enhance acetylcholine release, and improve age-related memory deficits (14-16). Several animal models are being used to study the role of the basal forebrain cholinergic neurons in learning and memory, and to study the molecular mechanisms underlying cholinergic neuronal plasticity (8, 10, 17, 18). Electrolytic lesions of discrete brain areas have been used to destroy specific neuronal groupings to determine the effect of their destruction on a particular behavioral paradigm, and thus elucidate their involvement in the behavior. Similarly, injections of toxic excitatory amino acids, e.g., ibotenic acid, into discrete areas have been intended to destroy, specifically, the cholinergic neurons without killing other neuronal phenotypes or destroying fibers of

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Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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passage. These somally destructive models have also provided very useful information concerning the role of the cholinergic system in learning and memory. However, as the intent of these models is to destroy the neuronal cell body, they are less informative about the mechanisms regulating cholinergic neuronal homeostasis, plasticity, and disease, particularly neuronal atrophy and cholinergic transmitter dysfunction. Axotomy has been used as a method to model the response of the nervous system to injury. The majority of experiments have examined the plastic and regenerative potential of peripherally projecting neurons or centrally projecting sensory ganglionic neurons (19). Within the brain proper, few axonal pathways provide a discrete bundle of axons that can be accurately and precisely transected. The fimbria/fornix bundle from the basal forebrain to the ipsilateral hippocampus is one major exception. Transection of the rat fimbria/fornix and the response of the basal forebrain medial septal neurons to such axotomy was first described by Daitz and Powell in 1954 (20). Using a stereotaxic knife cut and Nissl histochemistry, in the medial septum they observed a rapid, i.e., weeks, disappearance of the large, magnocellular neurons characteristic of the normal medial septum, and concluded that axotomy of this pathway resulted in the retrograde death of the septal neurons projecting through the fimbria/fornix. The advent of acetylcholinesterase (ACHE) histochemistry enabled the association of this enzyme to the septal-hippocampal projection, and, in conjunction with stereotaxic transections of the fimbria/fornix, led to the mapping of the terminations of the septal neurons within the hippocampus (21). AChE histochemistry also enabled the discovery of the reactive synaptogenesis of the septal projection following entorhinal cortex ablation (6). Butcher (22) refined the cholinesterase histochemical procedure and enabled the visualization and identification of the neurons within the basal forebrain. Because of the profound enrichment of AChE within these neurons, they were presumed to be cholinergic neurons. The advent of choline acetyltransferase immunohistochemistry later confirmed this hypothesis (3). Kromer et al. (23) were the first to use cholinergic histochemistry to examine the septum and diagonal band following fimbria/fornix transection. Although their focus was more on regenerative events in the hippocampus, they observed a profound loss of AChE-positive neurons in the medial septum. Gage et al. (24) described the time course of the disappearance of the cholinergic marker and correlated the disappearance with the apparent loss of large Nissl-stained neurons in adjacent sections, confirming and extending the original observations of Daitz and Powell (20) 30 years before. The attention of several laboratories on the use of cholinergic histochemistry in conjunction with the fimbria/fornix axotomy model set the stage for the exciting discovery that, following axotomy, intracerebroventricular infusion

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PARADIGMS OF NEURAL INJURY

of NGF prevented the retrograde disappearance of AChE-positive and large Nissl-positive neurons in the septum and diagonal band (25, 26). The fimbria/fornix axotomy model is continually used to expand on these phenomena in descriptive attempts to elucidate the molecular mechanisms regulating the response of the basal forebrain cholinergic neurons to axotomy, and to describe the potent efficacy of neurotropic proteins on the sustenance and phenotypic stimulation of these neurons (e.g., 27, 28). One particular issue has been whether the transected neurons really die and disappear after axotomy (20, 29), or whether they down-regulate their cholinergic phenotype, atrophy, and hibernate (26). The second hypothesis was first tested by Hagg et al. (30). They found that neurotrophic treatment would restore the cholinergic neurons even when initiation of treatment was substantially delayed beyond the time when the axotomized neurons had lost their cholinergic phenotype and were supposedly surely dead. This led to experiments attempting to label the neurons with dye before axotomy, and to obsevations that the dye quickly disappeared and thus the neurons must quickly die (29). Subsequent experiments (31-33) observed dye administered preaxotomy within axotomized neurons that were still alive in a shrunken atrophied state, supporting the hypothesis of Hagg et al. (30). The current hypothesis held by many is that the axotomized basal forebrain neurons very quickly (within days) downregulate cholinergic trnasmitter function and atrophy (34, 35). Although some of these neurons do die, many of these neurons appear to be able to stay alive indefinitely (11, 30). An axotomy procedure has been described that extends experimental observations to the nucleus basalis-neocortical system. Cuello and co-workers (36, 37) reported that devascularization lesion of the cerebral cortex results in a retrograde atrophy of cholinergic neurons in the nucleus basalis. They have also observed effects of neurotrophic factors on both survival of the nucleus basalis neurons and sprouting of cholinergic axons into viable cortical tissue.

Aspirative Transection of the Fimbria/Fornix The fimbria/fornix of a given hemisphere contains not only the efferent axons from the cholinergic neurons of the medial septum and horizontal limb of the diagonal band of Broca, which project across the full width of the fimbria/ fornix in a topographic organization, but also cholinergic efferents from the contralateral basal forebrain, noncholinergic axons from the basal forebrain, as well as efferents from the hippocampal pyramidal neurons (see Ref. 38 for a more detailed discussion). The axonal contents of the fimbria/fornix and the interpretation of the results of axotomy must thus be considered in

[7]

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109

the design of each experiment. Access to the right and/or left fimbria/fornix for surgical manipulation is confounded by their medial location and the fact that their most medial passage is overlaid by the superior sagittal venous sinus. Several methods have been devised to transect the fimbria/fornix, each with a rationale in support of the experimental purpose. Hefti, for example, has purposely performed a partial transection of the fimbria/fornix using a stereotaxic knife procedure (14, 25, 39). This not only prevents damage to the superior sagittal venous sinus (improving postoperative animal survival), but deliberately spares undamaged projections, the response of which can be studied (e.g., 39). In an attempt to achieve complete fimbria/ fornix transection using a stereotaxic knife procedure, Williams et al. (38) designed a knife, modified from Springer et al. (34), that theoretically could avoid damage to the sagittal sinus, yet reach and trasect the most medial fibers of the fimbria/fornix. However, the dependence on stereotaxic manipulation and the resiliency of brain parenchyma prevented complete transection of the fimbria/fornix. The aspirative transection of the fimbria/fornix under visual inspection is the only method currently identified that can achieve reproducible, complete transections of the septal-hippocampa| pathway without injury to the venous sinus. The aspirative fimbria/fornix transection procedure evolved out of experiments begun in the laboratories of Anders Bj6rklund and Ulf Stenevi (40). A major interest of their group was to determine the growth potential of transplanted nervous tissue into host parenchyma. The aspirative fimbria/ fornix transection not only deafferented the hippocampus, providing an "empty" place for the transplant to sprout, but also provided a cavity within the fimbria/fornix wide enough to contain a piece of embryonic tissue. The procedure described here is for the aspirative transection of the right fimbria fornix. The technique can be modified appropriately for left unilateral or bilateral aspirations (41).

Materials

Recommended anesthesia is a mixture (4 ml/kg) of ketamine (25 mg/ml), rompun (1.3 mg/ml), and acepromazine (0.25 mg/ml) Animal clippers Microsurgical dissecting scope Stereotaxic device for skull stability Squeeze bottle containing 70% (v/v) ethanol Beaker of 70% (v/v) ethanol Scalpel with No. 11 blade Scalp retractors, e.g., two Jones 2-inch towel clamps

110

PARADIGMS OF NEURAL INJURY

2-inch • 2-inch gauze, preferably Versalon all-purpose sponges Squeeze bottle containing 7% (v/v) hydrogen peroxide Dental or hobby drill with #6 or #8 drill bit Fine jewelers forceps 23-Gauge needle 3Fr (1-mm) Baron suction tube connected to vacuum flask with either house vacuum or a vacuum pump; have extras available Gelfoam surgical foam cut into small pieces and soaked in sterile saline Wound clips or suture

Surgical Procedure 1. Sterilize all surgical instruments and gauze sponges. 2. Anesthetize a rat with a subcutaneous or intramuscular injection of the ketamine mixture, 4 ml/kg. Clip the hair on the head with animal clippers. 3. Mount the rat in the stereotaxic device with the head of the animal directed away from you. Visibility of the surgical site is greatly facilitated if the nose bar is elevated to its highest position, i.e., 6 mm above horizontal. As experience is gained, the surgeon can perform the fimbria/fornix aspiration with the nose bar in a more stereotaxically correct position, for example, if an intracerebroventricular cannula is to be implanted (42). 4. Drench the scalp with ethanol, taking care to keep the solution away from the eyes of the rat. The ethanol keeps the dander and fur dust to a minimum as well as providing an antiseptic treatment to the surgical site. 5. With the scalpel, make a skin incision from between the eyes to the nape of the neck, and loosen the periosteum away from the midline by scraping with the scalpel blade. 6. Retract the scalp to left and right with the towel clips, and wipe the periosteum away with a gauze square. Cover the skull with a small squirt of hydrogen peroxide (it will bubble profusely on contact with red blood cell catalase), and wipe the skull clean with the sterile gauze. The hydrogen peroxide step is preferred because it acts to completely clean the skull, exaggerating the visibility of the skull bone sutures, to cauterize skull blood vessels and reduce skull bleeding, and to provide an antiseptic treatment to the surgical site. 7. The next objective is to cut an approximately 3-mm x 3-mm window through the rat skull using the coronal suture and the sagittal suture as the anterior and medial boundaries, respectively. Best results are obtained by using a fresh, sharp drill bit for each procedure. Begin by grinding parallel to the sagittal suture, just to the right of the suture. As the bone is eroded, the venous blue color of the superior sagittal venous sinus can be seen

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through the thinned bone (Fig. 1, see color plate). Adjust the position of the drilling lateral to the sinus to avoid injury, and drill completely through the bone as close to the sinus as possible without damaging it or the underlying dura mater. Then draw the remaining boundaries of the square, drilling along and over the coronal suture and closing the square (Fig. 2, see color plate). Carefully erode the bone filling the square down to a very thin transparent pane of bone covering the dura mater. This final sheet of bone is removed with the fine forceps, and all remnants are cleared up to the edge of the square (Fig. 3, see color plate). If a bilateral transection is the objective, two windows should be drilled, with a median sparing of the skull over the venous sinus. 8. Take the 23-gauge needle and tear an • shape into the dura. This should be started in one of the medial corners, again being very careful to insert the needle immediately lateral to the venous sinus so as to avoid puncture. Then move the needle diagonally across to the opposite corner (do not be concerned about damage to the underlying cortex). Then insert the needle at the other medial corner of the square, immediately adjacent to the sinus, and tear to the middle. Complete the • shape by inserting the needle at the remaining corner, and again, move to the middle. 9. Sterilize the suction tube by sucking an aliquot of ethanol out of the beaker. Use the suction to reflect each flap of dura to the sides, exposing the underlying cortex as fully as possible (Fig. 4, see color plate). The degree of vacuum can be controlled at the valve/pump and with the finger hole on the suction tube; the correct settings will be achieved empirically. It is important to minimize or prohibit the accumulation of blood clots within the suction tube, i.e., clogging will disable use of the tube; thus, have extra tubes available. Clogging can be minimized by frequent flushing of the tube with ethanol. Clogged tubes can be cleaned using ultrasound and careful, diligent reaming using a 1-mm wire, one of which comes with the new suction tube. 10. The next objective is to aspirate the overlying cerebral cortex and corpus callosum to enter the third ventricle and expose the fimbria/fornix. Visibility of structures is always impaired by the inevitable bleeding induced by aspiration of cortex. However, with the agility acquired by practice, the blood is aspirated with the cortex sufficiently to see what you are doing. Begin with a moderately paced sweep diagonally across the window from 2 to 8 o'clock. As the cortex is removed, continue deeper to the corpus callosum, which has a whitish, somewhat mottled, pin-holed appearance (Fig. 5, see color plate). Aspirate through the corpus callosum to expose the fimbria/ fornix (Fig. 6, see color plate). 11. Continue the diagonal sweep, which is essentially perpendicular to the axis of the fimbria/fornix, to aspirate across the fimbria/fornix. The

112

PARADIGMSOF NEURAL INJURY lateral edge of the bundle has distinctive white matter (Fig. 7, see color plate). Assure complete transection by aspirating through the final threads of this matter (Fig. 8, see color plate). The medial edge of the bundle is somewhat blinded by the overlying venous sinus. Complete transection of the medial edge is assured when pial vessels are observed to be pulled into the suction tube (Fig. 9, see color plate). Be careful not to overdo the medial aspiration to prohibit damage to the opposite hemisphere. Of course, if a bilateral transection is the objective, complete transection is assured if you see the suction tube in the contralateral window. 12. The cavity is now filled with saline-soaked Gelfoam, and the excess fluid is aspirated with the suction tube. The skin wound is closed with sutures or wound clips.

Acknowledgments I thank Dr. Anders Bj6rklund and Dr. Silvio Varon for making possible my visit to Lund, Sweden. I also thank Dr. Fred Gage for demonstrating the technique of fimbria/ fornix aspiration during that visit.

FIG. 1 A median hole is drilled through the skull just posterior to bregma (arrowhead) to identify and avoid the superior sagittal sinus (arrow). Bar: 1 mm. FIG. 2 A square (approximately 3 mm) is drilled extending across the coronal suture. FIG. 3 The skull bone is removed to the edges of the square. FIG. 4 An X-shaped incision is made through the dura and the edges (arrowheads) reflected by aspiration. FIG. 5 A diagonal aspiration of tissue is made through the cortex gray matter to expose the mottled-appearing white matter of the corpus callosum. FIc. 6 Aspiration through the corpus callosum exposes the lateral ventricle and the white matter of the fimbria/fornix. The arrowhead marks an inadvertent nick exposing the gray matter of the head of the hippocampus. Note the lateral vasculature on the floor of the ventricle and the most lateral location of the striatum. FIG. 7 Aspiration through the fimbria/fornix exposes the gray matter of the thalamus. The most lateral extent of the fimbria/fornix remains (arrowhead). FIG. 8 Complete lateral transection is assured by aspiration through the most lateral threads of the fimbria (arrowhead). FIG. 9 Assure complete transection of the medial extent of the fimbria/fornix by aspirating medial parenchyma until the median vasculature (arrowheads) becomes apparent. With care and practice, aspiration of the contralateral fimbria/fornix can be avoided.

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References

10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22.

23. 24. 25. 26. 27.

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[8]

Neurochemical Lesions" Tools for Functional Assessment of Serotonin Neuronal Systems J o a n M. L a k o s k i , B. J a n e K e c k , a n d A s h i s h D u g a r

Introduction Elucidation of the functional roles of a neurotransmitter system, including the serotonergic 5-hydroxytryptamine (5-HT) neuronal system, relies on a strategy of evaluating changes evoked in central nervous system (CNS) function following precise electrical or pharmacological stimulation. Alternatively, the removal of a given transmitter substance, whether by placement of electrolytic lesions, neurochemical depletion techniques, or development of transgenic animals with selective genetic deficiencies for the neurotransmitter under evaluation, is another frequently used paradigm for functional evaluation of a neurotransmitter system. In addressing changes in a given neurotransmitter system that are found during processes of injury or as a consequence of normal development and aging, the utilization of selective neurochemical degeneration techniques continues to provide a highly relevant approach. This chapter will focus on the application of selective neurochemical lesions of the serotonergic system, as provided by the discrete application of 5,7-dihydroxytryptamine (5,7-DHT), to aid in our understanding of nervous system function throughout the life span. The compensatory processes that occur from the induction of neurochemical degeneration, including plasticity and regeneration responses, will be reviewed in the context of serotonergic function with respect to both normal and pathological aging. The indole derivative 5,7-DHT selectively destroys serotonergic neurons following central administration to laboratory animals (1, 2). The rapid accumulation of this neurotoxin in the target neurons and its ability to inhibit 5-HT reuptake (2) point to a selectivity based on high-affinity uptake processes located on serotonergic neurons. The precise mechanism of the neurotoxicity resultant from 5,7-DHT intraneuronal accumulation is not yet known. However, the formation of a superoxide radical anion and cytotoxic hydroxyl radicals has been implicated in the mechanism of neuronal damage Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and cell death (3). Possible effects on intracellular serotonin-mediated signal transduction cascade processes have yet to be evaluated. The functional consequences of 5,7-DHT exposure were first evaluated in light of neurochemical and behavioral supersensitivity of the serotonergic system that developed over time following administration of the neurotoxin. Nelson et al. (4) demonstrated that intracerebral application of 5,7-DHT resulted in an 80% decrease in the level of hippocampal 5-HT within 4 days and a compensatory increase in the density of hippocampal [3H]5-HT highaffinity binding sites by 8-18 days. Potentiation of behavioral responses to administration of the serotonin precursor 5-hydroxytryptophan was also observed in adult rats treated intracisternally with 5,7-DHT during development (5). Cellular electrophysiological recording studies of serotonergic function in the amygdala (6, 7) similarly revealed a supersensitivity to 5-HTmediated responses following pretreatment with this neurotoxin. However, these studies also revealed site-specific differences in the development of compensatory changes to the loss of serotonergic innervation (7). Changes in receptor density and sensitivity, as well as 5-HT levels, also correlate with enhanced levels of tryptophan hydroxylase (TH) activity, suggesting that induction of this enzyme is a compensatory response from 5-HT nerve terminals spared by the neurochemical lesion (8). These observations have been extended to the molecular level and demonstrate an 85% decrease in the level of 5-HT~A receptor mRNA in a brain region-selective manner (9) and significant increases in tryptophan hydroxylase mRNA of serotonergic dorsal raphe neurons that survived the neurochemical insult (10). These observations of compensatory responses of the serotonergic system following a 5,7-DHT neurotoxic insult have prompted our laboratory to utilize neurochemical lesions to investigate the functional role of this neurotransmitter system with aging. The present chapter summarizes this methodological approach as applied to clarification of adaptive or plasticity changes in neuronal cell function that may be induced in midbrain and brainstem serotonin-containing raphe nuclei as a function of age. Utilizing a model of intraventricular (i.v.t.) administration of 5,7-DHT, cellular electrophysiological responses to serotonergic agents are evaluated in postsynaptic terminal projection regions of these nuclei. Using an appropriate model of aging, the female Fischer 344 rat of 3 to 20 months of age, the ability of the aged nervous system to adapt and demonstrate compensatory changes in response to the neurochemical stressor is being evaluated in the hippocampal pyramidal neurons, which are extensively innervated by the raphe nuclei. Similarly, a selective denervation of the serotonergic input to the hippocampus via placement of 5,7-DHT in the ascending indoleamine-containing pathway at the level of the fimbria fornix and cingulum bundle (11, 12) provides a model for precise manipulation of hippocampal function. This cytotoxic lesion strat-

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egy, as applied to age-dependent changes in hippocampal function, can serve to establish the role(s) of the serotonergic neuronal system in both normal and pathological nervous system function.

Serotonergic Lesion Strategies

Overview of Applications Neurochemical lesions of the serotonergic neuronal system by intraventricular, intracisternal, or intracerebral application of 5,7-DHT have been utilized to evaluate functional changes in the cellular and molecular activity of serotonin and its receptors in identified brain regions (2, 4-6, 8-11). This technique has been successfully applied to address critical questions of the role of serotonergic systems in (a) nervous system development (13), (b) regulation of neuroendocrine function (14-16), (c) degeneration induced by abused substances (17), (d) differential contribution of multiple serotonin receptor subtypes (18), as well as (e) brain region-selective regulation of serotonergic function (11, 19, 20). Selective neurochemical denervation of 5-HT neuronal systems has also been utilized to elucidate functional interactions with other neurotransmitter systems, including muscarinic receptor function (21).

Experimental Design The successful induction of a selective neurotoxic lesion requires attention to several methodological concerns. First, the agent selected for induction of the selective degeneration should meet the criteria of selectivity for the transmitter system under investigation. Second, the agent should be delivered to the relevant site(s) of action in a biologically active form. Finally, the critical issues of pharmacokinetics for the cytotoxic agent need to be addressed with respect to both the rate and volume of drug infused as well as the time chosen for evaluation of a functional index following the lesion. The selectivity of 5,7-DHT for targeted serotonergic neurons involves utilization of reuptake or transporter sites for 5-HT. Animals must be pretreated with a reuptake inhibitor with high selectivity for both noradrenergic (NE) and dopaminergic (DA) transporters in order to avoid nonspecific degeneration of other biogenic amine-containing neuronal systems (2, 3); desipramine (10 or 25 mg/kg, i.p.) is often administered 30-45 minutes prior to the infusion of the neurotoxin in order to fully occupy NE and DA transporter sites (22, 23). Additionally, care must be taken to avoid oxidation-induced inactivation of 5,7-DHT by preparing solutions in 0.01% ascorbic acid and

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keeping solutions ice cold and shielded from light until use. Our laboratory makes fresh stock solutions of this agent daily and stores it on ice in a foilwrapped container until the microinjection syringe is filled; the syringe is additionally wrapped in foil to retard any light-induced oxidation. The site chosen for administration of the drug will affect the choice of dose and volume of 5,7-DHT to be infused. For example, should a relatively nonselective depletion of serotonin be desired, i.v.t, administration is the route of choice; administration of drugs into the lateral ventricles can typically be executed rapidly and with a high degree of accuracy. The dose of 5,7-DHT infused to produce >90% depletion of 5-HT can range as high as 80-150 /xg administered in a 10-/xl volume per ventricle. Care should be taken to provide a constant rate of drug infusion in order to avoid mechanical damage to the ventricles or physical damage to the subcellular structures of the ependymal cell lining. Should a direct placement of 5,7-DHT into a component of the serotonergic system be desired (raphe nuclei, see Ref. 15; fimbria fornix/cingulum bundle, see Refs. 11, 12, and 22), the volume of drug to be infused must be significantly reduced to the 0.5 /xl level to avoid mechanical artifacts produced by the infusion. As the intracerebral injection of the neurotoxin is placed precisely in the target region, less drug is typically needed to produce the required depletion of 5-HT in the target region. It is also important to recall that the nature of the neurochemical insult is a dynamic process. Once 5,7-DHT is taken into the serotonergic neuron and degeneration ensues, a cascade of events follows, beginning with initial changes in 5-HT and TH levels (hours, days) followed by a continuing effect that lasts for several weeks; the extended effect is related to the up-regulation of postsynaptic receptor function (4, 10, 16, 18-20). Clear variations in the behavioral effects produced by 5,7-DHT administration are seen with respect to the time at which the testing ensues following placement of the lesion (24). Detailed protocols are now provided for both intraventricular administration of 5,7-DHT as well as its placement in the fimbria fornix/cingulum bundle.

Protocol 1: Intraventricular 5,7-Dihydroxytryptamine Lesions The methods described below are in reference to virgin female Fischer 344 rats ranging from 2 to 18 months of age (body weight range 140-280 g). 1. Each rat is anesthetized using sodium pentobarbital [40 mg/kg, intraperitoneally (i.p.)] and centered onto a small-animal stereotaxic unit using dull ear bars (in order to protect the eardrums from injury). Body temperature is maintained throughout the stereotaxic surgery by placement of a heating pad underneath the rat (Deltaphase Isothermal Pad, Model 39DP; Braintree

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Scientific, Inc., Braintree, MA) or use of a thermostatically controlled heating unit (Fintronics, Orange, CT). 2. Nomifensine (15 mg/kg, i.p.; Research Biochemicals International, Natick, MA) is administered 30 minutes prior to administration of the neurochemical lesioning agent or vehicle. Pretreatment with nomifensine or other reuptake inhibitors selective for both noradrenergic and dopaminergic presynaptic transporters provides an adequate protection of these catecholamine-containing neuronal systems. 3. The skin over the skull is carefully retracted to expose the skull. Coordinates for bregma are 0 mm anterior/posterior, 0 mm lateral/medial (25). A Hamilton syringe is used for bilateral infusion of the neurochemical agent or vehicle into the lateral ventricles; a 10-/A syringe (Model #84877; Hamilton Co., Reno, NV) with a 28-gauge needle is placed in a syringe holder that is directly attached to the stereotaxic arm. Following determination of coordinates 1.5 mm posterior and 1.3 mm lateral (left and right of midline), a handheld drill is used to expose the dura. Care is taken to minimize the size of the hole (typically 2 mm). Any remaining bone fragments may be removed with a dental pick. 4. A 7.5 ~g/~l solution of 5,7-dihydroxytryptamine (Research Biochemical Int.) is freshly prepared daily in vehicle (saline in 0.01% ascorbic acid); the preparation is wrapped in foil to minimize exposure to light and kept on ice. Once the syringe is filled with the 5,7-DHT solution, the syringe is lowered 3.0 mm ventral from the surface of the brain. The drug solution is infused in the right hemisphere over a 4-minute period at a rate of 2.5 ~l/min (total volume of 10 ~l/side); the syringe is maintained in position an additional 5 minutes to limit nonspecific drug diffusion. This procedure is repeated in the left hemisphere with a total of 150 ~g of 5,7-DHT delivered to each animal. The same procedure is used for vehicle-treated animals using identical placement, volume, and rate of vehicle infusion. The surgical site is sutured and animals are monitored during recovery (from anesthesia) before being returned to their home cages. 5. Animal body weights are monitored on a daily basis following surgery. Both lesioned and control groups are used in cellular electrophysiological studies at 21 days postlesion.

Experimental Controls In developing an experimental protocol for implementation of selective degeneration of serotonergic neurons, proper control groups should be included to facilitate pairwise comparisons with the treatment groups. For example, the control group for the above outlined infusion of 5,7-DHT into the lateral ventricles includes a treatment group receiving the identical anesthesia, pretreatment with nomifensine (NE and DA reuptake inhibitor) as well as vehicle

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infusion (identical with respect to rate and volume) into the target region. When conducting age-related comparisons of central nervous system function, control groups should be used for each relevant age group.

Protocol 2: Intracerebral 5,7-Dihydroxytryptamine Lesions The following procedure provides a discrete neurochemical lesion of serotonin-containing input to the hippocampus. The neurotoxin is placed at the site of the ascending serotonergic input to the hippocampus at the level of the fimbria fornix and cingulum bundle (FF/CB; 11, 12, 22). Note: although this protocol is virtually identical to the methods for bilateral intraventricular administration of 5,7-DHT (see above; Protocol 1), a strategy of multiple injections at multiple sites has been incorporated into the experimental design. The methods described below are in reference to virgin female Fischer 344 rats ranging from 2 to 18 months of age (body weight range, 140-280 g). 1. Anesthesia and maintenance of body temperature throughout protocol are as described above under Protocol 1, step 1. 2. Pretreatment with nomifensine (15 mg/kg, i.p., 30 minutes prior to injection of neurotoxin) is as described above under Protocol 1, step 2. 3. The skin over the skull is carefully retracted to expose the skull. Stereotaxic coordinates are obtained as described above under Protocol 1, step 3. The syringe is placed at a 15~ angle toward the midline and the tip aligned with bregma (25). The bone is removed to exposure the dura at coordinates 1.3 mm medial (right and left sides) and 1.0 mm posterior to bregma as described above. 4. A 10/xg//zl solution of 5,7-dihydroxytryptamine is prepared fresh daily in the vehicle (saline with 0.01% ascorbic acid) and is kept on ice and wrapped in foil to minimize exposure to light. Once the syringe is filled with the 5,7DHT solution, the syringe is lowered to the fimbria fornix at 4.5 mm vertical and the drug is infused over a period of 1 minute at a rate of 0.25 /zl/30 seconds (total volume of 0.5/xl); the syringe is maintained in position for an additional 3 minutes to limit nonspecific drug diffusion. Along this injection track, the needle is then raised so that the needle tip is placed in the right cingulum bundle at 2.5 mm ventral from the surface. The drug is then infused over a period of 1 minute at a rate of 0.25/zl/30 seconds (total volume of 0.5/zl); the syringe is maintained in place for an additional 3 minutes. The contralateral hemisphere is then treated in the same manner with an infusion placed at 4.5 mm and 2.5 mm vertical in the fimbria fornix and cingulum bundle, respectively, with a total of 20/xg of 5,7-DHT being delivered to each animal. The procedure is repeated for the vehicle-treated groups with

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identical placement, volume, and rate of vehicle infusion. The surgical site is sutured and animals are monitored during recovery. 5. Animal body weights are determined on a daily basis following surgery. Lesion and control groups are used in cellular electrophysiological studies at 21 days postlesion.

Functional Assessment of Serotonergic Denervation At the time point chosen for evaluation of the functional impact of denervation of serotonergic input to the hippocampus or other target region, several tools are available to confirm the selectivity and characteristics of the lesion. First, whenever possible, a histological evaluation of the site of 5,7-DHT injection (i. v. t or FF/CB) should be ascertained using 50-~m sections counterstained with a cell-specific dye. Histological verification of the correct placement of the lesion is critical in establishing the validity of the technique and to ascertain correct placement of the neurotoxin. Second, although not possible with every experiment (for example, with cellular physiological recording studies), expected depletion of 5-HT or tryptophan hydroxylase levels should be verified by high-performance liquid chromatography determination. Should a biochemical assessment not be feasible, a change in behavior, physiological sensitivity, or receptor subtype expression should be evident as compared to controls. In studies of cellular physiological responses of hippocampal pyramidal neurons recorded in 2- and 17-month-old rats, age-dependent changes in neuronal sensitivity to microiontophoretic drug application of 5-HT and related agonists are evident. However, comparison of age-matched controls to 5,7-DHT treatment groups are required to confirm the proper placement of the neurochemical insult.

Applications and Caveats The application of selective neurochemical degeneration of the serotonergic neuronal system continues to be an important approach useful in the identification of compensatory processes found with neuronal injury or as a consequence of normal development and aging. We have applied this cytotoxic lesion strategy to address fundamental questions of physiological and pharmacological changes in hippocampal function with aging (26). Indeed, this approach can successfully integrate cellular electrophysiological studies of spontaneously active CA1 and CA3 hippocampal subfield neurons with their adaptive responses to modulatory factors such as nerve growth factor. As clearly established for the developing serotonergic system by Sachs and

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PARADIGMS OF NEURAL INJURY Jonsson (27), multiple compensatory mechanisms emerge in response to alteration of 5-HT neuronal development following administration of 5,7DHT during the neonatal period. As applied to models of aging, a role for serotonin in mediating age-dependent changes in the activity of dietarily obese rodents has been identified using this lesion technique (28). Although the morphological changes in serotonergic neurons resulting from 5,7-DHT lesions appear comparable in young and old rats (29), the complex processes of reinnervation that may result in axonal regeneration and sprouting (30) have yet to be investigated fully in aged animals. Certainly the intriguing observation of an enhancement of spatial discrimination learning demonstrated in rats following 5,7-DHT lesions in the FF/CB (31) leads to the question of possible age-dependent changes in the neuromodulatory role of serotonergic systems in the hippocampus for learning and memory processes. As our studies of the physiological and pharmacological aging of serotonergic neuronal function in the hippocampus progress (32), the continued use of neurochemical lesion techniques will be valuable tools for functional assessment of this transmitter system.

Acknowledgments We thank Jane E. Smith, Lynn Maines, Dr. Melvin S. Billingsley, and Dr. Louise E. LeDuc for careful reading of this text. This research effort has been supported by PO1 AG10514 (Publication No. 25C; J.M.L.) and KO4 AG00450 (J.M.L.) from the National Institute on Aging.

References 1. H. G. Baumgarten and L. Lachenmeyer, Z. Zellforsch 135, 399 (1972). 2. H. G. Baumgarten, A. Bjorklund, L. Lachenmeyer, and A. Nobin, Acta Physiol. Scan. (Suppl.)391, 1 (1973). 3. T. Tabatabaie, R. N. Goyal, C. LeRoy Blank, and G. Dryhurst, J. Med. Chem. 36, 229 (1993). 4. D. L. Nelson, A. Herber, S. Bourgoin, S., J. Glowinski, and M. Hamon, Mol. Pharmacol. 14, 983 (1979). 5. G. R. Breese, R. A. Vogel, C. M. Kuhn, R. B. Mailman, R. A. Mueller, and S. M. Schanberg, Brain Res. 155, 263 (1978). 6. R. Y. Wang and G. K. Aghajanian, Brain Res. 120, 85 (1977). 7. C. de Montigny, R. Y. Wang, T. A. Reader, and G. K. Aghajanian, Brain Res. 200, 363 (1980). 8. M. K. Stachowiak, E. M. Stricker, J. H. Jacoby, and M. J. Zigmond, Biochem. Pharmacol. 35, 1241 (1986).

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10. 11. 12. 13. 14.

15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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C. Bendotti, A. Servadio, G. Forloni, N. Angeretti, and R. Samanin, Mol. Brain Res. 8, 343 (1990). M. C. Miquel, E. Doucet, M. Raid, J. Adrein, D. Verg6, and M. Hamon, Mol. Brain Res. 14, 357 (1992). M. Quik and E. Azmitia, Eur. J. Pharmacol. 90, 377 (1983). F. C. Zhou and E. C. Azmitia, Brain Res. 373, 337 (1986). E. Azmitia and P. M. Whitaker-Azmitia, J. Clin. Psychiatry 52(12), 4 (1991). L. D. Van de Kar, M. Carnes, R. J. Maslowski, A. M. Bonadonna, P. A. Rittenhouse, K. Kunimoto, R. A. Piechowski, and C. L. Bethea, J. Pharmacol. Exp. Therap. 251, 428 (1989). A.-L. Barofsky, J. Taylor, Y. Tizabi, R. Kumar, and K. Jones-Quartey, Endocrinology 113, 1884 (1983). M. Frankfurt, C. R. McKittrick, S. D. Mendelson, and B. S. McEwen, Neuroendocrinology 59, 245 (1994). G. Battaglia, in "Serotonin: From Cell Biology to Pharmacology and Therapeutics" (R. Paoletti, P. M. Vanhoutte, N. Brunello, and F. M. Maggi, eds.), p. 651. Kluwer, Dordrecht, The Netherlands, 1990. D. Verg6, M. Marcinkiewicz, A. Patey, S. E1 Mestikawy, H. Gozlan, and M. Hamon, J. Neurosci. 6, 3474 (1986). C. Manrique, A. M. Franqois-Bellan, L. Segu, D. Becquet, M. H6ry, M. Faudon, and F. H6ry, Brain Res. 663, 93 (1994). J. Sawynok and A. Reid, Eur. J. Pharmacol. 264, 249 (1994). R. Alonso and R. Soubrie, Synapse 8, 30 (1991). J. H. Williams and E. C. Azmitia, Brain Res. 207, 95 (1981). M. R. PranzateUi and S. R. Snodgrass, Psychopharmacology 89, 449 (1986). P. F. Gately, D. S. Segal, and M. A. Geyer, Behav. Neural Biol. 45, 31 (1986). P. Paxinos and C. Watson, "The Rat Brain in Stereotaxic Coordinates," 2nd Ed. Academic Press, Orlando, 1986. J. R. Perez-Polo, A. Dugar, G. Taglialatela, M. A. Micci, and J. M. Lakoski, Soc. Neurosci. Abstr. 21, 1373 (1995). C. Sachs and G. Jonsson, Med. Biol. 53, 156 (1975). K. T. Borer, R. Bonna, and M. Kielb, Pharmacol. Biochem. Behav. 31, 885 (1989). M. G. van Luijtelaar, H. W. M. Steinbusch, and J. A. Tonnaer, Exp. Brain Res. 78, 81 (1989). O. Bosler, G. Vuillian-Cacciuttolo, and H. Saidi, Neurosci. Lett. 143, 159 (1992). H. J. Altman, H. J. Normile, M. P. Galloway, A. Ramirez, and E. C. Azmitia, Brain Res. 518, 61 (1990). J. M. Lakoski, Neurobiol. Aging 4, 519 (1994).

[9]

Cerebral Glucose/Energy Metabolism" Valid Techniques in Humans and Animals Siegfried Hoyer

Introduction The healthy, mature, nonstarved mammalian brain uses glucose only by oxidizing it to obtain energy in the form of ATP, which is necessary to maintain cellular function. A study of the metabolic pathway of glucose and the energy pool in the brain is of interest in evaluating both normal function and pathological states. The normal supply of the substrates oxygen and glucose to the brain is guaranteed by the blood flow in the carotid and vertebral arteries. Cerebral blood flow (CBF) and cerebral oxidative metabolism have been found to be tightly linked functionally. Therefore, it seems necessary also to touch on the control mechanisms of CBF. The mammalian brain is a multiorgan organ; that is to say, it has a heterogeneous structure and consists of functionally different regions. This holds true for the gray matter in the cerebral cortex and for the diverse subcortical nuclei. Cerebral gray and white matter have to be regarded as distinct from one another in functional terms. It is as yet not known whether the white matter is also characterized by a regional diversity similar, in metabolic terms, to that of the gray matter.

Control Mechanisms of Cerebral Blood Flow There are two physiological parameters that control CBF: (1) the mean arterial blood pressure (MABP) and the cerebral perfusion pressure (CPP) and (2) the partition pressure of carbon dioxide in arterial blood (paCO2). Under normal conditions, MABP is around 100 mmHg in humans and in mammals (monkey, dog, cat, rat) commonly used for experimental purposes. Normal CBF is maintained over an MABP range of around 50 to around 150 mmHg (autoregulation of CBF). However, the cerebral metabolic rates of glucose, lactate, and CO2 change under moderate arterial hypotension (1). The relationship between MABP and CBF is valid only when the intracranial pressure (ICP) is normal (close to zero). When ICP is increased as a result

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of brain edema or other space-occupying lesions, the relationship between CPP and CBF becomes valid (CPP = MABP - ICP) (2). The physiological, normocapnic paCO2 is around 40 mmHg. Hypocapnia induces a fall and hypercapnia induces an increase in CBF in a rather linear manner between paCO 2 of around 20 and 80 mmHg. In this range, the cerebral metabolic rates of oxygen, CO2, glucose, and lactate are kept constant by an increase in the extraction rates of the substrates and vice versa (3).

Physiological Steady State In studies designed to investigate parameters of the cerebral glucose and oxidative energy metabolism, MABP and paCO 2 have to be maintained in physiological ranges. MABP can be monitored either by means ofa sphygmomanometer (preferred method for humans) or via an arterial catheter and pressure transducers (preferred method for experimental animals). Although the arterial partition pressures of oxygen (paO2) do not control CBF, a fall from normoxemic (around 100 mmHg) to hypoxemic values (around 80 mmHg or lower) has been shown to induce changes in cerebral glucose/energy metabolism (4-6). Another parameter that has to be kept normal is the arterial glucose concentration. Arterial hypoglycemia induces a more marked reduction in cerebral glucose consumption than in oxygen utilization (7), and drastic changes in energy metabolism in the cerebral cortex (8). Besides the arterial blood constituents pCO2, PO2, and glucose, further parameters are of importance because of their capability to vary CBF and thus, secondarily, cerebral oxidative metabolism: hematocrit, hemoglobin, and pH. To complete the steady-state parameters, body temperature (37~ has to be recorded either rectally (humans, experimental animals) or intraperitoneally (experimental animals). It is generally accepted that the maintenance of the above steady-state condition over an experimental period of at least 15 minutes guarantees the comparability of different studies. However, in human disease states, or in defined pathophysiological experimental conditions, both quality and quantity of the variations in the respective steady-state parameter will have to be strictly maintained.

Studies in Humans Various techniques are available for the investigation of global and local cerebral utilization rates of oxygen and glucose.

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Kety-Schmidt Technique Global measurements of substrate consumption of any organ are based on the Fick principle: the amount of a substrate that is used by that organ is represented by the difference in the concentrations measured in the arterial blood being supplied to it and in the venous blood released from its, i.e., the arteriovenous difference. However, there are conditions that have to be fulfilled for this principle to hold: the arterial and venous concentrations of the substrates and the blood flow through the organ have to be constant during the period of measurement (steady-state condition; see above), and one main vein only should drain the blood from the organ. The consumption or release of a substrate can then be calculated from the arteriovenous substrate difference and blood flow (9). It can be assumed that any substrate concentration is identical in the blood of all larger arterial vessels of the body. Therefore, there is no need to sample arterial blood from the carotid artery; it can be more conveniently sampled from the femoral artery, for example. The main venous drainage from the brain takes place via the internal jugular vein. However, this vein is joined by the facial, lingual, pharyngeal, and thyroid veins, so that the extracerebral contamination is considerable. Proximal to these veins, the superior bulb of the internal jugular vein is situated in the posterior part of the jugular foramen. Mixed cerebral venous blood, i.e., blood from both cerebral hemispheres, the brain stem, and the cerebellum, with only minor extracerebral contamination, can be collected from the superior bulb of the internal jugular vein by direct puncture. A modification (10) of the original Kety-Schmidt technique (9) facilitates blood sampling in that integral concentrations of blood substrates are collected over 10 minutes by motor syringes extracting 1 ml/ minute of blood, to avoid extracerebral contamination at the venous site in particular. With respect to the measurement of global CBF it must be borne in mind that the tracer used to calculate blood flow must be nontoxic, must not produce any side effects, must not be varied metabolically or itself induce variations in metabolism, should be insoluble or only slightly soluble in blood and adipose tissue, should diffuse rapidly into the brain, should be easily analyzed, should have a known brain-blood partition coefficient, and should be economical in use. Some candidates largely fulfill these prerequisites" nitrous oxide, argon, and both labeled krypton and xenon. Nitrous oxide in low concentration (15-20% in air) is most frequently used, although the calculation of CBF from a 10-minute saturation period is overestimated by 10-15%. This error can be minimized by prolonging the saturation period to 14 minutes and extrapolating to infinity (11). Although this Kety-Schmidt technique, which allows global measurements only, has some limitations, it

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has the advantage of providing data on both CBF and the utilization rates of substrates of interest. Several very sensitive analytical techniques are available for the measurement of cerebral arteriovenous substrate differences, such as oxygen and CO2 (gas chromatography), glucose, lactate, pyruvate, and ketone bodies (spectrophotometry), amino acids (high-performance liquid chromatography), free fatty acids (gas chromatography), etc. Uptake or release of these substrates and the shifts in the pattern of different metabolic pathways can be followed. Furthermore, the duration of one study is around 30-40 minutes from the puncture of the vessels to removal of the needles. It may be easier to maintain a steady state over this period than throughout longer lasting investigations (see following), which makes this method more practical for use in mentally deranged people. Although this technique is invasive, it can be applied almost daily if necessary without causing excessive stress to the patient. Finally, the costs are quite low.

Positron E m i s s i o n T o m o g r a p h y T e c h n i q u e s Positron emission tomography (PET) techniques are based on a modified Fick principle (see previous) in which radioactive tracers are used to determine the cerebral utilization rates of substrates. The direct measurement of radioactivity in the brain tissue replaces the determination of the substrate concentration in the mixed venous blood of the superior bulb of the internal jugular vein. Techniques are developed to investigate the utilization rates of glucose and oxygen. Tracers used are 18F-labeled a-fluoro-deoxyglucose (FDG), [llC]methyl-D-glucose, and 150, The FDG method is the one most commonly used in studies of regional glucose utilization by means of PET. This method is based on the principles outlined by Sokoloff et al. (12) and Reivich et al. (13). Deoxyglucose uses the same carrier at the blood-brain barrier for transport from arterial blood into the brain tissue. Here it is phosphorylated by hexokinase but not converted further into fructose 6-phosphate. Deoxyglucose 6-phosphate is trapped in the brain, and its resulting accumulation is the marker to be quantified by PET. Deoxyglucose 6-phosphate is not a substrate for glucose6-phosphate dehydrogenase, but can be hydrolyzed by glucose-6-phosphatase. For calculation of the local metabolic rate of glucose, the time interval between injection and PET measurement, the plasma concentrations of glucose and FDG during the study, the rate constants of the transmembranous carrier system, of the FDG phosphorylation step and the "lumped constant" must all be known. For studies under normal conditions, the rate constants and the lumped constant can be derived from animal experiments. However,

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under pathologic conditions, individual determination of the rate constants by dynamic PET scanning is thought to be more accurate (14, 15). The determination of local cerebral oxygen consumption is more crucial because of the very short half-life of oxygen-15, which is 2.1 minutes. Oxygen- 15 is inhaled and is almost entirely metabolized to labeled water (H2150). Three emission scans have to be carried out during the inhalation period for quantitative measurement of the metabolic rate of oxygen (16, 17). The immobility of the subject's head is a crucial factor for any PET study. Because the duration of one investigation either of local glucose utilization or of local oxygen consumption is around 1 hour, maintenance of the steady state may become difficult, especially because the patients concerned are often (mentally) restless and uncooperative (demented). Clearly, the PET technique can yield very precise information on regional oxidative metabolism and thus on pathobiochemical pathways in diseased states. Otherwise, an unequivocal disadvantage lies in the high costs (cyclotron, PET) and in the availability of highly trained specialists. Therefore, the availability of such equipment is restricted to a few research centers.

Nuclear Magnetic Resonance In recent years, high-resolution nuclear magnetic resonance (NMR) spectroscopy has been established as a noninvasive technique to investigate phosphorus resonances from brain tissue (18, 19). However, technical difficulties and instrumental limitations still restrict the wide application of NMR spectroscopy. Experimental data so far available on normal and diseased states of cerebral metabolism are characterized by considerable scatter.

Postmortem Studies Postmortem studies of cerebral glucose/energy metabolism are limited to compounds that do not vary at all, or hardly at all, during ischemia/anoxia of the brain in the agonal state. This precludes the reproducible investigation of labile phosphates. Apart from these, enzyme activities of the glycolytic and oxidative glucose breakdown processes (see following) can be validly studied during the first 2 days after death. However, interpretation of the results has to be adapted to allow for the postmortem changes occurring as a result of room temperature and the time lapse before tissue sampling after death. To this end, relevant animal experiments have to be performed to test how and to what extent room temperature and temperature in the cool

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chamber, both for different durations, affect the activities of the enzymes to be studied.

S t u d i e s in E x p e r i m e n t a l A n i m a l s For mammals to be used for studies of cerebral glucose/energy metabolism, maintenance of steady-state conditions (see previous) is a necessary condition. This holds for monkeys, dogs, cats, rats, gerbils, and mice, which have been shown to be appropriate for such studies. Substrate utilization rates, compound concentrations, and enzyme activities can be measured.

Utilization o f Substrates In principle, the same procedures are valid as have been noted previously for human beings. To study global cerebral metabolic rates of oxygen and glucose, the modified Kety-Schmidt technique (9, 10) can be applied in monkeys, dogs, and cats in the same way as in humans (see above), except that mixed cerebral venous blood has to be sampled from the exposed superior sagittal sinus in its distal part close to the confluence of the sinuses. In smaller animals (rats, mice, and gerbils, in particular), the extraction rates when blood is drawn either from an artery or from the superior sagittal sinus are 0.25 ml/min (rat) and 0.05 ml/min (mouse, gerbil) to avoid hemorrhagic shock situations. Local cerebral glucose consumption can be determined by autoradiography with [2-14C]deoxyglucose (6). The labeled tracer is injected intravenously, and arterial blood samples are collected at short intervals over 45 minutes to measure [2-14C]deoxyglucose and glucose concentrations. After the final arterial blood sample at 45 minutes, the animal is decapitated, and the brain is rapidly removed and frozen in 2-methylbutane chilled to - 4 0 to -50~ The frozen brains are coated with chilled embedding medium and sectioned at 20/xm in a cryostat at -22~ The tissue sections are thawmounted on glass coverslips and dried on a hot plate at 60~ Local tissue concentrations of 14C are determined by densitometric analysis of the autoradiograms with a densitometer equipped with a 0.2-mm aperture.

Tissue Studies in Vivo To investigate the metabolic compounds of the glucose/energy metabolism in cerebral tissue, another prerequisite has to be fulfilled: freezing of the brain in situ during maintenance of steady-state conditions (see previous).

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For this purpose, a scalp skin funnel is formed by a sagittal incision and blunt preparation from the skull. Before freezing, a wet, warmed (37~ sponge is put into the funnel to avoid cooling of the brain. Freezing is performed by pouring liquid nitrogen into the funnel for at least 3 minutes in rat, gerbil, and mouse, and for at least 5 minutes in monkey, dog, and cat. After disconnection of the respiratory tube, the whole animal (rat, mouse, gerbil) or only the head of the animal (monkey, dog, cat) is immersed in liquid nitrogen. This freezing technique and the subsequent preparation steps (see following) yield optimal conditions for blockade of catabolic steps in the metabolism of substrates and enzymes in cerebral glucose/energy metabolism; these procedures are also superior to freezing by immersion of the whole animal or the animal head in liquid nitrogen after disconnection of the ventilation tube, which leads to autolytic changes in metabolite concentrations (20, 21). During the subsequent preparation of the cerebral tissue, permanent cooling is necessary. Therefore, the brain as a whole is chiseled from the skull under liquid nitrogen. If regional determinations are to be performed, the brain can be sliced in a cryostat between - 2 0 and -15~ At temperatures higher than - 15~ the tissue metabolism is jeopardized by autolytic variations. The tissue samples can be stored at -80~ for months until biochemical analysis. This technique of brain tissue freezing and preparation is superior to the freeze-blowing technique (22), which does not allow separation of different brain areas or of gray and white matter. As was mentioned previously, the mammalian brain is a multiorgan organ, which means that different areas of the brain fulfill distinct functions because of a highly specialized metabolism. The freeze-blowing technique freezes brain tissue very rapidly, but it is mixed and contaminated with blood, blood vessels, and meninges.

Biochemical Analyses To determine the concentrations of the substrates of the glycolytic chain, the tricarboxylic acid cycle, and the energy pool, the frozen tissue samples are (rapidly) weighed at 4~ homogenized at -28~ in chloroform by an Ultraturrax, and deproteinated with 20 volumes of HC104/EDTA at -28~ The homogenate is centrifuged at 25,000 g for 10 minutes at 0~ Supernatants are neutralized to pH 7.2 with 0.4 M imidazole base, 1.5 N KOH, and 0.3 M KC1. Adenine nucleotides and creatine phosphate can be determined spectrophotometrically at 340 nm (23, 24) or by means of high-performance liquid chromatography (25). The latter technique allows the additional determination of guanine nucleotides and nicotinamide-adenine dinucleotide at 210 nm, but leaves the corresponding monophosphates undetermined. In the

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homogenation procedure of this technique, EDTA imidazole base and KC1 are not added, because these compounds are detected at 210 nm. The metabolites of the glycolytic chain and the tricarboxylic acid cycle are determined spectrophotometrically as described by Bergmeyer (23).

NMR

Spectrometry

As in studies in humans (see previous), energy-rich phosphates can be mapped in brain tissue of experimental animals using surface coils (26). In contrast to studies in humans, the coil can be directly positioned on the surface of the cerebral cortex, so that the spectra are not contaminated by spectra from the extracerebral tissues (skin, bone). This technique has the advantage that from one cerebral cortical area of an anesthetized animal with its head held in a stereotaxic apparatus, numerous data can be collected under different experimental conditions during the course of a single experiment. When studies in different areas are to be performed, the same experimental conditions of tissue freezing (see previous) must be observed. 3~p NMR signals can be detected by the ex vivo in vitro technique: briefly, deeply frozen cerebral tissue is chiseled from the skull under liquid nitrogen, and chipped into pieces of an appropriate size (2.5 g). The NMR signals can be detected at -10~ (27). When labeled [1-~3C]glucose is given by infusion, the glucose metabolism can be investigated in the same manner as can the 31p metabolism (28).

Enzyme Activities

The tissue is prepared in the same way as for substrate determination, i.e., steady-state conditions are maintained and the brain is frozen in situ. The activity rates of the enzymes measured ex vivo in vitro can therefore be assumed to represent the in vivo situation. Because liquid nitrogen abolishes the metabolic activity completely within a few seconds, the activity state of the enzymes may reflect the activity state at the time of tissue freezing. The simultaneous determination of the activities of enzymes and the concentrations of substrates makes it possible to evaluate what proportion of the substrate is complexed by the respective enzyme. For the measurement of enzymatic activities, the frozen tissue samples are homogenized in a 0.02 M Tris-HCl buffer (1 : 10, w/v) containing 0.1 mM EDTA, 0.1 mM DTT, 250 mM sucrose, 100 txl of 10% Triton X-100 at pH 7.5 and 0~ The homogenate is centrifuged at 100,000 rpm for 15 minutes at 2~ The enzymatic activity is determined by continuous optical tests at

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340 nm and 30~ by use of microcuvettes with a final volume of 1 ml. (For the biochemical tests used for the individual determination of the enzyme activities, the reader is referred to Ref. 23.)

Miscellaneous Aspects Anesthesia The maintenance of steady-state conditions (see previous) and the avoidance of painful stimuli, operation stress, and autonomic reflexes require adequate anesthesia in studies of experimental animals. Intraperitoneal administration of anesthetic drugs may influence the cerebral glucose/energy metabolism in an undesirable way, because the stage of anesthesia can hardly be controlled. Intravenous anesthesia with barbiturates, neuroleptics, or ketamine can be better regulated, allowing better maintenance of the steady-state conditions. However, these compounds have been found to cause variations in both CBF and the cerebral oxygen utilization rate in different ways, which means that undesirable and incalculable effects can occur in cerebral glucose/energy metabolism. Inhalation anesthesia with 0.5% (or less) halothane and nitrous oxide/ oxygen (70 : 30, v/v) may be assumed to be appropriate in animal experiments requiring steady-state conditions. The anesthetics are controlled best by a respirator adaptable to frequency and volume of the inspired gas mixture, which is applied via a tracheal tube. In such cases the animal is immobilized by a muscle-relaxing drug because of the restraint. Nitrous oxide in the concentration mentioned above has not been found to influence either cortical CBF or overall glucose utilization of the brain (29, 30). Although halothane in as low a concentration as 0.6% (v/v) reduces the cerebral metabolic rate of oxygen by 25%, no variations in glycolytic flux or in cerebral energy state, except for a decrease in the glucose concentration, could be observed under anesthesia with 1% (v/v) halothane (31). It is even possible to discontinue halothane anesthesia with the onset of steady state without inducing variations in the steady-state parameters.

Aging Process Before the effect of the aging process on cerebral glucose/energy metabolism can be evaluated, the term "age" has to be clearly defined. Rodents may be designated as aged when their strain has a 50% survival rate and when their survival curve is more or less rectangular (32). This definition is also

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valid for other species of experimental animals. It has to be taken into account that the deflection point of any survival curve differs from strain to strain of a species and between the sexes. General guidelines for age-related studies are recommended (33).

Conclusion Various techniques are available for the study of cerebral glucose/energy metabolism in vivo in humans and experimental (mammalian) animals in a reproducible manner. With due consideration for a few limiting conditions, such as steady-state parameters, anesthesia, and the methodologic limitations, important information can be gained on the basis of cellular work in normal and diseased states.

References 1. S. Hoyer, J. Hamer, E. Alberti, H. Stoeckel, and F. Weinhardt, Pfliigers Arch. Ges. Physiol. 351, 161-172 (1974). 2. J. Hamer, S. Hoyer, H. Stoeckel, E. Alberti, and F. Weinhardt, Acta Neurochir. 28, 95-110 (1973). 3. E. Alberti, S. Hoyer, J. Hamer, H. Stoeckel, P. Packschiess, and F. Weinhardt, Br. J. Anaesth. 47, 941-947 (1975). 4. J. Hamer, S. Hoyer, E. Alberti, and F. Weinhardt, Acta Neurochir. 33, 141150 (1976). 5. K. Norberg and B. K. Siesj6, Brain Res. 86, 31-44 (1975). 6. B. K. Siesj6 and L. Nilsson, Scand. J. Clin. Lab. Invest. 27, 83-96 (1971). 7. P. Della Porta, A. T. Maiolo, V. V. Negri, and E. Rossella Metabolism 13, 131-140 (1964). 8. L. D. Lewis, B. Ljunggren, R. A. Ratcheson, and B. K. Siesj6, J. Neurochem. 23, 673-679 (1974). S. S. Kety and C. F. Schmidt, J. Clin. Invest. 27, 476-483 (1948). 10. A. Bernsmeier and K. Siemons, Pfliigers Arch. Ges. Physiol. 258, 149-162 (1953). 11. N. A. Lassen and O. Munck, Acta Physiol. Scand. 33, 30-49 (1955). 12. L. Sokoloff, M. Reivich, C. Kennedy, M. H. DesRosiers, C. S. Patlack, K. D. Pettigrew, O. Sakurada, and M. Shinohara, J. Neurochem. 28, 897-916 (1977). 13. M. Reivich, D. Kuhl, A. Wolf, J. Greenberg, M. Phelps, T. Ido, V. Casella, J. Fowler, E. Hoffman, A. Alavi, P. Som, and L. Sokoloff, Circ. Res. 44, 127-137 (1979). 14. A. Gjedde, Brain Res. Rev. 4, 237-274 (1982). 15. K. Wienhard, G. Pawlik, K. Eriksson, H. W. Ilsen, H. Herholz, and W. D. Heiss, J. Cereb. Blood Flow Metab. 3(1), $474-$475 (1983). .

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PARADIGMSOF NEURAL INJURY 16. R. S. J. Frackowiak, G. L. Lenzi, T. Jones, and J. D. Heather, J. Comput. Assist. Tomogr. 4, 727-736 (1980). 17. A. A. Lammertsma, T. Jones, R. S. J. Frackowiak, and G. L. Lenzi, J. Comput. Assist. Tomogr. 5, 544-550 (1981). 18. D. I. Hoult, S. J. W. Busby, D. G. Gadian, G. K. Radda, R. E. Richards, and P. J. Seeley, Nature (London) 252, 285-287 (1974). 19. J. W. Prichard, J. R. Alger, K. L. Behar, O. A. C. Petroff, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 80, 2748-2751 (1983). 20. U. Ponten, R. A. Ratcheson, and B. K. Siesj6, J. Neurochem. 21, 1121-1126 (1973). 21. U. Ponten, R. A. Ratcheson, L. G. Salford, and B. K. Siesj6, J. Neurochem. 21, 1127-1138 (1973). 22. R. L. Veech, R. L. Harris, D. Veloso, and E. H. Veech, J. Neurochem. 20, 183-188 (1973). 23. H. U. Bergmeyer, "Methods of Enzymatic Analysis," 2nd Ed. Academic Press, New York, 1974. 24. J. Folbergrova, V. MacMillan, and B. K. Siesj6, J. Neurochem. 19, 24972505 (1972). 25. E. Harmsen, P. P. H. De Tombe, and J. W. De Jong, J. Chromatogr. 230, 131-136 (1982). 26. J. J. H. Ackerman, T. H. Grove, G. G. Wong, D. G. Gadian, G. K. Radda, Nature (London) 283, 167-170 (1980). 27. B. Chance, Y. Nakase, M. Bond, J. S. Leigh, Jr., and G. McDonald, Proc. Natl. Acad. Sci. U.S.A. 75, 4925-4929 (1978). 28. R. G. Shulman, T. R. Brown, K. Ugurbil, S. Ogawa, S. M. Cohen, and J. A. den Hollander, Science 205, 160-166 (1979). 29. N. Dahlgreen, M. Ingvar, H. Yokoyama, and B. K. Siesj6, J. Cereb. Blood Flow Metab. 1, 211-218 (1981). 30. M. Ingvar and B. K. Siesj6, J. Cereb. Blood Flow Metab. 2, 481-486 (1982). 31. L. Nilsson and B. K. Siesj6, J. Neurochem. 23, 29-36 (1974). 32. C. F. Hollander, M. J. van Zwieten, and C. Zurcher, in "Aging of the Brain" (W. H. Gispen and J. Traber, eds.), pp. 187-196. Elsevier, Amsterdam, 1983. 33. P. Coleman, C. Finch, and J. Joseph Neurobiol Aging 11, 1-2 (1990).

Heavy Metal Effects on Glia Evelyn Tiffany-Castiglioni,* Marie E. Legare, Lora A. Schneider, Edward D. Harris, Rola Barhoumi, Jan Zmudzki, Yongchang Qian, and Robert C. Burghardt

Introduction Heavy metals can contribute to neurologic and mental dysfunction, either as a result of metabolic trace metal imbalances, as in the case of brain copper (Cu) deficiency in Menkes' disease (1), or as a result of central nervous system (CNS) exposure to environmental toxicants, such as lead (Pb). Most of the research efforts from our laboratory concerning interactions of heavy metals with glia have focused on the effects of Pb on astroglia in culture. We have also investigated some interactions of Cu and other metals with these cells. This chapter will describe the methods used by the authors to measure heavy metal accumulation by rat astroglial cells in culture, as well as the effects of metal exposure on cell function. Methods described will include atomic absorption spectroscopy, determination of influx and efflux kinetics constants, and use of cellular fluorescence imaging techniques for analysis of cell functions. Data from this laboratory indicate that neural cells in culture respond to a variety of toxic or viral insults by a limited number of functional alterations, including disruptions of cytosolic glutathione content, mitochondrial membrane potential, C a 2+ homeostasis, and gap junctional intercellular communication (GJIC). Therefore we shall describe the imaging techniques we use to analyze those events. Inorganic lead is recognized to be a cause of neurobehavioral deficits in children at blood Pb levels exceeding 10-15 /xg/dl (2). Two goals of lead neurotoxicity research are to define mechanisms of Pb uptake and tolerance in CNS cells that accumulate Pb and to identify molecular and cellular alterations that underlie behavioral deficits. Cell and tissue cultures are practical tools with which to pursue these goals, offering such advantages over in vivo models as defined cell types, an extracellular environment that can be precisely manipulated, and direct observation. Cell culture studies of Pb neurotoxicity have been reviewed recently (3). In early cell and tissue culture studies of Pb neurotoxicity, investigators attempted to identify organ and

* To whom correspondence should be addressed. Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tissue targets of Pb toxicity and employed rudimentary measurements of cytotoxicity, such as viability and cell counts. Pb doses studied were typically high and of short duration. The collective data from these studies suggest differential sensitivity to Pb toxicity among various types of cultured neural cells, ranked as follows from most to least sensitive: myelinating cells (oligodendroglia and Schwann cells), neurons, and astroglia. In addition, astroglia were shown to take up and store large amounts of Pb intracellularly, a phenomenon resembling the Pb-sequestering ability hypothesized for mature astroglia in vivo. Subsequent studies have been more specific in their objectives, seeking to identify organelle and enzyme targets of Pb. Prelethal subcellular effects, such as blockage of ion channels and inhibition of enzymes by Pb, were measured as toxic end points, and the effects of low, more toxicologically relevant, Pb levels were addressed. Current research is now attempting to characterize alterations in discrete molecular targets, particularly those whose effects in the cell may be metabolically amplified. Putative cellular defense mechanisms are an important focus of this research effort, particularly in astroglia, which can survive and function in the presence of high intracellular lead concentrations. Given the slow turnover of Pb in the brain, mechanisms for tolerance are of considerable importance. Astroglial cultures prepared from immature rodent brain are very well characterized, possessing many of the features of astroglia in vivo (4). Several procedures have been described for culturing astroglia. We use a modification of the method of McCarthy (5) to prepare primary astroglial cultures from the cerebral hemispheres of 0 to 3-day-old Spraque-Dawley rat pups. Cultures produced in this manner have previously been shown by immunocytochemical staining for glial fibrillary acidic protein (GFAP) to be nearly pure astroglia (6). Primary cultures, first-passage cultures, and glial cell lines such as the C6 rat glioma (American Type Culture Collection, Rockville, MD) are all amenable to the analytical methods described below.

Analysis of Metal Content" Atomic Absorption Spectroscopy

Rationale for Analysis by Atomic Absorption Spectroscopy Several studies have shown that astroglia accumulate Pb from the culture medium and store it intracellularly (reviewed in Ref. 3). This finding is in agreement with morphological observations in vivo that astroglia take up Pb (7, 8). Pb accumulation by astroglia in culture is accompanied by transient alterations in intracellular Fe and Cu levels (6). Furthermore, astroglia in vitro also accumulate Fe from the culture medium when exposed to high extracellular concentrations (6). These findings suggest a role for astroglia

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in heavy metal sequestration in cases of abnormally high exposure to certain metals. The most commonly used method for trace element determination is atomic absorption spectroscopy (AAS). AAS operates on the principal that atoms of an element absorb radiation from an atomic emission light source at characteristic ground-state energy levels. As a result, the decrease in intensity is directly proportional to the concentration of the element in the sample. Atomization of the sample is necessary for elemental detection. Two major methods of atomization are available: the flame method and the furnace method, the latter of which is also called electrothermal atomization (ETA). The flame method requires a sample volume of at least 1 ml and can detect lead down to the level of parts per million (ppm). The ETA method, on the other hand, can operate with a smaller sample volume (1-100/A) and can detect at the level of parts per billion (ppb). We use ETA-AAS because of its more sensitive detection limit; furthermore, sample preparation for this method is amenable to cell culture. Other analytical methods are available for measuring metals in biological samples, such as inductively coupled plasma atomic emission spectroscopy (ICP-AES) and neutron activation analysis (NAA). The main advantage of ICP-AES and NAA is that multielement analyses can simultaneously be performed with the same sample. However, there are several constraints with these methods that may reduce their application, particularly for routine procedures, including cost of analysis, complicated preparation procedures, and limited detection ability for some elements (methods reviewed in Refs. 9 and 10).

A t o m i c Absorption M e t h o d s Primary or first-passage cultures of astroglia are seeded in T-75 tissue culture flasks (Corning, Oneonta, NY). The seeding density we use for first-passage astroglia is 2 x 10 6 cells/flask. C6 rat glioma cells have also been studied successfully. We typically culture astroglia in Waymouth' s 705/1 MD medium (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; various vendors, depending on growth characteristics of the serum lot), and C6 cells in a mixture (1:1, v/v) of Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/F12; GIBCO/BRL) supplemented with 10% FBS. Cultures are exposed to Pb in cell culture medium at desired concentrations for lengths of time that vary from less than 1 day to several weeks, after which the cells are harvested from the flasks by the following steps. First, the medium is removed and the cell monolayer is washed one time in situ with 4-5 ml HEPES-buffered saline solution containing EDTA (HBS) by gently tilting the flask from side to side. HBS consists of the

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following components (g/liter): NaCI, 8.0; KC1, 0.4; N a z H P O 4 , 0.1; HEPES buffer, 2.38; dextrose, 1.0; and EDTA, 0.29225 (Sigma, St. Louis, MO). The resulting solution is adjusted to pH 7.2-7.4 and filter sterilized (0.2 /xm VacuCap-90; Gelman Sciences, Ann Arbor, MI). Next, the HBS solution is removed and the cells are dislodged with trypsin. For trypsinization, we add 1.5-2.0 ml trypsin, 0.25%, to the flask, tilting it to bathe all the cells. Trypsin is prepared from 2.5% stock solution (Sigma) freshly diluted 1:9 with HBS. The flask is then incubated at 37~ for 3-5 minutes. Next we add 10 ml HBS solution to the flask, dislodging the cells into the solution by means of a pipette or by rapping the flask on the counter. The suspended cells are collected by pipette into one 15-ml disposable polystyrene centrifuge tube per flask. The cells are centrifuged at 800-1000 g for 5 minutes at room temperature to form a pellet. The cells are then washed twice in the same tubes by resuspending the pellet with a pipette in 10 ml fresh HBS, centrifuging, and removing the HBS with a Pasteur pipette connected to a vacuum hose and bottle. After the second wash, and prior to the last centrifugation, three small aliquots of well-suspended cells (total about 300/zl) are counted with a hemocytometer, and an average cell number per flask is calculated. Results are expressed as nanograms Pb/2 • 106 cells, although other methods of expression may also be useful (e.g., nanograms Pb per unit of protein or DNA). After the final centrifugation step, the HBS is aspirated with a Pasteur pipette and the cell pellet is allowed to dry overnight in the uncapped tubes placed on their sides in the tissue culture hood under fluorescent light. Once dried, the samples may be immediately processed for ETA-AAS or capped and stored at -20~ It is best to process and analyze as soon as possible to prevent loss of metal from leeching into the tube. The cell pellet is now ready for digestion and subsequent atomic absorption spectroscopy. Concentrated nitric acid (200/zl; Ultrex, J. T. Baker, Phillipsburg, NJ) is added to each tube and the suspension is vortexed for 20 seconds, then allowed to sit overnight at room temperature. The digested cells are diluted with 1.8 ml of matrix modifiers. The matrix modifiers contain 0.5% nitric acid and 1% ammonium phosphate in a 2:1 ratio (v/v). The diluted cell solution is vortexed for 20 seconds and centrifuged for 5-6 minutes at 2000 rpm (800 g). Total lead, copper, iron, and zinc are measured by atomic absorption spectroscopy with a Thermo Jarrell Ash Smith-Heiftje 12 spectrometer with furnace atomizer, model 188. Determination is by injection of 10-20/zl of digestion solution with drying, ashing, and atomization in accordance with optimum parameters for each element as suggested by the "Methods Manual for Furnace Operation," Vol. II (11). The parameters, such as ashing times and temperatures and slit width, are not given in this chapter because they vary with type of instrumentation used. All materials that come in contact with the samples, such as flasks and tubes, must be Pb

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free. We therefore recommend the use of disposable plasticware and glass Pasteur pipettes for all procedures.

Interpretation and Limitations of Lead Measurement by Atomic Absorption Spectroscopy The limit of detection for Pb in our laboratory by the ETA-AAS method described is 5/zg/1 (ppb). The technique may be insufficient for some applications, such as determinations of influx and efflux kinetics or detection of Pb levels in cells exposed to very low levels of Pb (e.g., <0.1 /xM total Pb in the medium). Furthermore, a large number of cells (about 4 x 106) is needed for each determination, even in the case of astroglia and C6 glioma cells, which readily accumulate Pb in culture. Therefore, the technique is not amenable to measurements of Pb in rat oligodendroglia or neurons, which are much more difficult to culture in large numbers. The technique measures total Pb (or other metal) concentration, but does not provide a measurement of the form in which the metal is found (e.g., free ion, protein bound, salt precipitate), nor does it indicate the specific site of lead in the cell (e.g., nuclei, mitochondria, lysosomes).

Analysis of Metal Transport" Influx and Effiux Kinetics C o n s t a n t s

Rationale for Measurement of Kinetics Constants Several possible mechanisms can be invoked for metal entry into astroglia, such as receptor-mediated transport (vis-~t-vis Fe 2§ transport by transferrin), endocytosis and/or pinocytosis, ion channels, or an anion-exchange transport system. These areas might be fruitfully explored by characterizing the kinetics of metal transport into astroglia in the presence of various chemical blockers or metabolic inhibitors. Our investigations of kinetics constants for metal transport in astroglia have thus far focused on copper. As previously mentioned, copper accumulates in cultured astroglial cells exposed to Pb. Intracellular accumulation of copper has also been described for fibroblasts from patients with Menkes' disease. One of the main clinical manifestations in Menkes' disease is neurological degeneration (1). In this lethal genetic disorder, copper metabolism is greatly disrupted, resulting in Cu accumulation by the intestinal epithelium and Cu deficiency in the brain. The intracellular Cu content of Menkes' fibroblasts is more than five times that of normal cells (12). Astroglial cells cultured from

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the macular mouse, an animal model of Menkes' disease, also accumulate an excessive amount of copper (13). The overall Cu deficiency in brain, accompanied by Cu accumulation in astroglia, suggests a defect in the transport or release of Cu from astroglia to neurons. Thus, the astroglial cell is of interest for its propensity to accumulate Cu in both Menkes' disease and lead toxicity. To date, the mechanism of copper transport into astroglia is not understood. Consequently we have no knowledge of transport parameters, particularly the kinetic constants that relate to the rates of uptake and efflux. Here we describe a method that uses 67CH to determine the K m and Vmax for Cu in cultured astroglial cells.

Methods f o r Measurement o f Kinetics Constants Time Dependence of 67Cu(II) Uptake Glial cells (first-passage astroglia or C6 glioma cells) are seeded at high density in 35-mm tissue culture dishes and grown for 2-3 days to a final cell density of about 3 x 10 6 cells/dish. Prior to the analysis, the medium is removed from the cultures and the cultures are washed with 2 ml of Dulbecco's phosphate-buffered saline (DPBS, Irvine Scientific, Santa Ana, CA) at room temperature for 30 seconds. The cultures are refed with 2 ml of a mixture (1 : 1, v/v) of Dulbecco's modified Eagle's medium/Ham's F12 medium. This medium is serum free to minimize interference by plasma proteins that bind copper. A final concentration of 50 nM 67CHC12 (Brookhaven National Laboratory, Raton, NY; specific activity 256 Ci/mmol), carrier-free, is then added to the cultures. The radioactive medium is removed after various incubation times (e.g., 10, 20, and 50 minutes at 37~ and the cells are washed with 2 ml of 150 mM NaC1 (adjusted to pH 4.0 with 0.1M HC1) at room temperature for 30 seconds. The cells are harvested by scraping the dish bottom with a cell scraper after adding 1 ml of 0.5 N NaOH. The cell lysate is completely transferred to a 4-ml vial for counting the quantity of 67Cu(II) retained in the cells with a gamma counter. The uptake of 67C11 is expressed as picomoles Cu/mg protein. Protein is assayed with bicinchoninic acid (BCA; Pierce, Rockford, IL) according to Pierce's assay protocol. A typical time-course analysis shows a progressive uptake for the 50-minute incubation period. No uptake is observed at 4~ The 67CH in 4~ cells can be used to correct for radioactivity that adheres to the cell membrane.

Determination of Kinetic Parameters (Km and Vmax): Uptake of 67Cu(II) The cells are plated as described above and washed with 2 ml DPBS for 30 seconds at room temperature. Fresh, serum-free DMEM/F12 medium containing the equivalent of 10, 20, 50, 100, and 200 nM of 67CuCI2 is carefully

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i

0.12

ll[Cu(ll)] (llnM) FIG. 1 Double-reciprocal plot of copper uptake in cultured C6 rat glioma cells. For this plot, y = 0.28 + 143.56x; r 2 = 0.99. Kinetics constants for copper influx are Km = 512 nM and Vmax - 3.57 pmol Cu/mg protein/minute. Each point represents the mean _+ standard deviation from quadruplicate determinations.

pipetted over the cell layer, 2 ml/dish. After a 10-minute incubation at 37~ the radioactive medium is removed and cells are washed with 2 ml of 150 mM NaC1 (adjusted to pH 4.0) at room temperature for 30 seconds. Copper67 uptake for each concentration of 67Cu is expressed as picomoles Cu/mg protein/minute. A double reciprocal plot of velocity v e r s u s 67Cu concentration is determined to asses the apparent K m and gmax parameters. These data are shown in Fig. 1. A straight-line relationship is obtained by plotting 1/pmol Cu/mg protein/minute v e r s u s 1/[67Cu]. Values for apparent K m and gma x ar e calculated on the basis of the straight-line relationship of the Michaelis-Menten equation: llv-

1/Vma x +

Km/Vmax(1/S),

which graphically is equivalent to y = b + m x , where b is the intersecting point on the y axis (1/Vmax), and m is the slope, equal to K m / V m a x . The calculation of x when y equals zero sets x equal to - 1 / K m, which is an alternative and perhaps more convenient method for determining the K m parameter. Using the equations, one is able to derive values of K m -- 512 nM and Vmax = 3.57 pmol Cu/mg protein/minute for Cu transport into astroglial cells. Moreover, the linearity of the plot suggests the cells contain either

142

PARADIGMS OF N E U R A L I N J U R Y

a single binding site for copper in the membrane or multiple sites with identical binding affinities.

Time Dependence of 67Cu(II) Efflux Cultures are prepared as described, and washed with 2 ml DPBS, after which 2 ml of fresh DMEM/F12 serum-free medium containing 50 nM 67CUC12 is carefully pipetted over the cells. After a 50-minute incubation at 37~ the radioactive medium is removed and the cells are rinsed gently with 2 ml DPBS at room temperature for 30 seconds. Fresh DMEM/F12 medium (2 ml) is then pipetted over the cells and incubation is allowed to continue. After 5, 15, or 30 minutes of incubation at 37~ the cells are collected as above with 0.5 M NaOH and a cell scraper, and 67Cu retained in the cells is determined and expressed as picomoles Cu/mg protein. Copper-67 appearing in the medium with time is also monitored. Effluxing ceases in about 30 minutes. Any 67Cu remaining within the cells at 50 minutes is, therefore, regarded as nonexportable. The difference between cell-retained 67Cu at 50 minutes and time zero is used to calculate the exported fraction.

Determination of Kinetic Parameters (K m and Vmax):Efflux of 6ZCu(II) Plated cells are preloaded with 10, 20, 50, 100, or 200 nM of 67CUC12 in fresh DMEM/F12 serum-free medium at 37~ for 50 minutes. According to the Darwish assumption (14), the concentration of copper in the cell at the efflux site is equivalent to the concentration of copper in the medium once equilibrium has been established. After the incubation, the radioactive medium is removed and the cells are washed with 2 ml DPBS at room temperature for 30 seconds. Fresh DMEM/F12 medium (2 ml) is layered over the cells as before. Cell-retained 67Cu is determined at time zero and after a 5minute incubation at 37~ The efflux velocity is obtained from the difference between the two readings for each 67Cu load and expressed as picomoles Cu/mg protein/minute. Graphic analysis of the velocity versus 67Cu concentrations in the medium gives the kinetic curve of 67Cu eff~ux. The values for Km and Vmaxof 67Cu efflux are determined with a double-reciprocal plot (see preceding). The results of the kinetic analysis are shown in Fig. 2. Based on the values shown, astroglial cells have an efflux Km = 67.7 nM and an efflux gmax = 0.677 pmol Cu/mg protein/minute.

Interpretation and Limitations o f Kinetics Constants Determinations The above procedure estimates Km and Vmax transport constants by standard kinetic analysis and employs standard assumptions. Rates of uptake must be performed rapidly, preferably when only unidirectional flow of 67Cu into

[10] HEAVY METAL EFFECTS ON GLIA

143

12 C o,...

E

r o=.,,. 0

10

_

0 LD.

o= E 0

-

6

-

4

-

2

-

0

E

o. ,i,,,,. >

0

/

-0.02

'

0.00

I

'

0.02

I

0.04

llICu(ll)!

'

I

'

0.06

I

0.08

'

I

0.10

'

0.12

(llnM)

FIG. 2 Double-reciprocal plot of copper efflux in cultured C6 rat glioma cells. For this plot, y = 1.47 + 100.15x; r 2 = 1.00. Kinetics constants for copper efflux are K m = 67.7 nM and Vmax = 0.677 pmol Cu/mg protein/minute. Each point represents the mean of duplicate determinations. the cell is occurring. Binding of 67Cu to the membrane must also occur rapidly, but depends on the concentration of 67CHin the medium, i.e., below the level that would saturate the membrane binding sites. Transport is viewed as a two-step process with binding preceding the actual activation of the mobilizing factor. The method gives no insight into how the transport mechanism or carrier is functioning. It is essential that the 67Cu in the medium have free and open access to the membrane carrier and that other competing ligands be eliminated from the medium. Because serum contains albumin, and albumin has at least one high-affinity site for copper on the protein, the albumin must be removed. Cells, therefore, are suspended in serum-free medium for the duration of exposure to 67CH. Our interpretation of the data presented in Figs. 1 and 2 is that the import and export of copper by astroglia require two systems. The efflux system appears eight times more sensitive. This conclusion can be interpreted to mean that cells export copper with a greater affinity than they take it up. The imbalance is met by having Vmax for efflux only one-fifth as rapid (at saturation) as input. Because of the strong binding affinity of the efflux system, internal systems that require copper for metabolic function must compete with a highly efficient efflux system designed to keep the cell in homeostasis and to protect the cell from transient high environmental exposure. The remainder of the chapter will address methods for analyzing disruptions in cell homeostasis that result from exposure to metals in culture.

144

PARADIGMS OF NEURAL INJURY

A n a l y s i s o f Cell F u n c t i o n : C e l l u l a r F l u o r e s c e n c e I m a g i n g

Rationale for Use of Imaging Techniques Over the past several years, the convergence of several technologies has led to the development of new experimental approaches for investigating mechanisms of chemical toxicity. Innovations in cell culture strategies and a renaissance in light microscopy centered around fluorescence techniques have led to the development of a new generation of in vitro toxicity assays based on the quantification of fluorescent probes in living cells. The authors have been exploiting the powerful new technology of microscopic image analysis with vital fluorescent probes and interactive laser cytometry for the development of highly sensitive, mechanistically relevant assays for neurotoxicants. This technology has many potential advantages over traditional in vitro toxicity assays. First, the assays, which are based on the quantitation of fluorescent probes in individual cells and cultures, are noninvasive and are typically carried out with living cells in the culture dish. Second, the assays are highly sensitive and quantitative. Third, data can be collected from both individual cells and cell populations. This feature is valuable for screening out variant cells (such as those in mitosis) that may respond differently from the majority of cells to a toxin. Fourth, in many cases repeated measurements can be taken from individual cells as well as from cultures. Appropriate controls may also be run in the same culture dish because of the amenability of the system to repeated measurements. Fifth, the types of measurements obtained are highly mechanistic, and thus provide not only a screening device for the presence of a toxicant, but also information on the mechanisms of toxic action. Sixth, once assays have been developed and validated with an instrument such as the Meridian ACAS interactive laser cytometer (as described further in the next section), they should be readily adaptable to automated cytofluorometric assay systems.

Fluorescent Probes and Analytical Instrumentation Fluorescent probes have been created (and continue to evolve) that are designed to react with biomolecules under conditions of relatively low temperature (i.e., temperatures compatible with living cells) and near-neutral pH (15). Many fluorescent indicators of cellular function are uniquely suitable for probing living cells because of a combination of five properties (16): specificity, the ability to detect the probe in a complex mixture of biomolecules; sensitivity, the potential for detection of few molecules in a given volume; spectroscopy, differences in absorption or emission caused by sensi-

[10] HEAVY METAL EFFECTS ON GLIA

145

tivity to their immediate physicochemical environment; temporal resolution, the potential for fluorescence measurements over time points longer than the excitation/emission of the fluorescent probe, i.e., longer than 10-s seconds; and spatial resolution, defined by the resolving power of the microscope lens used to image the fluorescent probe. Many of the probes, such as the acetoxymethyl (AM) ester derivatives of a number of fluorescent indicators, may be noninvasively delivered (without microinjection or damage to the plasma membrane) into cells because of their membrane permeability properties. After entry into the cell the ester derivatives are subsequently cleaved by nonspecific cytosolic esterase activity to yield charged, membrane-impermeant probes. Other noninvasively delivered probes are membrane permeable but partition into cells based on charge, or are nonfluorescent until covalently bound to molecules within the cytoplasm. Coinciding with advances in fluorescent probe technology are developments in analytical microscopy instrumentation that permit the analysis of diverse molecular targets on or within cells. Technical improvements in microscopy include the development of lasers with emission lines suitable for use with many fluorescent probes, computer automation and control over the intensity and duration of sample illumination, and digital imaging photometric systems coupled with computer-controUed stage positioning, all of which combine to permit spatially resolved fluorescence signals. Modern fluorescence detection systems provide a means to balance the need for sufficient illumination with sensitivity, permitting excitation irradiance levels low enough to avoid damage to the sample while providing detectability equivalent to micromolar concentrations of fluorophores. There are a number of commercially available instruments capable of exciting and detecting fluorescent signals from living cells. The following description is restricted to our experience with an instrument manufactured by Meridian Instruments, Inc. (Okemos, MI), referred to as an ACAS 570 Interactive Laser Cytometer (ACAS is an acronym referring to the Adherent Cell Analysis and Sorting capabilities of the instrument). Relevant features of the instrument have been previously described (17) and include a Coherent Innova 90-5 5 W argon ion laser that produces ultraviolet (UV) illumination in the range of 351.1-363.8 nm and several visible lines throughout the range of 457.9-528.7 nm. The selected laser illumination line is directed through the rear illumination port of an Olympus IMT-2 inverted microscope equipped for epifluorescence. Fluorescence excitation with minimal destruction of fluorophores is optimized by regulation of the intensity and duration of the laser spot. Attenuation of the laser beam is controlled with two separate components. Neutral-density filters are inserted into the optical path to reduce beam intensity. The second component of beam attenuation employs an acoustooptic modulator (AOM). The AOM is used to separate the zero

146

PARADIGMS OF NEURAL INJURY

and first diffracted order of light. Only the first diffracted order light is admitted to the optical system and the amount of first-order light is regulated by activation of the AOM. By application of a high-frequency, high-voltage signal to the AOM, changes in the Bragg angle of the crystal diverts the firstorder beam from the optical axis to attenuate the beam with potentially rapid (_<2 ~sec) on/off transitions. Sample image scanning is obtained by stage stepper motors, which allows the laser beam to be kept along the center of the optical system. Emitted fluorescence is passed through a dichroic element and barrier filter, and is detected and amplified by a photomultiplier tube. The output of the fluorescence-measuring circuits is digitized with a 12-bit analog-to-digital converter and transfers this to the computer under software control. Individual image scans are held in a digital frame store and are readily available for computer analysis. A diagram of the Meridian ACAS 570 is shown in Fig. 3. The ACAS instrumentation has recently been upgraded (Ultima) by the incorporation of a Coherent Enterprise laser capable of providing simultaneous or sequential UV (351.1-363.8 nm) and visible (488 or 514 nm) illumination. In addition to motorized X and Y stage scanning, combination of Xaxis galvanometric mirror and Y-axis stage scanning provides more rapid imaging capabilities. Both the ACAS 570 and Ultima instruments are equipped with confocal capabilities, which add a third spatial dimension (i.e., volume analysis) to fluorescence imaging. Even without confocal capabilities, the additional dimension of time (i.e., kinetics) combined with image analysis of living cells expands the analytical potential of the instruments dramatically. Although the quantitative analyses described in the following sections have been adapted for use with the ACAS 570, the methods are also valid for other digital imaging fluorescence instruments. Additional general considerations of this technology for in vitro toxicology testing have been reviewed

(18).

Analysis of Cytosolic Glutathione Content and Mitochondrial Membrane Potential

Rationale for Analyzing Glutathione Content and Mitochondrial Membrane Potential In view of the ability of astroglia to take up Pb from the culture medium and store it intracellularly, coupled with their resistance to overt Pb toxicity, it would be reasonable to postulate mechanisms of Pb tolerance in these cells.

147

[10] HEAVY M E T A L EFFECTS ON GLIA

i

Video Signal

I I

l rt ', F~I

Laser Power Supply

I

I

NDF

Inverted Microscope

I

Detector

Argon Ion Laser

A

=

FIG. 3 Generalized configuration of Meridian ACAS Interactive Laser Cytometer. The diagram illustrates the major components of the instrumentation, including the host computer, laser illumination, microscope, motorized stage, and detector systems. The instrument control system is a microcomputer-based unit that also supports fluorescence intensity data acquisition and analysis. The argon ion laser can be tuned to provide one of the several useful wavelengths of light (UV or visible) that are used to excite fluorescent probes on or within cells. The laser beam is attenuated by an acoustooptic modulator (AOM) that can regulate the intensity and duration of laser illumination (duty cycle). Neutral-density filters (NDF) are also used to reduce the intensity (amplitude) of irradiation as needed. Laser light enters the inverted microscope through the epiillumination port of the microscope and, after passing through an excitation filter (EF), is reflected into the objective lens and onto cells on the stage of the microscope by a dichroic mirror (DM). Because the objective lens is fixed in position, fluorescent images are generated by moving the stage of the microscope in X and Y directions with precision stepper motors. Another stepper motor can control Z axis position for confocal imaging applications. Emitted fluorescence is collected by the microscope objective (OB) lens and passes through the dichroic mirror due to its longer wavelength and proceeds to the detector, which contains photomultiplier tubes (PMT). Isolation filters (IF) and the dichroic mirror divide light into separate wavelength bands, each of which can then be amplified for fluorescence intensity images. Dashed lines indicate the light path. Solid lines indicate electrical circuits. FSM, Front surface mirror; BF, barrier filter; VC, video camera.

148

PARADIGMS OF NEURAL INJURY

The astroglial cell is an excellent candidate for examining the concept of tolerance, i.e., adaptation by a cell to the presence of Pb by adjustment of cellular homeostatic mechanisms. In this section, the measurement of intracellular glutathione (GSH) is considered as a potential indicator of cell tolerance to the accumulated Pb. Another cellular function, the maintenance of a high electrochemical membrane potential in mitochondria, is considered as a site for cell damage that may ensue from the failure of the GSH system to protect the cell, and also as a direct target for Pb-induced damage. Intracellular GSH is an important component of cellular homeostasis. This abundant tripeptide, which is synthesized from constituent amino acids (glutamate, cysteine, and glycine), comprises the principal component of a cellular detoxification system, capable of scavenging reactive oxygen species and maintaining the redox state of cellular thiols (19). Astroglia are of particular interest with regard to GSH function because they appear to be involved in GSH compartmentation in brain (20). Chemically induced GSH depletion has been shown to cause mitochondrial degeneration in brain cells, apparently because GSH-dependent reactions are critical for reducing the significant levels of hydrogen peroxide produced in mitochondria (21). It may therefore be useful to measure mitochondrial membrane potential at the same time points for which one measures GSH levels. Mitochondria, the organelles that generate cellular ATP, are postulated targets for Pb-induced injury in astroglia (22) and other cells (reviewed in Ref. 23). The inner mitochondrial membrane contains the enzymes for electron transport and oxidative phosphorylation. An electrochemical gradient, which can be detected as a membrane potential difference and a pH gradient, exists across this membrane to couple energy released during electron transport to the phosphorylation of ADP to ATP. Chemically induced dissipation of the electrochemical gradient would decrease the rate of oxidative phosphorylation and deplete cellular energy levels. Temporal correlations can be studied by fluorescence cytometry for Pb-induced alterations in GSH content and mitochondrial membrane potential.

Methods for Glutathione Measurement The ACAS 570 described above is used for the quantification of cellular fluorescence. Primary or first-passage cultures of astroglia have been used for these assays, and are examined in culture on days that correspond temporally with postnatal development ages of interest. Monochlorobimane (mBC1; Molecular Probes, Eugene, OR) is currently the fluorescent probe of choice for intracellular GSH measurement due to its low reactivity to GSH and

149

[10] HEAVY METAL EFFECTS ON GLIA

other thiols, and its ability to form a fluorescent adduct with GSH in a reaction catalyzed by glutathione S-transferase (GST) (reviewed in Ref. 24). In this assay, cells are initially plated in T-75 tissue culture flasks, in which they are treated with Pb at desired concentrations and exposure times. Because the fluorescent adduct of mBCI requires ultraviolet excitation, cells must be subcultured on poly(L-lysine)-coated coverglass chamber dishes (Nunc, Naperville, IL) for at least 12-24 hours prior to analysis. Cultures are washed once with CaZ+/MgZ+-free phosphate-buffered saline (pH 7.4) and then loaded with 40 IxM mBC1 (stock solutions are 10 mM in ethanol) in serum-free, phenol red-free medium. Cultures are then washed four times with serum-free medium without phenol red and analyzed. In all fluorescence imaging assays carried out in our laboratory, we use HEPES-buffered DMEM/F12 medium without serum or phenol red (Sigma) because serum may interfere with dye loading and phenol red may interfere with fluorescence light detection. HEPES adequately buffers the medium at pH 7.2-7.4 in the absence of CO2 for the time period (usually 20-30 minutes) during which the culture is on the microscope stage. Because GST activity can vary from one cell type to another, loading parameters must be optimized by the performance of a kinetic analysis of probe loading into cells. Typically two culture dishes per treatment are loaded on the stage and the fluorescence intensity in clusters of about 30 cells is recorded at 1-minute intervals. Once loading kinetics are determined, fluorescence data may be analyzed by means of a curve-fitting regression analysis program and extrapolated to identify equilibrium loading and the rate constant from the equation F (t) = Feq(1 -

e-kt),

where F (t) is the fluorescence at any time, t, Feq is the fluorescence intensity at equilibrium (i.e., GSH level), and k is the estimated rate constant for mBC1 conjugation to GSH (i.e., k = GST activity) (25). Emitted fluorescence (461 nm) is detected with a barrier filter (BP 485/45) with the ACAS 570 at an excitation wavelength of 351-363 nm. Figure 4 shows a digital image of astroglia labeled with mBC1. Typical analysis of cells is performed by defining the area occupied by individual cells. Borders of the cell are identified by drawing a polygon around the area to be analyzed. The integrated and average fluorescence intensity of each cell can then be determined. Background fluorescence detection values of dishes containing serum-free medium with and without cells are used to set threshold sensitivity for the photomultipliers. Control GSH levels are determined by scanning at least 10 cells in four areas from each control dish. Two or more dishes per treatment group are tested in each experiment. GSH values are expressed

150

PARADIGMSOF NEURAL INJURY Color -

Values -

3880t

-

2813

-

2626

-

2440

-

2253

-

2867

~ -

1 8 8 8

- 1694

-~

i

1587 1320

1134 947 761 574 388 281 151

FIG. 4 Digital image of cellular monochlorobimane fluorescence; this image was reproduced from the pseudocolor intensity image on black and white film. The pseudocolor scale provides a visual index of fluorescence intensity within each area of the cell. In this image, lighter areas within cells reflect higher fluorescence intensity (i.e., more GSH-mBC1 conjugate). The image shows a control culture of astroglia, which exhibits a characteristic mBC1 fluorescence pattern identifying normal cytoplasmic levels of GSH. as the mean + S E M for each treatment group and can cally for the analysis of treatment effects. Figure 5 two concentrations of Pb on G S H content in cultured expressed as a percentage of control fluorescence in alterations as a function of time (26).

be compared statistishows the effects of astroglia. Values are order to detect G S H

Methods for Measurement of Mitochondrial Membrane Potential The potentiometric fluorescent dye rhodamine 123 (Molecular Probes, Eugene, OR) is used in this assay to provide a relative assessment of electrochemical potential across the mitochondrial membrane. The incorporation of rhodamine 123 is dependent on the maintenance of an electrochemical potential across the mitochondrial membrane (27), and dissipation of the

151

[10] HEAVY METAL EFFECTS ON GLIA 200 I-I control !~1 0.1 pMPb

m 1.0 pM Pb

,=_,

100

I

0 0 tO

e~

0.3

!

i

1

1

2

3

6

9

days of treatment

FIG. 5 Astroglial glutathione levels after low-level lead acetate treatment plotted as a percentage of control glutathione levels. Astroglial cultures corresponding in age to postnatal day 21 were treated daily with 0, 0.1, or 1.0/zM lead acetate in Waymouth's 705/1 medium with 10% FBS. After an initial decrease in the astroglial content of glutathione in response to Pb treatment, glutathione content increased compared to control values in a dose-dependent manner. ,, Differs significantly from control, same day (p < 0.01). **, Differs significantly from control and other Pbtreated groups, same day (p < 0.01). Reproduced from Ref. 26, with permission.

electrochemical potential is indicated by a decrease in fluorescence (28). Cells previously treated with Pb in T-75 flasks are subcultured at low density (75,000 to 100,000 cells per dish) into 35-mm plastic tissue culture dishes at least 12 hours prior to assay. Mitochondrial loading with rhodamine 123 is performed by washing cultures in situ once with PBS, and then incubating the cultures with 5 /zg/ml rhodamine 123 in serum-free medium (prepared from 2 mg/ml stock in ethanol). Incubation time is based on kinetic analysis of rhodamine 123 loading, which is typically 30 minutes in astroglia. Cells are then washed four times in serum-free medium without phenol red before scanning on the ACAS 570 (488 nm excitation wavelength). Up to eight different areas of the dish can be used to record mitochondrial fluorescence intensity, as well as several dishes per treatment group. The intensity of

152

PARADIGMS OF NEURAL INJURY 200 [ ] control [ ] 0.1 pM Pb

9 1.0 pM Pb

l-

I1) O

o -o

_~

100

T

-r

-1-

tO 0 tO L_

(I)

I

0.5

1

6

12

14

days of treatment

FIG. 6 Mitochondrial membrane potential in astroglia after low-level lead acetate treatment as a percentage of control values. Astroglial cultures corresponding in age to postnatal day 21 were treated with Pb as described for Fig. 5. A significant, nearly maximal decrease in mitochondrial membrane potential was seen after a 1day exposure to 1 /xM Pb. By day 12 of treatment, both doses of Pb resulted in a decrease in mitochondrial membrane potential to the same degree. ,, Differs significantly from control, same day (p < 0.01). Reproduced from Ref. 26, with permission. (Additional timepoints have been added to the previously published figure.)

labeling is quantified as a function of metal dose, time of treatment, and length of posttreatment recovery. The average fluorescence intensity is recorded from at least 100 cells per treatment group. Data are treated as described for GSH. Figure 6 shows the effect of Pb on mitochondrial membrane potential in astroglia as a function of treatment time.

Interpretation and Limitations of Glutathione and Mitochondrial Membrane Potential Measurements Analysis of the fluorescence intensity of the G S H - m B C 1 conjugate by digital fluorescence imaging is a useful assay of cellular GSH content. Other thiolspecific probes have been described that are suitable for visible-wavelength laser excitation, including 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethyleosin diacetate (CMEDA) (29). C M F D A and C M E D A

[10] HEAVY METAL EFFECTS ON GLIA

153

are not as specific for GSH as mBC1, and therefore these visible probes may be more suitable for monitoring the total level of free intracellular thiol than GSH specifically. Both mBC1 and CMFDA are nontoxic agents that may also be used intentionally to deplete GSH within cells (25). With the interactive laser cytometer, there is no signal overlap of the two probes and therefore it is possible to deplete available GSH with one fluorescent probe and monitor new synthesis with the other at the appropriate wavelength. Fluorescence intensity values of the conjugated GSH are then monitored at appropriate intervals (ranging from 1 to 5 minutes over 1 hour). The assay for mitochondrial membrane potential does not distinguish between toxic insults that act directly on mitochondria and extramitochondrial events that damage mitochondrial membrane integrity, such as decreased GSH concentrations or increased intracellular C a 2+ concentrations. Therefore, mitochondrial membrane potential should be interpreted in the context of other indicators of cellular injury. Thus the data shown in Fig. 5 indicate a transient decrease in cytosolic GSH concentration in astroglia exposed to 0.1 to 1.0/zM lead acetate, followed by recovery to normal levels, and then a subsequent elevation to levels in excess of normal. Within the same time period (Fig. 6), mitochondrial membrane potential decreases and remains decreased even after GSH levels have recovered. This result suggests that additional mechanisms are involved in the toxicity of Pb at the later time points. The relationship between GSH depletion and decreased mitochondrial membrane potential has not yet been clarified in this system but could be approached by testing the ability of exogenous GSH or a membranepermeant GSH ester to protect mitochondria from Pb-induced damage. In any case, the period of GSH depletion in astroglia may represent a period of vulnerability to cell injury, both to the astroglia and to neurons that depend on them for amino acids and other supporting functions.

A n a l y s i s o f I n t r a c e l l u l a r C a 2+ C o n t e n t

Rationale for Measuring [Ca2+] C a 2+ has well-established roles as a second messenger in signal transduction. The extracellular C a 2+ concentration is high, typically 1.2-1.3 mM. In contrast, the Ca 2+ concentration in the cytoplasm is narrowly buffered to around 10 -7 M by C a 2+ homeostatic mechanisms, including plasma membrane transport systems and CaZ+-sequestering systems. These mechanisms are important potential targets for the toxic effects of Pb and other metals. Although Pb is considered a nonphysiologic metal, the ionized form (Pb 2+) apparently can enter a number of metabolic pathways by replacing other ions, particu-

154

PARADIGMS OF NEURAL INJURY larly Ca 2+, which is a divalent cation with a similar hydrated diameter. The interaction of Pb 2+ and Ca 2+ at cellular sites has recently been reviewed (3,

30). This interaction takes place at three sites: the plasma membrane, where Pb 2+ and Ca 2+ compete for transport systems that regulate their entry or exit, such as Ca 2+ channels and the CaZ+-ATPase pump; intracellular Ca 2+ storage depots, such as the endoplasmic reticulum, mitochondria, and Ca 2+binding proteins; and effector proteins whose activity is regulated by Ca 2+,

such as protein kinase C and calmodulin. One of the earliest and most discrete toxic effects of Pb common to different cell types and numerous cellular processes is perturbation of the Ca 2+ intracellular signaling system. Pb is hypothesized to affect Ca 2+ signaling by two mechanisms: replacement of Ca 2+ or elevation of intracellular free Ca 2+ levels. A variety of fluorescent probes has been developed to detect very small, rapid changes in intracellular free calcium concentrations, [Ca2+]i, including indo-1, fluo-3, fura-2, and quin-2 (31). These probes can be loaded noninvasively into living cells as membrane-permeant acetoxymethoxy derivatives and can indicate changes in [Ca2+]i on a millisecond time scale because of a characteristic change in their individual spectra on binding to Ca 2+. When cells are treated with agents that alter Ca 2+ homeostasis in a predictable fashion, such as Ca 2+ ionophores, sophisticated analysis of Ca 2+ homeostatic mechanisms is possible. The choice of probes is based on the wavelength selection capabilities of the available instrumentation and the type of analysis being performed. Indo- 1 is well-suited for measuring small, rapid changes in [Ca2+]i with instruments that generate a single excitation wavelength (such as the ACAS 570). Indo-1 requires UV excitation (351-363 nm) but provides the advantage of permitting measurement of [Ca2+]i because there is a shift in the emission spectrum from 485 to 405 nm on binding Ca 2+. The two emission wavelengths are separated with a dichroic mirror and are monitored simultaneously with separate detectors. Intracellular [Ca 2+] is calculated by comparing the ratio of emissions to that of a standard curve. Ratiometric analysis of emission wavelength pairs is independent of absolute changes in fluorescence intensity at a given wavelength. Thus, quantification of [Ca2+]i is independent of the extent of dye loading, leakage, cell thickness, or photobleaching. In addition, indo-1 has great sensitivity below 1/zM. Fluo-3 has two major advantages, its ease of use due to its visible excitation at 488 nm and emission at 520 nm and its great sensitivity in the micromolar range. Fluo-3 is limited, however, in its ability to m e a s u r e [Ca2+]i, because of its single emission wavelength. Quantitative measurements are possible, however, if cells can be uniformly loaded. Fura-2 requires the use of dual excitation wavelengths in the UV range (380 and 340 nm), which is not available with the ACAS 570. Ca 2+ binding shifts the excitation spectrum about 30 nm to the shorter wavelengths,

[10] HEAVY METAL E F F E C T S ON GLIA

155

so that the ratio of intensities obtained from 340/380-nm excitation pairs provides a good measure of [Ca2+]i . Quin-2, which has less specificity for C a 2+ than newer generation probes, is most efficiently excited at relatively shorter wavelengths (340 nm), lacks the brightness of newer probes, and does not show the useful CaZ+-induced wavelength shift in either excitation or emission spectrum needed to generate a ratio signal (31). The similarity of physical and chemical properties between Pb 2+ and C a 2+ that allows them to interact with the same cellular substrates unfortunately may also allow both cations to interact with the same fluorescent probes. Though the specificity of newer generation Ca 2+ probes over other metals is generally good, for those probes with which Pb 2+ interactions have been studied, the results have shown a strong interaction with Pb 2+. Solutions to the technical problems arising from this circumstance are still under development, and will be examined below. The description of methodology in this section is limited to the use of indo-1 for the analysis of intracellular [Ca 2+] in cultured astroglia.

Method for Measuring [Ca2+] The Ca2+-sensitive fluorophore indo-1 (Molecular Probes) is used to quantify intracellular C a 2+ in astroglia with the ACAS 570. Because the excitation wavelength of indo-1 is in the UV range, cells are subcultured onto a glass substrate 12-48 hours prior to analysis, just as described for the measurement of glutathione content. Cells are noninvasively labeled with the acetoxymethyl ester of indo-1. A stock solution of 1 mM indo-1/AM is prepared in dimethyl sulfoxide (DMSO) and diluted with serum-free, phenol red-free medium to 1 /zM (0.05% final DMSO concentration) for loading cells in culture dishes. Typically, loading of astroglial cells with indo-1 requires 1 hour. Following incubations, cells are washed once with PBS followed by three washes with serum-free, phenol red-free medium. Cells are then scanned with an excitation wavelength of 351.1-363.8 nm and emitted fluorescence is monitored simultaneously at 485 and 405 nm. Figure 7 shows a paired emission wavelength scan of astroglial cells in culture labeled with indo-1. Fluorescence intensity values are then compared to a calibration curve generated by monitoring fluorescence of indo-1 free acid and Ca 2+ added to known concentrations in a physiological buffer solution (10 mM MOPS, 115 mM KC1, 20 mM NaCI, 1 mM M g S O 4 , and 1 mM EGTA). A standard 0.1 M calcium solution (Orion) is used to generate the calibration curve. Fluorescence measurements are collected from 10 cells per dish and four culture dishes for each experimental treatment group.

156

PARADIGMS OF NEURAL INJURY Col

Detector

1

Data

or

Values

-

2500

-

2362

t

-:

.....

-

2224

-

-

2087

-

-

1 9 4 9

-

-

1 8 1 2

-

-

1674

-

-

1537

-

-

1399

-

-

1261

-

-

1 1 2 4

-

-

986

-

-

849

-

-

711

-

-

574

-

-

436

-

-

2995

-

....

Detector

2

Data

........... ....

FIG. 7 One of a temporal series of digital fluorescence images of the Ca 2+-sensitive ratiometric probe, indo- 1, excited with UV light (351-363 nm) and recorded simultaneously at 485 nm (detector 1, image at left) 405 nm (detector 2, image at right). Unbound indo-1 emits at 485 nm and CaZ+-bound indo-1 emits at 405 nm. By computing the ratioed fluorescence (detector 2/detector 1 = 405/485), quantification of free Ca 2+ within the cell can be determined. Direct quantification of Ca 2+ content in cells is then performed by comparing the ratio of emissions to that generated in a standard curve. More rapid detection of changes in intracellular [Ca2+] can also be performed by monitoring ratioed fluorescence from line scans through cells or by monitoring ratioed fluorescence changes within a single point in a cell.

Interpretation and Limitations of[Ca e+] Measurements The image analysis procedure discussed provides a method for measuring basal [Ca2+]~ with indo-1 that is rapid, noninvasive, and can be applied to many cells simultaneously. In addition, the same procedures may be used to measure the ability of cells to recover from deliberate perturbations in Ca 2+ homeostasis as a function of treatment with a toxic metal. For example, the Ca 2+ ionophore ionomycin (final concentration 1.0 ~ M in <0.05% DMSO) causes a rapid influx of Ca 2+ into the cytosol, which peaks within 30 seconds after addition to the medium (Fig. 8). [Ca2+]i returns to basal levels within several minutes in normal astroglia as a result of Ca2+-ATPases pumping Ca 2+ out of the cell or into organellar depots. The basal level of indo-1 ratioed fluorescence ( F 0) and the peak level of ratioed fluorescence (Fp) in control

157

[10] HEAVY METAL E F F E C T S ON GLIA 1.2 1.1

1.0

0.9

0.8

0.6

t i i i I i i i i ! i i i i i i i i i i i i i i i i I

0

1O0

200

300

400

500

Calcium (Units) vs. Time (sec)

600

FIG. 8 An illustration of changes in indo-1 ratioed fluorescence over time within a single astroglial cell. Basal indo-1 fluorescence was recorded for the first 60 seconds. Next, 1 /xM of the Ca 2+ ionophore, ionomycin, was added at the time indicated by the vertical line, resulting in a rapid increase in intracellular [Ca2+], which returned to baseline over the next 6-7 minutes. Although the data shown are for a single cell, recordings from multiple cells are typically collected simultaneously.

cells can be expressed as a ratio (Fp/F0) and used as a reference point for altered C a 2+ homeostasis. Previous work has shown that this ratio is a more sensitive indicator of cell injury than [Ca2+]i, because a significant depression of Fp/Fo can be detected in toxin-treated cells before significant changes in [Ca2+]i are observed (32). A second modified application of the indo-1 procedure is to add the ionophore to cultures in calcium-free Tris-buffered saline, rather than medium, in order to assess fluxes of internal calcium stores. A third application is to a s s e s s C a 2+ permeability across cell membranes by measuring [Ca2+]i immediately after the addition to the culture of agents that open or block C a 2+ channels, as well as agents that release or prevent the release of C a 2+ from intracellular depots. In each case cellular responses can be compared between control cultures and cultures treated with metals as a potential indication of cell injury. As suggested earlier, the presence of Pb in cells presents special problems when measurements of [Ca2+]i are attempted with fluorescent probes. In the case of indo-1, we have recently found that Pb 2+ reacts strongly with indo-1 under the experimental conditions described (Fig. 9). However, the emission

158

PARADIGMS OF NEURAL INJURY .6.5

I

0 O9

.4 0

CD

mM

.3

TPEN uM

uM

EK

.2 .1 0.0

i

0.4

0.8 1.2 1 5 i0 0 CONCENTRATION

i

i

i

I00

FIG. 9 A ratiometric calibration curve that examines indo-1 fluorescence during sequential addition of increasing concentrations of Ca 2+, Pb 2+, and the heavy metal chelating agent N, N, N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). The ratio of bound-to-unbound indo-1 fluorescence was monitored during addition of increasing [Ca 2+] (0.2-1.2 mM in 0.2 mM increments) until saturation. Once saturation of indo-1 fluorescence was obtained, Pb 2+ was added (1.0, 5, and 10/zM). Addition of Pb 2+ resulted in an increase in the 405/485 fluorescence ratio. Subsequent addition of TPEN (100/xM) returned the fluorescence ratio to the pre-Pb 2+ treatment level, i.e., the Ca 2+ saturation level.

spectra of pb2+-indo-1 and CaZ+-indo-1 differ sufficiently from each other that it may be possible to detect Pb 2+ entry into cells with indo-1 (33). Several experimental approaches have been used to analyze PbZ+-Ca 2+ interactions in cells, particularly the perturbation of Ca 2+ stores and the ability of Pb 2+ to act as a Ca 2+ surrogate. Before the advent of methods to investigate changes in intracellular Ca 2+ concentrations by the use of fluorescent probes, radioisotopes were used to label internal divalent cation pools (both Ca 2+ and Pb 2+) and their efflux was measured. This method was not sensitive to rapid, quite small changes in cytosolic [Pb 2+] or [Ca2+]. More recently, two CaZ+-binding indicators, 5F-1,2-bis(o-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid (5F-BAPTA) and fura-2, have been used to measure changes in both intracellular Ca 2+ and Pb 2+. 5F-BAPTA binds many metals, but each produces a different chemical shift that is detectable by 19F nuclear magnetic resonance (NMR) spectroscopy. As previously mentioned, fura-2 is a commonly used Ca 2+ indicator that can be analyzed by dualexcitation spectrofluorometry because the excitation spectra of the free fura2 molecule and its Ca 2+ complex are different. 5F-BAPTA, like fura-2, may

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159

be introduced into living cells as membrane-permeable acetoxymethoxy derivatives. Schanne et al. (34) exploited the capacity of 19F NMR spectroscopy to quantitate distinct signals simultaneously emitted by complexes formed between the 5F-BAPTA and C a 2+ or Pb 2+ in 5F-BAPTA-Ioaded cells. These investigators demonstrated the twofold elevation of [Ca2+]i in a mouse neural cell line after exposure for 2 hours to 5/xM lead acetate, with a concomitant increase of [pb2+]i to 30 pM. This study has been the only one to date to m e a s u r e [pb2+]i and to demonstrate that [Ca2+]i is sensitive to Pb treatment in a cell line of neural origin. 19F NMR is an attractive technique for measuring intracellular [pb2+], except for the considerable expense of the technology, which limits the availability of the required instrumentation. Tomsig and Suszkiw (35) used fluorescence cytometry with fura-2 to explore whether subsequent Pb-induced events are mediated by [Ca2+]i or by [PbZ+]i, given that Pb treatment elevates intracellular C a 2+. These investigators showed that Pb 2+ can trigger the release of norepinephrine from isolated bovine chromaffin cells, apparently acting as a potent Ca 2+ surrogate. Cells were loaded with fura-2, which binds C a 2+ with high affinity (Kd = 2 x 10 -7 M ) , but was also shown to bind 1 : 1 with Pb 2+ at a much higher affinity (Kd = 4 x 10 -12 M ; Kd values are from Ref. 30). The excitation spectra of fura-2 in the presence of saturating concentrations of either Pb 2+ or C a 2+ are largely overlapping but sufficiently distinct to be analyzed by recording the 340/380-nm ratio of fluorescence. Spectra recorded from fura-2-1oaded cells that were incubated in Pb 2+ buffer solutions indicated the entry of Pb 2+ into the cells at a level (1-10 pM) proportional to extracellular [pb2+]. Extracellular Pb 2+ also triggered [3H]norepinephrine release from intact fura-2 loaded cells. The possibility that Pb 2+ induces secretion through recruitment of Ca 2+ from internal stores in intact cells could not be excluded by this experimental technique. Furthermore, Simons (30) has subsequently shown that fura-2 also forms a complex with Z n 2+ (K d = 1 x 10 -9 M ) with a fluorescence excitation spectrum similar to that of Pb 2+ and a stronger fluorescence yield. Nevertheless, fura-2 should be useful for measuring changes in intracellular [pb2+], though not for determining absolute Pb 2+ values.

Analysis of Gap Junctional Intercellular Communication

Rationale for Measuring Gap Junctional Intercellular Communication Gap junctions are communication channels that allow the diffusion of small molecules (up to 1000 Da, or 1.5 nm in diameter) or electrical signals from the cytoplasm of one cell to another. A gap junction is composed of aggregates

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PARADIGMS OF N E U R A L INJURY

of connexons located on the plasma membranes of two closely apposed adjacent cells. Each connexon consists of six polypeptides (connexins) that span the plasma membrane and line a central pore. The connexons of two cells align coaxially to form a cytoplasmic continuity between the interiors of apposed cells, thus facilitating direct intercellular communication. The effects of neurotoxicants on gap junctional intercellular communication (GJIC) are of interest because of the importance of gap junctional contact in cellular homeostasis. Astroglial gap junctions are believed to be important in the regulation of ionic environment (36) and cellular signal transduction (37), and as participants in a pulsatile calcium communication network throughout the central nervous system (CNS) (38). Therefore, gap junctional communication may be used in chemical signaling as well as in a metabolic buffering capacity by passage of low molecular weight substances from one cell to another. Most gap junction channels are rapidly closed by cytoplasmic acidification (36) and by nonphysiological levels of [ C a 2 + ] i . Closure of gap junctions is thought to provide a general mechanism to seal off unhealthy or injured cells from healthy members of a physiologically coupled cellular community (39).

Method for Measurements of Gap Junctional Intercellular Communication Gap junctional intercellular communication can be measured by methods based on the transfer of low molecular weight fluorescent dyes between adjacent, communicating cells. The method described is a modification of the fluorescence recovery after photobleaching (FRAP) technique (40). FRAP applied to gap junctions (gap FRAP) makes use of 5-carboxyfluorescein diacetate (CFDA; Molecular Probes) for loading cells. CFDA is a nonfluorescent, membrane-permeable dye that is hydrolyzed in the cytoplasm by nonspecific esterases to yield a fluorescent, membrane-impermeant dye, 5carboxyfluorescein (CF). Cells are subcultured at a medium density (to yield a growth pattern consisting of small groups or pairs of cells) into 35-mm plastic tissue culture dishes. After 12-24 hours GJIC is measured by dye coupling in the culture dish with the ACAS 570. Loading of the cells with dye is performed by incubating the cultures in situ with 10/~g/ml CFDA (prepared from a stock solution that is 2 mg/ml in DMSO) in HEPES-buffered medium without serum or phenol red for 10 minutes at 37~ Cultures are then washed four times with serum-free medium without phenol red. A microscopic field containing aggregates of cells is selected for analysis. Several abutting cells in

[10] HEAVY METAL E F F E C T S ON GLIA

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groups or in pairs are selected from each field for monitoring of fluorescence transfer at an excitation wavelength of 488 nm. Cellular fluorescence is imaged by scanning the cells on the motorized stage of the microscope in a two-dimensional raster pattern. Laser strength for scanning is adjusted to a level sufficient to excite CF fluorescence without causing significant photobleaching of the cells. One cell is photobleached in each pair or group by a series of higher intensity point bleaches produced by focusing the 488-nm argon ion laser beam through the microscope objective. The point bleaches photochemically reduce the amount of CF fluorescence in the cell. Laser strength of the point bleaches and number of bleaches per cell are carefully controlled so as to reduce fluorescence enough to measure recovery without causing visible cell damage at the light microscopic level. Isolated cells, which should not recover fluorescence, may be bleached as negative controls and other groups of cells left unbleached and demarcated as positive controls. A series of five postbleach image scans is generated at 1-minute intervals to measure subsequent redistribution of intracellular fluorescence through gap junctions. Figure 10 shows a typical analysis for astroglial-astroglial GJIC in culture. At least three analyses from each of three dishes per treatment group are conducted. Data (percentage of the prebleach fluorescence level recovered) are expressed as the mean _+ SEM. Fluorescence recovery can be compared between treatment groups at user-determined time points after photobleaching to assess the approximate level of communication. Dose responses can be determined in this manner. In experiments with astroglial cultures, we do not exceed 4 minutes postbleaching because of artifacts generated in subsequent measurements. Figure 11 shows a comparison of the effects of two Pb concentrations on GJIC in cultured astrog|ia, where Pb was found to produce no change from control values. Treatment groups can be compared more precisely by use of a curve-fitting regression analysis to extrapolate fluorescence recovery over time and thus determine the rate constant for fluorescence recovery. The rate constant k may be obtained from the following equation: F (t) = Feq(1 - e -kt) + F (0), where t is time after photobleaching, Feq is the percentage of fluorescence recovery of the bleached cell at equilibrium, e is the constant (the base of the natural system of logarithms), and F (0) is the percentage of fluorescence intensity immediately after photobleaching. The value of Feq depends on the number of cells in contact with each other and the initial level of photobleaching, F (0) (18).

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PARADIGMS OF N E U R A L INJURY

FIG. 10 Analysis of gap junction-mediated intercellular communication by fluorescence recovery after photobleaching (gap FRAP). The pseudocolor images produced by the ACAS 570 were reproduced on black and white film. Upper left: Image of cellular carboxyfluorescein fluorescence in an astroglial culture prior to photobleaching. Lighter areas within the cells reflect higher fluorescence intensity. Upper right: Cellular fluorescence intensity immediately after photobleaching cells 1 and 2. Cell 3 was selected as a nonphotobleached control to monitor potential background photobleaching from fluorescence image scanning. Cellular fluorescence was recorded after 4 minutes (lower left) and revealed an increase in fluorescence in cells 1 and2 that was contributed by contacting cells via gap junctional contacts. Lower right: Kinetics of fluorescence recovery for the three cells. Note the increase in fluorescence in cells 1 and 2 over time, whereas the unbleached control cell 3 did not lose fluorescence.

Interpretation of Gap Junctional Intercellular Communication Measurements The experimental approach for direct monitoring of cell-cell communication by means of gap FRAP offers many advantages over other direct physiological methods of junction permeability measurement. Unlike the " s c r a p e load-

[10]

163

H E A V Y M E T A L E F F E C T S ON G L I A 40

] / /

I"1

control

9 II.IMPb 30

E"

8 (D tQ) O (/)

20

c O ',1--

10'

0

1

2

3

4

time (minutes after photobleaching

FIG. l 1 Gap FRAP analysis of astroglial gap junctional communication after exposure to lead acetate (1.0/~M). No effect on junctional cell-cell communication was seen after 5 days of repeated daily exposure to Pb, either in terms of total recovery of fluorescence or percentage of recovery 1-4 minutes after photobleaching. Reproduced from Ref. 26, with permission.

ing" method with Lucifer Yellow or fluorescent dye microinjection, gap FRAP is noninvasive. Furthermore, there is no loss of temporal resolution as with microinjection methods. Dual voltage clamping is a very sensitive method for evaluating junctional communication, but its use is restricted to the measurement of the electrical properties of gap junctions in paired cells. Perhaps the greatest advantages of the gap FRAP method are that appropriate controls can be run in the same microscopic field of scanning (and hence the same culture dish) and that multiple measurements can be conducted on the same cell without traumatic manipulations. As many as five consecutive gap FRAP assays on the same cells is possible (41). As shown in Fig. 11, GJIC in astroglial cultures is not affected by exposure over several days to micromolar Pb concentrations. This finding is consistent with an earlier report that several heavy metals at sublethal concentrations (Ni, Cd, Pb, and Cr) are not potent inhibitors of communication in Syrian hamster embryo primary cultures (42). However, we have also found that

164

PARADIGMS OF N E U R A L INJURY 50,

40

> o O tD

rr

30

20

10 -

=

Fe4 Fe5 control

0

Time (rain) FIG. 12 Gap FRAP analysis of astroglial gap junctional communication after exposure to 0, l0 (FeS), or 100/~M (Fe4) FeC12 . In this experiment, cells were exposed daily for 7 days to the Fe dose indicated. For each datum 15-19 cells were measured. Error bars represent standard errors. At each time point measured (1-4 minutes) the mean percentage of recovery was significantly higher in the Fe4 group than in the other two groups. The rate constant (k) for diffusion of CFDA into the photobleached cell was calculated, which is proportional to the permeability of the channel. The k value was calculated to be 0.3425, 0.3784, and 0.5275 for the 0, FeS, and Fe4 groups, respectively. Thus the permeability of the gap junctional channels was increased by over 40% in cells treated with 100/zM Fe (Fe4) compared to the other treatment groups (p < 0.01).

Fe up-regulates gap junctional communication, as shown in Fig. 12. The dose of Fe (100/~M) selected was that which produces the same amount of metabolic injury in astroglia as 1 /~M Pb [as measured by reduction of glutamine synthetase (glutamate-ammonia ligase) specific activity]. The finding that Pb and Fe affect GJIC differently supports the concept that metals damage cells by distinct pathways, leaving a unique set of molecular end points, which we term their "signatures." It should be possible to develop a battery of sensitive assays to detect and characterize these unique signatures, particularly by the use of cellular fluorescence imaging techniques.

[10] HEAVY METAL EFFECTS ON GLIA

165

Acknowledgments Work in the laboratories of E.T.-C., R.C.B., and E.D.H. has been supported by grants from the NIH (R01-ES05871, R01-HD26182, and R01-HD29959) and an NIH Superfund Grant to Dr. Stephen Safe (P42-ES04917). M.E.L. and L.A.S. are recipients of Physician Scientist Awards (K11-ES00251 and K11-ES00279) from the NIH. We thank Jeff Bowen and Michelle Nyberg for excellent assistance in the preparation of the manuscript.

References

.

9. 10.

11. 12. 13. 14. 15.

16.

D. M. Danks, in "The Metabolic Basis of Inherited Disease" (C. R. Scriver, A. I. Beaudet, W. S. Sly, and D. Valle, eds.), 6th Ed., p. 1411. McGraw-Hill, New York, 1989. M. J. Davis, NeuroToxicology 11, 285 (1990). E. Tiffany-Castiglioni, NeuroToxicology 14, 513 (1993). S. W. Levison and K. D. McCarthy, in "Culturing Nerve Cells" (G. Banker and K. Goslin, eds.), p. 309. MIT Press, Cambridge, Massachusetts, 1991. K. D. McCarthy, J. Pharmacol. Exp. Therap. 226, 282 (1983). E. Tiffany-Castiglioni, J. Zmudzki, J. N. Wu, and G. R. Bratton, Metab. Brain Dis. 2, 61 (1987). D. Holtzman, C. DeVries, H. Nguyen, J. I. Olson, and K. Bensch, NeuroToxicology 5, 97 (1984). J. A. Thomas, F. D. Dallenbeck, and M. Thomas, J. Pathol. 109, 45 (1973). C. Vandecasteele and C. B. Block (eds.), "Modern Methods for Trace Element Determination." Wiley, New York, 1993. T. J. Kneip and L. Friberg, "Handbook On The Toxicology of Metals" (L. Friberg, G. F. Nordberg, and V. B. Vouk, eds.), 2nd Ed., Vol. I, p. 44. Elsevier, New York, 1986. Thermo Jarrel Ash Corp., "Methods Manual for Furnace Operation," Vol. II, 1993. T. J. Goka, R. E. Stevenson, P. M. Hefferan, and R. R. Howell, Proc. Natl. Acad. Sci. U.S.A. 73, 604 (1976). H. Kodama, Y. Meguro, T. Abe, M. H. Rayner, K. Y. Suzuki, and S. Kobayashi, J. Inherit. Metab. Dis. 14, 896 (1991). H. Y. Darwish, R. C. Schmitt, J. C. Cheney, and M. J. Ettinger, Am. J. Physiol. 246, 648 (1984). R. P. Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals" (K. D. Larison, ed.). Molecular Probes Inc., Eugene, Oregon, 1992. D. L. Taylor and E. D. Salmon, in "Fluorescence Microscopy of Living Cells in Culture" (Y. Wang and D. L. Taylor, eds.), Part A, p. 207. Academic Press, San Diego, 1989.

166

PARADIGMSOF NEURAL INJURY 17. M. Schindler, M. H. Allen, M. R. Olinger, and J. F. Holland, Cytometry 6, 368 (1985). 18. R. C. Burghardt, R. Barhoumi, D. Doolittle, and T. D. Phillips, in "Principles and Methods of Toxicology" (A. W. Hayes, ed.), 3rd Ed., p. 1231. Raven Press, New York, 1994. 19. A. Meister, Pharmacol. Therap. 51, 155 (1991). 20. S. P. Raps, J. C. K. Lai, L. Hertz, and A. J. L. Cooper, Brain Res. 545, 312 (1989). 21. A. Jain, J. Martensson, E. Stole, P. A. M. Auld, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 88, 1913 (1991). 22. D. Holtzman, Toxicol. Appl. Pharmacol. 89, 211 (1987). 23. P. Goering, NeuroToxicology 14, 45 (1993). 24. P. S. Rabinovitch, C. H. June, and T. J. Kavanagh, in "Clinical Flow Cytometry: Principles and Application" (U. D. Bauer, R. E. Dunque, and T. V. Shanley, eds.), p. 505. Williams and Wilkins, Baltimore, Maryland, 1993. 25. R. Barhoumi, J. A. Bowen, L. S. Stein, J. Echols, and R. C. Burghardt, Cytometry 14, 747 (1993). 26. M. E. Legare, R. Barhoumi, R. C. Burghardt, and E. Tiffany-Castiglioni, NeuroToxicology 14, 267 (1993). 27. B. Ehrenberg, V. Montanta, M. D. Wei, J. P. Wuskell, and L. M. Loew, Biophys. J. 53, 785 (1988). 28. J. R. Bunting, T. V. Phan, E. Kamali, and R. M. Dowben, Biophys. J. 56, 979 (1989). 29. M. Poot, T. J. Kavanagh, H. C. Kang, R. P. Haugland, and P. S. Rabinovitch, Cytometry 12, 184 (1991). 30. T. J. B. Simons, NeuroToxicology 14, 77 (1993). 31. R. Y. Tsien, Rev. Neurosci. 12, 227 (1989). 32. R. C. Burghardt, R. Barhoumi, E. Lewis, R. H. Bailey, K. Pyle, B. Clement, and T. D. Phillips, Toxicol. Appl. Pharmacol. 112, 235 (1992). 33. M. E. Legate, R. Barhoumi, E. Hebert, R. C. Burghardt, and E. Tiffany-Castiglioni, in preparation. 34. F. A. X. Schanne, T. L. Dowd, R. K. Gupta, and J. F. Rosen, Proc. Natl. Acad. Sci. U.S.A. 86, 51 (1989). 35. J. L. Tomsig and J. B. Suszkiw, Am. J. Physiol. 259, C762 (1990). 36. J. J. Anders, Glia 1, 371 (1988). 37. E. L. Hertzberg and R. G. Johnson (eds.), "Gap Junctions of Modern Cell Biology." Alan R. Liss, New York, 1988. 38. A. H. Cornell-Bell, S. M. Finkbeiner, M. S. Cooper, and S. J. Smith, Science 247, 470 (1990). 39. W. R. Loewenstein, Am. Rev. Respir. Dis. 142, $48 (1990). 40. M. H. Wade, J. E. Trosko, and M. Schindler, Science 232, 525 (1986). 41. L. S. Stein, J. G. Boonstra, and R. C. Burghardt, In Vitro Cell Dev. Biol. 28A, 436 (1992). 42. S. O. Mikalsen, Carcinogenesis 11, 1621 (1990).

[11]

Source, Metabolism, and Function of Cysteine and Glutathione in the Central Nervous System David K. Rassin

Introduction In recent years there has been an explosion of interest in the role of glutathione; this tripeptide (y-glutamylcysteinylglycine) has been associated with antioxidant function (reviewed in Refs. 1-3), immunologic functions (4, 5), the disease process associated with human immunodeficiency syndrome (HIV) infection (6, 7), and modulation of neurotransmitter function in the central nervous system (CNS) (8, 9). Of particular interest with respect to neural injury are the antioxidant and excitatory amino acid modulatory roles of glutathione. Glutathione may directly modify oxidant stress or it may modulate release of excitatory amino acids by influencing the function of the N-methyl-D-aspartate (NMDA) receptor (8, 9). In addition, glutathione and cyst(e)ine may prevent excitatory amino acid-induced apoptosis by protecting against intracellular glutathione depletion (10, 11). Glutathione is readily synthesized in mammalian cells from its three precursor amino acids, glutamate, cysteine, and glycine. However, glutathione does appear to be most dependent on the availability of cysteine in order to maintain organ pools (12, 13). Cysteine may fill many of the functions supported by glutathione, reflecting the common structural feature of these two compounds, the sulfhydryl group. Cysteine is usually synthesized from the essential amino acid methionine via the transsulfuration pathway (Fig. 1); however, there are several circumstances, particularly during early development, wherein cysteine may have to be supplied in the diet due to the low hepatic activity of cystathionase, the enzyme responsible for catalyzing cysteine synthesis (12, 13). In the following presentation the origin and metabolism of cysteine and glutathione will be discussed with attention to some of the methods that have been particularly useful in investigating their characteristics.

Cysteine Cysteine is a sulfur-containing amino acid that is synthesized from the essential amino acid methionine via the transmethylation and transsulfuration pathways (Fig. 1). Cysteine may be made available to the central nervous Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

167

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PARADIGMS OF N E U R A L INJURY

PROTEIN <

METHIONINE

~L I MethioninAdenosyl e transferase I~

8 - ADENOSYLMETHIONINE I

MethylTransferase~E

S-ADENOSYL~OMOCYSTEI

]

\

N-Methyltetrahydrofolate: Homocysteinmethyl e transferase

~ [ SAHHydrolase] ~ HOMOCYSTEINE

J/ ~ Cystathionin13-Synthase e ]

CYSTATHIONINE PROTEIN <

J/ i Cystathionase]

CYSTEINE

>

GLUTATHIONE

/+ [ CysteineDioxygenaseI

CYSTEINESULFINIC ACID

kj/ [ CysteinesulfinDecarboxyl ic aseI

HYPOTAURINE

J/ ] HypotaudneOxidase]

TAURINE

FIG. 1 The metabolic pathway of methionine metabolism leading to cysteine synthesis. system either by transport from the periphery or by endogenous synthesis. The presence of significant amounts of cystathionine, the sulfur-ether precursor of cysteine, and relatively low activities in brain of the enzyme responsible for catalyzing cysteine synthesis, cystathionase, have suggested that transport into the brain is a primary source of cysteine (16-18). However, more recent studies have found cystathionase activity in brain, though less than that observed in other tissues (19). In general, cysteine concentrations are very low in central nervous system tissue (20). Most of the available cysteine may be utilized for glutathione synthesis, thus glutathione may serve as a cysteine reservoir (21-22). Complicating the evaluation of the role of cysteine in the CNS is the reactivity of its sul~ydryl group resulting in the ready formation of disulfides. Cysteine will form a variety of mixed disulfides with proteins and glutathione in addition to the homogeneous disulfide, cystine (Fig. 2). Thus, total cyst(e)ine [this terminology has been used to reflect all forms (sum of sulfhydryl and disulfides) of cyst(e)ine] may be more reflective of cysteine status than the usual measurements of the oxidized form, cystine, by amino acid analysis.

169

[11] CSH AND GSH IN THE CNS H2N-CH-COOH I

HOOC-CH-CH2-CH2-CO-NH-CH-CO-NH-CH2-COOH I

CH2I

I

NH2

CIH2

SH

SH

Cysteine (CSH)

7-Clutomylcysteinylglycine (Clutothione) (GSH)

2 G S H -- G S S G

Oxidized Glutathione

2 C S H -~ C S S C

Cystine

GSH + CSH - GS-SC

Glutothione-Cysteine Mixed Disulfide

G S H + Protein-SH -- GS-SProtein

Glutathione-Protein Mixed Disulfide

C S H + Protein-SH -- CS-SProtein

Cysteine-Protein Mixed Disulfide

FIG. 2 The various chemical forms in which cyst(e)ine and glutathione may be found in vivo. A similar group of reduced and oxidized forms of cysteine, glutathione, and protein may be formed with homocysteine.

To address this issue we have developed a procedure to measure total and bound cyst(e)ine (23). This procedure has affirmed that the body pools of cyst(e)ine are considerably greater than usually appreciated; for example, previous studies indicated about 7/~mol/100 g of cyst(e)ine in brain (24-26); however, when our methodology was applied, amounts in the order of 75 /~mol/100 g of brain were found (27). The following section details the application of this procedure.

Assay o f Total and Fractions of Cyst(e)ine Total cyst(e)ine and the protein-bound fraction are measured by the method of Malloy and co-workers (23, 27, 28) utilizing the colorimetric assay of Gaitonde (29). Tissue is homogenized in water (1 : 5, wv) and an aliquot (1 : 5, vv) treated with 10% trichloroacetic acid (TCA) to precipitate the proteins and bound cysteine. A pellet is prepared by centrifugation at 17,000 g for 10 minutes. Protein-bound half-cystine is determined by resuspending the protein pellet in TCA and recentrifuging as above. This supernatant wash is discarded and the protein pellet resuspended in 1.0 ml of 10 mM dithiothreitol (DTT). The suspended pellet is adjusted to pH 8-9 with 0.2 ml 1 N KOH and allowed to incubate at room temperature for 10 minutes. The reaction

170

PARADIGMSOF NEURAL INJURY

is stopped by addition of 0.2 ml 15% sulfosalicylic acid (SSA), the precipitate is removed by centrifugation as above, and the supernatant solution is then filtered through a 0.45-/~m Millipore syringe filter. The filtrate is then adjusted to pH 8-9 by addition of 0.2 ml 1 N KOH. Cysteine is determined in the original supernatant solution [free cyst(e)ine] and in the supernatant solution prepared from the pellet (bound cysteine) by the ninhydrin reaction. Samples (0.5 ml) and a standard curve and reagent blank are incubated on ice for 30 minutes with the addition of 0.5 ml (5/~mol) of DTT. After incubation, 1 ml glacial acetic acid and 1 ml acid ninhydrin reagent (250 mg ninhydrin in 4 ml concentrated HC1 plus 6 ml glacial acetic acid) are added and the tubes heated in a boiling water bath with caps for 10 minutes. Tubes are removed from the boiling water, placed in an ice bath, and 7 ml of cold 95% ethanol is added and then well vortexed. Absorbance is read at a wavelength of 560 nm and concentrations are calculated from the standard curve. The total cyst(e)ine may be expressed as the sum of the free and the percent bound.

Assay of Cystathionase Activity of the enzyme responsible for catalyzing cysteine synthesis, cystathionase, may be determined (14, 15, 30) in brain tissue by incubating aliquots of homogenates (usually 1 g tissue in 5 ml of pH 6.9, 0.03 M potassium phosphate buffer) at 37~ for 30 minutes in a total volume of 0.5 ml containing 50/~mol Tris-HC1 buffer, pH 8.4; 0.125 /~mol pyridoxal phosphate; and 2 /~mol L-cystathionine. A killed enzyme (30 seconds in a boiling water bath) blank is included to account for endogenous cyst(e)ine. The reaction is halted by cooling in an ice-water bath. Dithiothreitol (ice-cold), 5/~mol in 0.5 ml H20, is added and allowed to stand for 30 minutes to reduce all the cyst(e)ine to cysteine. The amount of cysteine produced is then determined by the acid-ninhydrin method described above (23, 29) and enzyme activity is calculated as nanomoles of cysteine produced per hour per milligram of protein.

Glutathione Glutathione is an important component in the detoxification of xenobiotics in the liver, usually involving the glutathione S-transferases to catalyze the formation of excretable conjugates. Glutathione S-transferase activity exists in the central nervous system (3 l) but is of unknown importance. Glutathione appears to function as an intracellular reducing agent (2), as an antioxidant

171

[11] C S H A N D G S H I N T H E C N S

GSSG

8

7

GSH

GammaGlu-AA + Cys-Gly

Oxoproline

3

>

Giu ~

4

FIG. 3 Some aspects of the metabolism of glutathione. 1, y-Glutamyltransferase; 2, y-glutamylcyclotransferase; 3, 5-oxoprolinase; 4, y-glutamylcysteine synthetase (glutamate-cysteine ligase); 5, glutathione synthetase; 6, dipeptidase; 7, glutathione reductase; 8, peroxidases.

(2), as a modulator of the N-methyl-D-aspartate receptors (8, 9), and as a component of the 7-glutamylamino acid transport cycle in the CNS (32) (see Fig. 3). The role of glutathione in protecting against antioxidant damage has become more appreciated with the implication of oxidant stress in the neuropathology associated with hypoxia, hyperoxia, Alzheimer's disease, Parkinson's disease, and aging (33-39). The relative amounts of reduced (GSH) and oxidized (GSSG) glutathione may serve as markers of oxidant stress, thus in situations of oxidative attack the GSSG/GSH ratio increases as an index of such stress. Protection against oxidative stress depends on maintenance of intracellular GSH concentrations, and such maintenance depends on the availability of precursor cysteine (21). Glutathione may be protective both as a function of its role in modulating the excitotoxic effects of glutamate (10-11) and as a function of its role in protecting against the metabolic continuum, from oxidant attack to energy depletion to neurotrophin disruption to cell death in the CNS (40). These considerations have emphasized the need for measures of glutathione, oxidized and reduced, in the CNS. Such methodology has been reviewed

172

PARADIGMS OF NEURAL INJURY

(41-42), and the two most useful techniques are the spectrophotometric method developed by Tietze (43) as modified by Griffith (44), and highperformance liquid chromatographic methodology using dual-cell electrochemical detection (45).

Spectrophotometric Assay of Glutathione Total and oxidized glutathione are measured using an enzymatic recycling method (based on glutathione reductase) and the GSH blocking agent, 2vinylpyridine (43, 44). Tissues are homogenized directly in 3% sulfosalicylic acid (w/v) (preferable technique) or first in a phosphate buffer and then treated with sulfosalicylic acid. Rapid preparation is necessary to minimize autoxidation of glutathione to the disulfide form. The protein precipitate is removed by centrifugation (17,000 g for 15 minutes) at 5~ and the supernatant solution is used for the glutathione assay. Four tubes are prepared for each sample; 200/zl of unknown is added to each tube. Two tubes have 4/zl of 2-vinylpyridine added (this should be a clear liquid; obtain fresh material if an amber color is present). Add 12/~1 of triethanolamine to all tubes to raise the pH to 7-7.5. Mix for 1 minute and let stand at room temperature for 60 minutes. Place 200 ~1 of sample into a 1.5-ml cuvette, add 700/zl of NADPH (0.3 mM in EDTA-phosphate buffer, pH 7.5), 100/zl of DTNB (6.0 mM in EDTA-phosphate buffer, pH 7.5), and 10/zl of glutathione reductase (50 units/ml), invert cuvette two or three times to mix, place immediately into a recording spectrophotometer set at a wavelength of 412 nm, and measure change in absorbance for 120 seconds. The slope of the line is calculated and compared to a standard curve prepared from slopes given by different known amounts of glutathione. Glutathione measured in the tubes with added 2-vinylpyridine represents the oxidized form and is subtracted from tubes without 2-vinylpyridine (total glutathione) to calculate the amount of reduced glutathione.

HPLC Assay of Glutathione In cases in which direct measurement of oxidized and reduced glutathione in the presence of cysteine and cystine is desired at high sensitivity, highperformance liquid chromatography with dual-cell electrochemical detection is useful. Tissue samples may be prepared as described above in sulfosalicylic acid. These samples are then injected onto a 5-~m C18 reversed-phase cartridge column (22 cm • 4.6 mm; Pierce, Rockford, IL) with a 3-cm guard column packed with the same stationary phase. Compounds are eluted with

Ill]

173

CSH A N D G S H IN T H E CNS

A

B

=

-lto

to co

,+ -r to

cO to to

!

03

UPSTREAM ELECTRODE OFF

to to

-r" to (.9 r

, 9

. L'~

UPSTREAM ELECTRODE ON

F I G . 4 The separation and detection of glutathione and cyst(e)ine by HPLC with dual-cell electrochemical detection. (A) Upstream electrode is off, so only the already reduced (sulfhydryl) compounds are detected; (B) upstream electrode is on, and all four forms of the two compounds are detected.

96% monochloroacetic acid at pH 3.0 and 4.0% methanol containing 1.0 mM sodium octyl sulfate as an ion-pairing agent. Dual, serially assembled mercury-gold electrodes are prepared (46). Cysteine, cystine, oxidized glutathione, and reduced glutathione separate on the column and then are all modified to their reduced forms by the first (upstream) electrode set at - 1 . 0 V. The detector (downstream) electrode, set at 0.15 V, then is used for quantitation based on the oxidation potential of the reduced compounds (Fig. 4).

Inhibitory Agents Agents that inhibit the synthesis of glutathione have been particularly useful in elucidating the metabolism of this tripeptide (47). The most widely used inhibitor has been buthionine sulfoximine (BSO), an analog of the glutamine synthetase (glutamate-ammonia ligase) inhibitor methionine sulfoximine. Administration of BSO effectively inhibits y-glutamylcysteine synthetase by binding to the enzyme. BSO effectively reduces GSH synthesis in brain of newborn animals, but is not as effective in adult animals, presumably due

174

PARADIGMS OF N E U R A L INJURY

to poor transport of the agent across the blood-brain barrier (48). Administration of BSO directly to the brain and in solutions of dimethyl sulfoxide (DMSO) represent alternative techniques to bypass the problem of transport into the CNS, and are effective in reducing glutathione synthesis (49, 50). A second pharmacologic agent that has been useful in the manipulation of both cysteine and glutathione is the cystathionase inhibitor, propargylglycine (PPG). PPG inhibits cystathionase noncompetitively and irreversibly, limiting the conversion of cystathionine to cysteine (51, 52). Adult rats treated with PPG (40/xmol/day) for 15 days had about a 50% reduction in plasma cystine with a 200-fold increase in plasma cystathionine, the immediate cysteine precursor (53). Brain glutathione was reduced by approximately 20% in these studies; unfortunately the cystathionine and cysteine in brain were not reported. Other studies have shown that animals treated with increased dietary methionine accumulate cystathionine in the central nervous system, reflecting a possible metabolic block and indicating that peripheral cysteine synthesis may be required to support transport of cysteine into the CNS followed by in situ synthesis of glutathione in the CNS (27, 28, 54). Several repletion agents are available to assess the specificity of the inhibitory agents noted above. Poor absorption of glutathione and cysteine oxidation to the more insoluble cystine are factors that complicate direct use of these metabolites. However, glutathione esters, particularly the monoester, appear to be readily absorbed and efficiently replete glutathione when it is depleted by BSO treatment (55). In like manner the cysteine analog, Nacetylcysteine, is readily absorbed and converted to cysteine, in which form it also can further promote glutathione synthesis (56-58). Thus, the tools to deplete and replete cysteine and glutathione are available for further investigation of the roles of these compounds in the central nervous system.

Conclusion Glutathione and cysteine have important roles as antioxidant protective agents in the CNS. The increasing evidence that protection of CNS tissue from oxidative stress is an important mechanism in preventing neuronal cell death is leading to better understanding of some of the degenerative processes that attack the CNS. Two studies have investigated mechanisms of oxidatively induced cell death or apoptosis in cultured embryonic cortical neurons (10, 11). Hypothesizing that oxidant cell death is mediated through excitatory amino acids, these studies explored the role of glutathione and cysteine in these cells on treatment with homocysteate or glutamate (11). The protective effect of N-acetylcysteine and depletion of glutathione by homocysteate led

[11] CSH AND GSH IN THE CNS

175

to the suggestion that cells are protected by cystine uptake into the cell and conversion to glutathione (11). The above described protective mechanisms suggest that if indeed diseases such as Alzheimer's and amyotrophic lateral sclerosis involve oxidative stress and cell death, antioxidant administration may offer some hope of ameliorating the damage. Antioxidant maintenance in the CNS appears to require cyst(e)ine transport followed by glutathione synthesis in situ. Thus, agents that promote CNS glutathione synthesis may be important in altering the cascade of events that is initiated by any form of oxidative stress, and maintaining homeostasis requires an appropriate supply of cyst(e)ine.

References 1. A. Meister, in "Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects" (A. Larsson, S. Orrenius, A. Holmgren, and B. Mannervik, eds.), pp. 1-22. Raven Press, New York, 1983. 2. A. Meister, in "Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, O. Avramovi6, and R. Poulson, eds.), Part A, pp. 1-48. Wiley, New York, 1989. 3. M. Taniguchi, K. Hirayama, K. Yamaguchi, N. Tateishi, and M. Suzuki, in "Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, R. Poulson, and O. Avramovi6, eds.), Part B, pp. 645-727. Wiley, New York, 1989. 4. W. Dr6ge, H. P. Eck, H. GmOnder, and S. Mihm, Am. J. Med. 91, 140S144S (1991). 5. M. K. Robinson, M. L. Rodrick, D. O. Jacobs, J. D. Rounds, K. H. Collins, I. B. Saporoschetz, J. A. Mannick, and D. W. Wilmore, Arch. Surg. 128, 29-35 (1993). 6. R. Buhl, K. J. Holroyd, A. Mastrangeli, A. M. Cantin, H. A. Jaffe, F. B. Wells, C. Saltini, and R. G. Crystal, Lancet 2, 1294-1297 (1989). 7. S. Mihm, J. Ennen, U. Pessara, R. Kurth, and W. Dr6ge,AIDS 5,497-503 (1991). 8. D. I. Levy, N. J. Sucher, and S. A. Lipton, NeuroReport 2, 345-347 (1991). 9. N. J. Sucher and S. A. Lipton, J. Neurosci. Res. 30, 582-591 (1991). 10. R. R. Ratan, T. H. Murphy, and J. M. Baraban, J. Neurosci. 14, 4385-4392 (1994). 11. R. R. Ratan, T. H. Murphy, and J. M. Baraban, J. Neurochem. 62, 376-379 (1994). 12. A. Meister, Nutr. Rev. 42, 397-410 (1984). 13. A. Meister, in "Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, O. Avramovi6, and R. Poulson, eds.), Part A, pp. 367-474. Wiley, New York, 1989. 14. J. A. Sturman, G. E. Gaull, and N. C. R~iih~i, Science 169, 74-76 (1970). 15. G. E. Gaull, J. A. Sturman, and N. C. R. R~iih~i,Pediatr. Res. 6, 538-547 (1972). 16. H. H. Tallan, S. Moore, and W. H. Stein, J. Biol. Chem. 230, 707-716 (1958). 17. T. L. Perry, K. Berry, S. Hansen, S. Diamond, and C. Mok, J. Neurochem. 18, 513-519 (1971).

176

PARADIGMSOF NEURAL INJURY 18. S. H. Mudd, J. D. Finkelstein, F. Irreverre, and L. Laster, J. Biol. Chem. 240, 4382-4392 (1965). 19. J. A. Sturman, D. K. Rassin, and G. E. Gaull, Int. J. Biochem. 1, 251-253 (1970). 20. R. K. Shaw and J. D. Heine, J. Neurochem. 12, 151-155 (1965). 21. N. Tateishi, T. Higashi, A. Naruse, K. Nakashima, H. Shiozake, and Y. Sakamoto, J. Nutr. 107, 51-60 (1977). 22. E. S. Cho, N. Sahyoun, and L. D. Stegink, J. Nutr. 111, 914-922 (1981). 23. M. H. Malloy, D. K. Rassin, and G. E. Gaull, Anal. Biochem. 113,407-415 (1981). 24. T. L. Perry, S. Hansen, K. Berry, C. Mok, and D. Lesk, J. Neurochem. 18, 521-528 (1971). 25. T. L. Perry, H. D. Sanders, S. Hansen, D. Lesk, M. Kloster, and L. Gravlin, J. Neurochem. 19, 2651-2656 (1972). 26. H. H. Tallan, D. K. Rassin, J. A. Sturman, and G. E. Gaull, in "Handbook of Neurochemistry" (A. Lajtha, ed.), 2nd Ed., Vol. 3, pp. 535-558. Plenum, New York, 1983. 27. M. H. Malloy and D. K. Rassin, Pediatr. Res. 18, 747-751 (1984). 28. M. H. Malloy, D. K. Rassin, W. C. Heird, and G. E. Gaull, Am. J. Clin. Nutr. 34, 1520-1525 (1981). 29. M. K. Gaitonde, Biochem. J. 104, 627-633 (1967). 30. G. E. Gaull, D. K. Rassin, and J. A. Sturman, Neuropdediatrie 1, 199-266 (1969). 31. M. Das, R. Dixit, P. K. Seth, and H. Mukhtar, J. Neurochem. 36, 1439-1442 (1981). 32. A. Meister, Science 180, 33-39 (1973). 33. J. D. Adams, Jr., L. K. Klaidman, I. N. Odunze, H. C. Shen, and C. A. Miller, Mol. Chem. Neuropathol. 14, 213-226 (1991). 34. G. Benzi, F. Marzatico, O. Pastoris, and R. F. Villa, J. Neurosci. Res. 26, 120-128 (1990). 35. A. Meister, Biochem. Pharmacol. 44, 1905-1915 (1992). 36. K. Kramer, H.-P. Voss, J. A. Grimbergen, C. Smink, H. Timmerman, and A. Bast, Gen. Pharmacol. 23, 105-108 (1992). 37. A. Pean, G. B. Steventon, R. H. Waring, H. Foster, S. Sturman, and A. C. Williams, J. Neurol. Sci. 124, 59-61 (1994). 38. M. A. Verity, NeuroToxicol. 15, 81-92 (1994). 39. S. Fahn and G. Cohen, Ann. Neurol. 32, 804-812 (1992). 40. Z. Pan and R. Perez-Polo, J. Neurochem. 61, 1713-1721 (1993). 41. R. C. Fahey, in "Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, O. Avramovi6, and R. Poulson, eds.), Part A, pp. 303-307. Wiley, New York, 1989. 42. M. E. Anderson, in Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, O. Avramovi6, and R. Poulson, eds.), Part A, pp. 339-365. New York, 1989. 43. F. Tietze, Anal. Biochem. 27, 502-522 (1969). 44. O. W. Griffith, Anal. Biochem. 106, 207-212 (1980). 45. L. A. Allison, J. Keddington, and R. E. Shoup, J. Liq. Chromatog. 6, 17851798 (1983). 46. Anonymous, LCEC Application Note No. 53, pp. 1-3 Bioanalytical Systems Inc., West Lafayette, Indiana, 1983.

[111 CSH AND GSH IN THE CNS

177

47. A. Meister, Pharmacol. Therap. 51, 155-194 (1991). 48. O. W. Griffith and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 76, 5606-5610 (1979). 49. E. Pileblad and T. Magnusson, Neurosci. Lett. 95, 302-306 (1988). 50. R. Steinherz, J. M~trtensson, J. Wellner, D. and A. Meister, Brain Res. 518, 115-119 (1990). 51. W. Washtien and R. H. Abeles, Biochemistry 16, 2485-2491 (1977). 52. R. H. Abeles and C. T. Walsh, J. Am. Chem. Soc. 95, 6124-6125 (1973). 53. E. S. Cho, J. Hovanec-Brown, R. J. Tomanek, and L. D. Stegink, J. Nutr. 121, 785-794 ( 1991). 54. D. K. Rassin, in "Absorption and Utilization of Amino Acids" (M. Friedman, ed.), Vol. II, pp. 71-85. CRC Press, Boca Raton, Florida, 1989. 55. J. M~trtensson, R. Steinherz, A. Jain, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 86, 8727-8731 (1989). 56. R. J. Flanagan and T. J. Meredith, Am. J. Med. 91, 131S-139S (1991). 57. R. Ruffmann and A. Wendel, Klin. Wochenschr. 69, 857-862 (1991). 58. M. F. Banks and M. H. Stipanuk, J. Nutr. 124, 378-387 (1994).

[12]

Magnetic Resonance Spectroscopy of Neural Tissue R i c h a r d J. M c C l u r e , K a n a g a s a b a i P a n c h a l i n g a m , William E. K l u n k , a n d J a y W. P e t t e g r e w *

Introduction Magnetic resonance spectroscopy (MRS) is a powerful physical technique for examination of the quantity and structure of organic molecules in solution and in vivo to monitor metabolites noninvasively in intact animals and humans. MRS provides useful information about high-energy and phospholipid metabolism from observations of their characteristic metabolites. In this chapter we will (1) cover the key topics necessary to understand the MRS procedures used to examine neural tissue both in vitro and in vivo, (2) present 1H and 31p MRS methods to examine metabolite levels in autopsy brain tissue, and (3) discuss in vivo 31p MRS methods to monitor metabolite levels in Alzheimer's disease (AD) brain. The examples given to illustrate these procedures have been taken from studies done in this laboratory.

Terms and Theory This section provides an introduction to MRS terms and theory. More detailed explanations can be found in several reviews (1-3).

Nuclear Magnetic Moment The property of the atomic nucleus that allows it to produce an MRS signal in a magnetic field is its magnetic moment, a property arising from the intrinsic nuclear spin caused by an uneven number of protons and neutrons in the nucleus. In the nucleus, the proton and neutron spins combine to give a net spin (angular momentum) to the nucleus. In a number of nuclei, such as those in 1H, 13C, 19F, and 31p, the charges are distributed uniformly and

* T o w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

178

Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

[12] MRS OF NEURAL TISSUE

179

Spherical Non-Spinning Nucleus: !~ = 0 eQ = 0

Spherical Spinning Nucleus: I~ • 0 eQ = 0

Ellipsoidal (Prolate) Spinning Nucleus: I~ ~ 0 eQ>0

Ellipsoidal (Oblate) Spinning Nucleus: !~ • 0 eQ<0

FIG. 1 Representations of the different nuclear spin types. Reprinted from Neurobiol. Aging 15, Pettegrew, Panchalingam, Klunk, McClure, and Muenz. Alterations of cerebral metabolism in probable Alzheimer's Disease: A preliminary study, 117-132, Copyright 1994, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.

they act like spherical objects that spin like a top (Fig. 1). These nuclei have unpaired nuclear spins and are assigned spin values of 1/2. A large number of nuclei behave like spinning objects with nonspherical charge distribution. These nuclei are assigned spin values of 1 (2H and 14N) or integer multiples of 1/2 such as 3/2 (TLi, 23Na, 35C1, and 39K). The above two types of nuclei possess a nonzero angular momentum and are therefore MRS sensitive. Nuclei of biological interest with magnetic moments that can be measured by MRS are given in Table I. 1H and 31p are the nuclei most commonly utilized for biological applications. Nuclei that do not have a net spin are not MRS sensitive and do not give a signal.

Effect of External Magnetic Field and Applied Radio Frequency Pulse A magnetic resonance spectrometer consists of a strong magnet, a radio frequency (rf) transmitter, and an rf receiver. An rf pulse applied to MRSsensitive nuclei in the presence of an external magnetic field produces a signal. A group of spinning nuclei placed in a uniform magnetic field will

180

PARADIGMS OF NEURAL INJURY TABLE I

M R S - R e l a t e d Properties of Biologically U s e f u l N u c l e i a

m,,

Isotope 1H ZH 13C 14N 19F 29Si 31p 23Na 7Li 27A1 35C1 39K 55Mn 59Co

Spin

Natural abundance (%)

MRS frequency (MHz)

1/2 1 1/2 1 1/2 1/2 1/2 3/2 3/2 5/2 3/2 3/2 5/2 7/2

99.985 0.015 1.108 99.63 100 4.70 100 100 92.58 100 75.53 93.1 100 100

100.000 15.351 25.145 7.224 94.093 19.867 40.480 26.451 38.864 26.057 9.798 4.666 24.67 23.614

Relative receptivity b 1.000 0.00000145 0.00176 0.001 0.8328 0.000369 0.0663 0.0925 0.272 0.206 0.00355 0.000473 0.175 0.277

a From Ref. 34, with permission. b Calculated from the nuclear magnetogyric ratio, nuclear spin, and natural abundance of the isotope relative to ~H (33).

rotate (precess) around this field. The motion of each nucleus is very much like a spinning top or gyroscope in the earth's gravitational field. In Fig. 2 the total nuclear magnetic moment is represented by the vector M. This vector precesses around the applied magnetic field (H 0) at a rate proportional to the field. This precessional frequency (tOo) is referred to as the Larmor frequency and is given by too = y H 0, where 3' is the magnetogyric ratio constant, which is a physical constant characteristic of each atomic nucleus. For example, in an external field of 1.5 T, protons have a Larmor frequency of about 60 MHz. In a 11.7-T field, protons have a Larmor frequency of 500 MHz. The Larmor frequency of phosphorus at 1.5 and 11.7 T is about 26 and 202 MHz, respectively. Therefore, in order to get a signal from these precessing nuclei, one needs to apply a radio frequency electromagnetic field (rf pulse) that has the same frequency as the Larmor frequency too. This is called resonance, and under the resonance condition the atomic nuclei will optimally absorb the energy from the rf field, and go from ground state to excited state. This process is shown in Fig. 2 as rotating (nutating) the vector away from the direction of the external field H 0 (z axis) so that it lies along the x axis; this requires a 90 ~ pulse. The magnitude of the signal is proportional to the x component of the rotated vector, i.e., the magnitude is proportional to sin(0). A 90 ~ nutation will give a maximum positive signal, a 270 ~ nutation

[12]

181

MRS O F N E U R A L T I S S U E

z

~o= ~Ho ~, = YH1 0 = 'YH,t~ J

FIG. 2 Vector representation of total magnetic moment in an external magnetic field. Reprinted from Neurobiol. Aging 15, Pettegrew, Panchalingam, Klunk, McClure, and Muenz. Alterations of cerebral metabolism in probable Alzheimer's Disease: A preliminary study, 117-132, Copyright 1994, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.

will give a maximum negative signal, and nutations of 0~ or 180~ will give no signal. The reason for this is that the rf receiver coil is oriented in the MRS probe to detect rf signals perpendicular to the applied magnetic field, H 0. The length of the rf pulse and its stength (I-I~) will determine how far the angular momentum vector is rotated. The angle 0 through which the nuclear magnetic moment vector moves during the rf pulse time (tp) is given by 0 = yH~

tp.

At the end of the rf pulse, the nuclei will return to their ground state (relaxation) by interacting with fluctuating electromagnetic field gradients around them. These gradients are produced by other nuclei in the same molecule or nuclei in surrounding molecules. The signal produced during this relaxation process is referred to as the free induction decay (FID) (Fig. 3). If the rf pulse is repeated before the nucleus returns to its ground state, the vector will be rotated more and more until it has been rotated 180~ and no signal will be observable. This process is called saturation of the nuclear magnetization. The period between successive pulses is called repetition

182

PARADIGMS OF NEURAL INJURY

0.0

0.3

0.6

0.9

1.2

1.5

1.8

Seconds FIG. 3 1H MRS free induction decay signal from a Bruker AM500 spectrometer operating at a field strength of 11.7 T. Reprinted from Ref. 34, with permission.

time (TR) and should be long enough to prevent saturation effects when quantifying resonances in the MRS spectrum. Only fully relaxed spectrum can be used for accurate quantification of resonances. Fourier transformation of the FID produces a conventional MRS spectrum (Fig. 4).

Chemical Shift MRS spectral patterns are significantly affected by the chemical environment of nuclei. These effects are transmitted predominantly through covalent bonds. Although all nuclei of a given atom type have the same Larmor frequency in identical magnetic fields, the Larmor frequency is field dependent (Fig. 4). The circulation of electrons under the influence of a magnetic field will generate their own magnetic field opposing the applied field. This changes the magnitude of the effective magnetic field experienced by the nucleus in question and results in a shift of its nuclear resonance position. The electron distribution surrounding nuclei that are covalently bonded together will be characteristic for different types of chemical moieties. The chemical shift differences (chemical shift dispersion) increase with stronger magnetic fields, thus the resolution of spectral peaks is improved at higher field strengths.

183

[12] MRS OF NEURAL TISSUE H

H

H

-O-C-&-C-H

56A

o6A

H Loc

~

.~o

I

~

o

,

"O

w ,

o

,~o

<

'q.

i

4.0

"o

H Loc

0 c

u

H

3.5

3.0

(1) O

c-

_t-n

D

IX.

i

i

2.5

2.0

IX.

,

1.5

ppm

FIG. 4 1H MRS spectrum at 11.7 T of a perchloric acid extract of human brain. Identification of most of the MRS signals in this ppm range is provided. The appearance of multiple resonances with different splitting patterns for a given metabolite is frequently encountered; an example of the assignment of the lactate signals from its molecular structure is given in the top left and top right. The area of the doublet from the methyl resonance of lactate is three times that of the quartet from the single proton on the a carbon of lactate. Lac, Lactate; Ino, inositol; Cr, creatine; Glu, glutamate; Gin, glutamine; Gly, glycine; Tau, taurine; SIno, scyllo-inositol; PE, phosphoethanolamine; PC, phosphocholine; GPC, glycerophosphocholine; Cho, choline; 3APP, 3-aminopropylphosphonic acid; NAA, N-acetyl-L-aspartate; Asp, aspartate; Succ, succinate; GABA, y-aminobutyric acid; Ace, acetate; and Ala, alanine. Reprinted from Ref. 34, with permission.

Chemical shifts are useful for identification" of metabolites in samples of biological interest. For example, differences in the electron distribution surrounding the phosphorus nuclei in the a-,/3-, or y-phosphates of 5'-adenosine triphosphate (ATP) result in signals at different frequencies (chemical shifts) that are characteristic for each of the three phosphates (Fig. 5a). An example of changes in the chemical environment of protons giving rise to large separations of chemical shifts is seen in the proton resonances of lactate, illustrated in Fig. 4. Typical reference compounds are 3-(trimethylsilyl)propionic2,2,3,3-d4 acid (TMSP) for 1H MRS and 85% orthophosphoric acid for 31p MRS. Chemical shifts are usually reported in units of parts per million (ppm or 8), which are independent of field strength because they are obtained by

184

PARADIGMSOF NEURAL INJURY

Ionized Ends

PE Pi

~

7-ATP

IP

I

0

~-ATP

13-ATP

~

.... k.J____JL.__J~,:

ppm 5

Esterified Ends

.......... .... J ~ _ ~ = ~

-5

-10

......

-15

- : ...............................

-20

FIG. 5 Comparison of (a) ~3p MRS in vitro spectrum of perchloric acid extract of freeze-clamped rat brain tissue at a magnetic field strength of 11.7 T and (b) 31p MRS in vivo spectrum of human brain (dorsal prefrontal cortex) at a magnetic field strength of 1.5 T. In the in vivo spectrum, the ionized ends are y-ATP and fl-ADP; the esterified ends are a-ATP and a-ADP, and the middles are fl-ATP. PME, Phosphomonoesters; Pi, inorganic orthophosphate; PDE, phosphodiesters; PCr, phosphocreatine; IP, phosphoinositol; PE, phosphoethanolamine; PC, phosphocholine; GPE, glycerophosphoethanolamine; GPC, glycerophosphocholine; ATP, adenine triphosphate; ADP, adenine diphosphate; and UDP, uridine diphosphate. Reprinted from Ref. 34, with permission.

dividing the resonating frequency by the applied frequency and multiplying by 106. Chemical shifts also are reported in hertz (Hz), but the applied field strength must be designated because the chemical shift is dependent on magnetic field strength. Chemical shift dispersion is expected to increase with increases in I-I0, thus conducting experiments at higher field strengths will increase the separation of the spectral peaks in units of hertz.

[12]

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MRS OF N E U R A L T I S S U E

The resonance frequency of a nucleus in a MRS spectrum is given as the difference between the unknown nuclear resonance position and the resonance position of some arbitrarily chosen reference. Therefore the chemical shift 6 is usually expressed as 8 ---- ( H r e f -- A u X 1

Hunknown)/Hre f 0 6/Uinstrument,

where H r e f and Hunknow n a r e the resonance field strength for the reference and unknown nucleus, respectively; Av is the difference between the difference frequencies of the reference and unknown (in hertz); and Uinstrumentis the observed frequency (in hertz) characteristic of the spectrometers operating at different field strengths.

Spin Relaxation We mentioned earlier that at the end of the rf pulse, the nucleus will return to equilibrium by dissipating the energy absorbed from the rf pulse to its surroundings. This process is called spin relaxation. During relaxation, the excited nuclei exchange energy with each other and with other types of nuclei in the system. As a typical example, consider two protons A and B in a water molecule. The total field at A arises from the external field H0, the local field arising from proton B, and from the local static field of the surrounding bonding electrons (see chemical shifts above). If the water molecule rotates, the strength and the direction of the magnetic dipole field generated by the asymmetrical charge distribution in the water molecule will change with time (fluctuate). Therefore, there is coupling between the nuclear precession and molecular motion mediated by the interaction between the two protons. However, the relaxation of nucleus A takes place only if the local field at A fluctuates and only if the fluctuations take place at the Larmor frequency. There are two general mechanisms through which an excited nucleus can dissipate the acquired energy. One of these is called longitudinal or spin-lattice relaxation. The time constant that characterizes spin-lattice relaxation is called T1. It is a measure of how rapidly the z component of the nuclear magnetic moment reaches equilibrium after it is displaced from its equilibrium state. This mechanism is caused by the components of the rapidly fluctuating fields in the x and y directions. Therefore, if the magnetic moment is rotated by 90 ~ to a position along the y axis by a suitable rf pulse, it will recover to its equilibrium position according to the equation

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PARADIGMS OF N E U R A L INJURY

Mz(t ) = Mo[1 -

exp(-t/T1) ].

The other relaxation mechanism, called transverse or spin-spin relaxation, is characterized by the T 2 time constant and is produced by both rapidly and slowly fluctuating local magnetic fields in the x, y, and z directions. Therefore, there are more potential interactions giving rise to T2 relaxation compared to T1 relaxation and consequently T2 -< T1. T2 relaxation is a measure of how rapidly the magnetic moments in the x-y plane lose their phase coherence. If all nuclei have the same Larmor frequency, their magnetic vector would be precessing together. But if for some reason (field inhomogeneity, fluctuating local fields) the Larmor frequencies are not all the same, they soon lose phase coherence because some of the nuclei will be precessing faster and some slower. The net magnetization in the x-y plane soon will be zero. The distribution of Larmor frequencies for a particular resonance peak determines its linewidth. Therefore there is a direct relationship between the linewidth and T2. Again, if the magnetic moment is rotated by 90 ~ to a position along the x axis by a suitable rf pulse, the components along the x and y axis will decay according to the equation Mx(t) = M0 exp(-t/T2). In all relaxation mechanisms, two molecular parameters determine the rate of relaxation and therefore the values of T1 and T2. The first parameter is the magnitude of the particular interaction. The second is the correlation time, %, which can be defined as the average time to transverse one radian (i.e., 1/speed at which the molecule rotates). Such motion tends to retard the process of relaxation and increases T1 and T2 (because more slowly moving molecules experience more energy-depleting events). More precisely, it is the magnitude of rc in relation to the Larmor frequency that is most important. Thus, one can define three ranges of molecular motion (fast, intermediate, and slow) in terms of the value of rc times Larmor frequency. From these considerations one can define ranges of molecular motion as fast (extreme narrowing limit, too% ,~ 1, intermediate (tOotc = 1), or slow (tOorc > 1).

Information Contained in Magnetic Resonance Spectrum A resonance peak in the MRS spectrum contains at least four pieces of information. The chemical shift is characteristic of a particular chemical type of a given nucleus. The area under the peak is proportional to the number

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187

of nuclei giving rise to that peak, therefore MRS is a quantitative technique. The width of the peak indicates something else about the chemical environment of the nucleus. Although the chemical shift is affected predominantly by the chemical environment within a few bonds of the nucleus, intermolecular interactions can affect the peak width of the resonance. It also is important to realize that the major artifactual increases in peak width are caused by inhomogeneities in the applied magnetic field. If a given population of a certain type of nucleus experiences slightly different applied fields in different regions of the sample, they will show a distribution of Larmor frequencies. The peaks resulting from these chemically identical nuclei in different magnetic environments cannot be resolved and show up as a broader peak. Another structure-dependent MRS spectral characteristic is the splitting of resonance signals caused by indirect coupling of nuclear spins through intervening bonding electrons. This spin coupling is caused by the tendency of the bonding electron to pair its spin with the spin of the nearest nuclei; this affects the spin of adjacent bonding electrons, which in turn affect the spin of other nuclei. This effect is not usually significant beyond three bonds. The magnitude and pattern of the splitting are useful for identification of molecules in samples of biological interest. For example, the ~H MRS resonance for the methyl protons of lactate is split by a single proton on the a carbon to give a doublet splitting (Fig. 4); the proton on the a carbon is split by the three methyl protons to give a quartet (Fig. 4).

Sensitivity of Magnetic Resonance Spectroscopy A major limitation of MRS is its lack of sensitivity. The receptivity or MRS signal strength of a particular nuclei is dependent on its intrinsic magnetogyric ratio and the applied magnetic field strength. 1H gives the strongest MRS signal of all nuclei. Table I gives the MRS receptivity relative to ~H at a given field strength, also taking into account the natural abundance of the isotope. Note that in this definition of receptivity the smaller values denote decreased sensitivity. In terms of sample concentration, the sensitivity of MRS is approximately 1/zmol/ml for each individual phosphorous-containing molecule and 10 nmol/ml for each hydrogen-containing molecule in a mixture. The sensitivity of MRS can be increased by increasing the applied magnetic field strength. For example, signal strength is increased nearly 18 times by increasing the field strength from 1.5 to 10 T. The signal intensity also can be increased by enriching the sample with those nuclei whose MRSobservable isotope is not 100% naturally abundant (e.g., ~3C).

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PARADIGMS OF N E U R A L INJURY

Line-Broadening M e c h a n i s m s Field Inhomogeneity Inhomogeneity of the applied field is the major contributor to the linewidth of an MRS signal. If the applied field is spatially inhomogeneous, identical spins in different parts of the sample will have slightly different Larmor frequencies. These different frequencies will result in a distribution of unresolved spectral lines, which will appear as one broadened line. In addition, the spins will more quickly lose their synchronization of precession about the axis of the external field (lose phase coherence) and the FID will be much shorter than that determined by the natural/'2 of the sample.

Chemical Exchange Using the above principles, we also can understand how linewidth can give some indication about the chemical state or environment of nuclei giving rise to that resonance line. The following example applies to the situation wherein there is a consistent degree of applied field homogeneity and similar overlap of other unresolved peaks because of small differences in chemical shift; however, these conditions are not always met in in vivo studies. Essentially all molecules in a sample have a variety of local chemical environments in which they can exist. These local environments will have slightly different local magnetic fields due to shielding effects of the local electrons, as discussed previously. The process of "chemical exchange" describes the movement of a molecule between these chemical environments. If we consider only two possible environments (e.g., complexation with and dissociation from a cation), then we can consider the effects of correlation time on the rf signal emitted by an excited nucleus in that molecule. At one extreme (slow exchange), when the time spent in each environment is long compared to the time it takes the nucleus to precess once about the axis of the applied field (i.e., the reciprocal of the Larmor frequency), then that nucleus will stay in each environment long enough to emit a detectable signal in both environments. If the two environments have sufficiently different local magnetic fields, then two separate resonance signals will be generated (Fig. 6a). At the other extreme (fast exchange), the molecule is shifting between the two different environments so fast that the molecule moves between the two local magnetic fields many times before each rotation about the applied field axis is complete. In this case, only one highly averaged local magnetic field is experienced and a sharp resonance signal is produced (Fig. 6b). At an intermediate rate of exchange, whereby the time spent in each local magnetic field is comparable to the time of rotation about the applied axis, the resultant resonance is made up of resonances at the Larmor

189

[12] MRS OF N E U R A L TISSUE

A

VA

VB

FIG. 6 Theoretical spectra showing chemical exchange between two groups of nuclei in different chemical environments. (a) Slow chemical exchange, (b) fast chemical exchange, and (c) intermediate chemical exchange. Reprinted from Neurobiol. Aging 15, Pettegrew, Panchalingam, Klunk, McClure, and Muenz. Alterations of cerebral metabolism in probable Alzheimer's Disease: A preliminary study, 117-132, Copyright 1994, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK. frequencies of each local magnetic field and another resonance frequently located between the two local magnetic fields. All of these resonances are unresolvable and result in one broad resonance line (Fig. 6c). An example of the chemical significance of line broadening is the increase in the linewidth of the/3 resonance of ATP when complexed with aluminum (4) or magnesium (5). This increase in linewidth can be detected by MRS and it gives an indication of the degree of cation complex formation by ATP.

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PARADIGMS OF N E U R A L INJURY

Correlation Time Effects As stated in the discussion on spin relaxation, the correlation time ~'c of a molecule describes the speed of rotation of that molecule (faster rotation equals shorter correlation time). Molecules with short values of ~'c will have long values of T2and vice versa. The longer the T2, the narrower the linewidth. This can be seen in practical terms in the in vivo phosphodiester resonance. Glycerophosphocholine (GPC) is a small phosphodiester made up of a choline molecule attached to a phosphate group, which is in turn attached to a glycerol molecule. In solution and in the brain it is rapidly rotating and has a relatively short correlation time (long T2) and produces a narrow resonance signal. Phosphatidylcholine (PtdC) is a large phospholipid made up of two fatty acids attached to glycerophosphocholine. The PtdC present in membrane vesicles rotates much more slowly and has a longer correlation time (shorter T2). The MRS resonance peaks of these two compounds cannot be resolved in vivo by 31p MRS, because just the phosphate is detected and the chemical environments of the phosphate in GPC and PtdC are similar (same chemical shift). In any given distribution of chemical environments, the more mobile GPC will generate a more narrow line than the larger, more slowly tumbling PtdC in membrane vesicles, because PtdC will more closely approximate the condition of intermediate exchange. Each orientation (with respect to the applied field) of the electron cloud surrounding the phosphorus nuclei will give rise to a distinct local magnetic field and hence resonance frequency. The range of orientation of PtdC in the tumbling vesicle will in turn give rise to a range of resonance frequencies and a broad line. Therefore, all else being equal, variations in the in vivo ratio of GPC/PtdC will result in linewidth variations in the in vivo phosphodiester resonance.

Spatial L o c a l i z a t i o n f o r in Vivo M a g n e t i c R e s o n a n c e S p e c t r o s c o p y Spatial localization is required with in vivo MRS because only a portion of the neural tissue is of interest, in contrast with in vitro MRS of solutions, where the entire sample is placed in a very homogeneous magnetic field. There have been numerous discussions of the methods used to localize the "volume of interest," or VOI, but we will briefly discuss those used in our laboratory.

Depth-Pulse Localization The depth-pulse localization technique achieved by using a rf field gradient produced by a surface coil, as described by Bendall (6), but without phase cycling, has been used for our early in vivo 31p MRS experiments. This

[12] MRS OF NEURAL TISSUE

191

technique uses a straightforward pulse-acquire sequence to acquire the data, which minimizes the loss of spectral information due to delayed acquisition. With this technique two principles are simultaneously considered. The first principle is in the choice of the size of the surface coil. The surface coil contains both the transmitter coil (used to generate the Larmor frequency pulse) and the receiver coil (used to detect the small signal generated by the excited nuclei). The receiver coil is usually constructed to be concentric with and smaller than the transmitter coil, so that the signal from a small region of the sample is detected, although a larger VOI is excited by the rf pulse. The other consideration is the width of the rf pulse. An rf pulse applied to a living animal will dissipate in power as it penetrates the tissue. If this were not so, then a pulse applied for a certain duration (the pulse width) by a surface coil would equally excite a cylinder of tissue from immediately below the coil through the entire subject to exactly the same "flip angle." But because the power dissipates, a given pulse width will generate different flip angles at different distances from the transmitter coil. Therefore, for MRS study of the brain, the pulse width is chosen to produce a 180~ flip angle in the skin and muscle of the scalp, and 90 ~ in the region of most interest; the bone of the skull gives no detectable in vivo signal. There will be some signal from surrounding brain tissue, but this is usually very acceptable. Thus, the depth-pulse method makes use of variation in tissue flip angle to localize the VOI. Because the "shape" of the actual rf field generated by the transmitter coil is conical, the VOI is also conical, with its base near the surface of the coil. This localization technique is effective for studying 15- to 20-cm 3 volumes of brain areas that are close to the surface of the skull, but it is not suitable for studying brain areas more distant from the surface coil.

31p MRS Using Spin-Echo Pulse Sequence The spin-echo localization technique is conceptually more complex than the pulse-acquire sequence (described previously). As the term implies, spinecho techniques involve generation of a spin-echo MR signal (7). The spinecho signal is made up of two FIDs back to back. The echo is generated by applying two rf pulses (usually a 90 ~pulse followed by a 180~pulse) separated by the spin-echo time (TE) divided by 2. The echo signal will be formed at TE. This technique allows one to acquire a signal with a minimum loss of the initial part of the FID. It is important to acquire the initial part of the FID in 31p MRS experiments in order to avoid loss of long correlation time components. Based on this concept, a new pulse sequence for 31p MRS was published that allows for much shorter echo times (TE) in a multiple-voxel experiment (8). This pulse sequence allows for a TE as short as 2.5 msec. The short TE is important because of the short T2 (long correlation time)

192

PARADIGMS OF NEURAL INJURY

components of the in vivo 31p spectrum, especially the membrane metabolites, phosphomonoesters (PME) and phosphodiesters (PDE), that reflect membrane metabolism. The long correlation time (short T2) components of the PDE peak are of interest because they appear to arise from mobile vesicular lipids such as synaptic vesicles and may provide information on synaptic density. Longer TE values cause a major portion of the signal from these rapidly relaxing metabolites to be lost before data acquisition is begun. First-order phase correction is not necessary in this pulse sequence due to the fact that the acquisition is a spin-echo. This means the baseline distortion seen with common free induction decay techniques is avoided and only zero-order phase correction is needed. In addition to simplifying the processing of spectral data, this eliminates potential errors introduced by first-order phase correction in the quantitation of short T2 components. The use of a slice-selective composite spin-echo pulse sequence eliminates the need for three-dimensional phase encoding for volume selection. The two-dimensional acquisition mode employed is reported to produce better slice profiles with a relatively small number of phase encodes (8). This localization technique shows great promise and we now are implementing this technique for our in vivo 31p MRS studies. This sequence allows simultaneous acquisition of multiple spectra from several brain regions within a 35-mm slice, saving significant amounts of measurement time. In this composite spin-echo method, the echo time and slice profile are specified and the rf pulse that produces this echo is determined. The 180~ ~ (where a will usually be between 30~ and 60 ~) and 180~ pulses overlap at very short echo times. The composite spin-echo rf pulse is incorporated into a twodimensional phase-encoded spectroscopic imaging sequence using triangular phase-encoding waveforms in the X and Y gradients for an 8 x 8 imaging matrix. The Z gradient contains the slice selection gradient and is divided into two ramps in order to meet the constant integrated gradient requirements and the slew rate constraints of the coil. This new pulse sequence is now supplied by General Electric (GE; Milwaukee, WI) as the " S P I N E C H O " research pulse sequence. There are technical limitations present in all of the localization methods presently employed. Other recently developed gradient localization techniques such as STEAM (stimulated echo acquisition mode) and ISIS (imageselected in vivo spectroscopy) utilize magnetic field gradients to introduce a well-defined field inhomogeneity to select a VOI. These techniques require time for the gradients (which would interfere with the sample signal) to be turned off before the receiver coil can be turned on. During this "acquisition delay" some of the signal is lost. Because not all signals decay at the same rate, depending on the T~ and T2 of nuclei in different molecules, there will

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be a disproportionate loss of signal from short T2 compounds, such as the phospholipids. Therefore, the depth-pulse sequence or the spin-echo localization techniques discussed above have biochemical advantages for the 31p MRS study of brain, given the importance of membrane phospholipid metabolism. More detailed information concerning the various in vivo MRS localization methods can be found in the literature (9, 10).

Summary The basic principles discussed above apply to both in vitro and in vivo MRS. However, there are many more caveats to be considered when conducting in vivo experiments. These are introduced by the greater degree of field inhomogeneity, complex pulse techniques required for localization, contribution of different types of tissue fluids to the signal, different "magnetic susceptibilities" of various tissues, and several other factors. Thus, while we can extend basic MRS theory to in vivo experiments, results must be interpreted with these caveats in mind. Discussions should include all possible effects that could lead to a given change, in order to indicate a degree of certainty for the interpretation of each change.

Experimental Considerations

Identification of Resonance Signals The principal means of identification of MRS signals for both in vitro and in vivo experiments is the chemical shift of the nuclei. Examples are given in the following sections.

In Vitro MRS Spectra The molecular identities of individual resonance signals in extracts of biological samples must be carefully verified through the use of appropriate biochemical and spectroscopic procedures. The identification of the major signals in ~H and 3~p MRS spectra of perchloric acid (PCA) (Figs. 4 and 5a) of brain tissue is well documented (11-13) and should be a good starting point for most investigations. The following spectral properties can be used to verify these assignments or to identify new signals. The chemical shift of the nuclei is one of the most useful characteristics for identification because it is dependent on the chemical environment of

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the nuclei. Changes in chemical shift with pH are useful in identifying resonances from molecules containing acidic or basic functional groups (14). Titration of unknown complex samples can aid in identifying spectral signals and also indicates the pH value that gives the best separation of MRS signals (13). The characteristic splitting of signals by coupling of nuclear spins through covalent bonds can help to identify the molecular origin of signals and, through decoupling experiments, to identify signals originating from the same molecular component. Often the identity of the MRS signal can be confirmed by adding a relatively small amount of a known compound to the sample mixture. In Vivo M R S Spectra Resolution of in vivo MRS spectra of brain is diminished compared to in vitro MRS spectra of extracts (Fig. 5). This loss of resolution of spectral peaks is due in part to lower magnetic field strength (1.5 T) of in vivo studies compared to the in vitro studies (11.7 T). The production of high field magnets sufficiently large to accommodate humans is one limiting factor. In vivo MRS

spectral peaks also are broadened by T2 spin relaxation effects that reflect the environment of the molecules in living tissue versus the in vitro spectra of molecules in solution. Also, field inhomogeneity is an important contributor to line broadening. The identification of the origin of in vivo resonance signals requires detailed knowledge of in vitro spectra of brain extracts, because many of the in vitro identification procedures, such as titration and spiking the sample, are not possible in vivo. Comparison of chemical shifts of in vitro MRS spectra with those obtained from in vivo experiments is the primary means of identification. Despite the loss of detail seen in the in vitro spectra, the pioneering work of Ackerman et al. (15) has established that in vivo MRS yields meaningful data. The noninvasive nature of in vivo MRS opens a myriad of opportunities to study neuropsychiatric disease entities such as stroke (16), schizophrenia (17), AD (18, 19), and others (20). In vivo MRS can be used to monitor and detect changes in neural tissue resulting from neuronal injury.

Quantitation of MRS

Spectra

In Vitro MR S Spectra

Using appropriate routine instrumental conditions, the integrated area under an MRS signal peak corresponds to the number of atoms of that particular moiety in the sample. The quantitation of metabolites from brain tissue

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195

extracts can be accomplished by adding a known amount of an appropriate internal standard to the weighted tissue at the time of extraction and reporting the results in absolute units of micromoles/gram or by using tissue metabolites as their own internal reference and expressing the results in relative units such as mole%. These two approaches were investigated in a study of PCA extracts of brain tissue by 1H and 31p MRS and high-performance liquid chromatography (HPLC) in the same tissue sample (21). This study found that the results were essentially identical for the mole% and micromole/gram methods. It is important that as many metabolites be included as can be accurately determined. Although mole% values correlate well with absolute values, an appropriate internal standard, such as 3-aminopropylphosphoric acid (3-APP), should be used for in vitro 1H and 31p MRS. However, the results of this study should be equally applicable to in vivo MRS experiments when appropriate internal standards are not available. In Vivo M R S Spectra In vivo 3~p MRS of human brain provides spectral peaks for PME, inorganic

orthophosphate (Pi), PDE, phosphocreatine (PCr), and the ~-, /3-, and yphosphates of nucleotide triphosphates (predominantly ATP) and the c~- and /3-phosphates of nucleotide diphosphates such as 5'-adenosine diphosphate (ADP) (see Fig. 5b). Spectral peaks for the individual PME and PDE molecules are not resolved at the lower field strength (1.5 T) of the in vivo study. Also, the observed spectral peaks are broad, of variable linewidth, and poorly resolved. These spectral characteristics are due in part to variations in the spin-spin relaxation times (/2) for each metabolite and field inhomogeneity (10). Increased field strength, although feasible for small-animal studies, will not yield well-resolved spectra because of the field inhomogeneity contribution to the linewidth. Because the peaks generally overlap and have variable linewidths, areas of the peaks are usually obtained from curve-fitting software programs such as NMR1 of New Methods Research, Inc. (East Syracuse, NY), the GE/Nicolet curve analysis program, or the GE SAGE/ IDL software. Figure 7 gives an example of curve fitting an in vivo 31p spectrum using the NMR1 program. The best method of quantitation would be based on comparison of sample signal to a reliable internal standard; however, no reliable internal standard exists for in vivo MRS. In vivo MRS spectra are quantitated by both the mole% method (11, 12, 21-23) and the external standard method (24). The mole% method has the advantage of expressing data in units that are not sensitive to partial volume effects; partial volume effects can confound absolute quantitative methods. As discussed above, the mole% method has been shown to compare very favorably with absolute quantitation of in vitro

196

PARADIGMS OFNEURALINJURY PME Component

Lorenzians Simulated

Spectrum

PDE PCr Ionized Ends

~ .. /~x~"

~

Experimental ~ Spectrum__~.J

Middles

~

^ /

Esterified Ends

~

~z~

~,~

~' L

~

~

~

Difference '1o

....

5 ....

o ....

-5"

ppm

L10''"

-1 5 . . . .

-20: . . . .

-26:"

FIG. 7 Quantitation of the 31p MRS in vivo spectrum of brain. The experimental spectrum is shown after baseline correction; component Lorentzians (top trace) are then fit to the individual peaks. Addition of the Lorentzians produces the simulated spectrum. The goodness of fit to the experimental data is controlled by ensuring that the difference (bottom trace) between the experimental and simulated spectra is negligible. Reprinted from Neurobiol. Aging 15, Pettegrew, Panchalingam, Klunk, McClure, and Muenz. Alterations of cerebral metabolism in probable Alzheimer's disease: A preliminary study, 117-132, Copyright 1994, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.

samples (21). The mole% method of quantitation is used for our in vivo 31p MRS studies using the depth-pulse localization technique. In our in vivo 31p MRS studies using the spin-echo localization technique, quantitation of 3lp spectra is done by both mole% and the external standard method, because the field of view for this technique includes areas external to the skull. The external standard method (24) involves fixing three long vials of chromium acetyl acetonate-doped, 0.5 M phosphonitrilic chloride trimer solution in xylene around the head at regular intervals to intersect the section of interest. The ratio of the area of the integrated peak of the external standard to the area of the metabolite of interest is corrected for the relative volumes of the two compartments. This simple technique includes corrections for differences in T] or T2 of the metabolite compared to the external standard, as do other techniques (25). However, using long repetition times minimizes errors introduced by T~ differences, whereas using short TE values minimizes T2 differences. Another important measure that can be obtained from in vivo 3]p MRS spectra is the intracellular pH, which is related to the chemical shift difference between the PCr and Pi peaks (26).

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197

Sample Preparation MRS techniques are adaptable for studying brain tissues from a variety of sources, such as autopsy tissue, freeze-clamped tissue, and intact living brain. Each of these sources is important for examining neural tissue. Autopsy tissue is an important source of brain tissue for measuring certain metabolite levels found to be stable after death. It is important to determine the effect of autopsy time, etc., on the stability of the metabolites to be studied. High-energy metabolites, such as ATP and PCr, are not stable after death. Freeze-clamping brain tissue of animals with liquid nitrogen at the time of harvesting minimizes the loss of labile high-energy metabolites. PCA extraction is commonly used to extract the water-soluble compounds from the lipid and protein components of the tissue (12). The PCA extraction procedure (12) has been utilized for tissue weights of 100-500 mg. The weighed frozen tissue is pulverized at liquid nitrogen temperature in a liquid nitrogen precooled mortar. Standard aliquots of 60% PCA and 40 mM 3-APP, the internal standard (0.25 ml/g brain of both solutions added separately), are added to the liquid nitrogen precooled mortar, pulverized, and thoroughly mixed with the powdered frozen brain tissue. The mixed powders are rapidly transferred to a liquid nitrogen precooled polypropylene centrifuge tube and centrifuged at 43,600 g for 15 minutes at -4~ The supernatant is removed and the pH is immediately adjusted to pH 10.0-10.5 with 1 N K O H . The potassium perchlorate precipitate is pelleted by centrifugation at 43,600 g for 15 minutes. The supernatant is passed through a potassium Chelex column (1 x 10 cm), to remove polyvalent cations, and the column is rinsed with two bed volumes of glass-distilled water. The removal of the polyvalent cations improves the sharpness of the ATP signals in the 3~p MRS spectrum; however, in addition to removing inorganic cations, organic cations, such as choline, are removed as well. If these organic cations are of interest, ~H MRS spectra should be obtained both prior to and subsequent to the treatment with potassium Chelex. The eluant and washes from the Chelex column are pooled, lyophilized to a white powder, and stored at -20~ in a plastic vial until the sample is analyzed.

Instrumental Conditions In this section we provide examples of the routine experimental conditions used in our laboratory for obtaining MRS data.

31p MRS The lyophilized PCA extract is dissolved in 1 ml of H 2 0 and the pH adjusted to 9.6-9.7 for analysis by 3~p MRS. This pH is optimum for the resolution of signals from the PME and the stability of chemical shifts of phosphorus

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atoms in the titratable phosphate moieties. The samples are analyzed in a 1.0ml, 10-mm microcell at 27~ by a Bruker (Billerica, MA) AM500 spectrometer operating at a field strength of 11.7 T for a 31p observed frequency of 202.49 MHz and a 1H decoupling frequency of 500 MHz. The samples are analyzed under bilevel proton decoupling to maintain a constant nuclear Overhauser enhancement while spinning at 20 Hz to enhance signal resolution. The 31p chemical shifts are reported relative to 85% orthophosphoric acid. Typical experimental parameters are a single-pulse sequence with a 45 ~ pulse flip angle, 16K data points per FID, 7042.25-Hz sweep width, 1.16-second acquisition time giving a digital resolution of 0.42 Hz/point, and a 2-second interpulse delay. In addition, an exponential noise filter is used, introducing 1Hz line broadening, before Fourier transform. The number of scans range between 10,000 and 20,000, depending on the amount of tissue used. Using these conditions, it takes between 5 and 10 hours to analyze the PCA extract of one tissue sample by 31p MRS.

1n MRS ~H MRS analysis of PCA extracts in our laboratory are typically conducted using the following procedure. The lyophilized PCA extract is dissolved in 500/xl of 99.8% D20 containing 0.2 mM TMSP. TMSP is an internal reference for calibration of chemical shifts of the resonances. The pD (pD is the uncorrected pH meter readings in a D20 solution) is adjusted to 5.0-5.2 in a 5-mm MRS tube for analysis by 1H MRS. This pH is optimum for the resolution of signals from compounds measured in this extract. The samples are analyzed by a Bruker AM500 spectrometer equipped with deuterium lock and Fourier transform capabilities operating at a field strength of 11.7 T for a 1H observed frequency of 500.13 MHz. The sample is analyzed at 27~ spinning at 20 Hz to enhance signal resolution. Water suppression is achieved by presaturating the signal at very low decoupling power to ensure minimal effects on the neighboring signals. 1H MRS chemical shifts are reported relative to the TMSP signal, which is assigned a chemical shift of 0.00 ppm. Typical experimental parameters are single pulse with solvent suppression, 3-/xsec pulse width (35 ~flip angle chosen to prevent overdriving the preamplifier), 140-/zsec acquisition delay, 3.6-second acquisition time, 4545.5-Hz sweep width, 2-second repetition delay, 32K FID size, and 100 acquisitions. In addition, an exponential noise filter is used, introducing 0.3Hz line broadening, before Fourier transform. It usually takes less than 30 minutes to analyze a PCA extract of a tissue sample by 1H MRS.

In Vivo 31p MRS Using Depth-Pulse Localization Technique The experiments are performed on a Signa system (Medical Systems Operations, General Electric Co., Milwaukee, WI) at a field strength of 1.5 T. The subject is supine within a horizontal superconducting magnet, which

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generates the static magnetic field. A surface coil, which operates both as a source of the radio frequency and as a receiver, is placed just above the scalp over the brain region of interest. All subjects have routine T~-weighted ~H MRI scans in the sagittal, coronal, and axial planes immediately preceding or following the 31p MRS spectroscopy studies. A 20-cm 31p surface coil and a coplanar 7.5-cm surface coil dual-tuned at both the 31p and 1H frequencies are used to acquire 1H images to identify the location and approximate volume of the brain sampled by spectroscopy. The 1H images for spectral localization are obtained by transmitting with a Helmholtz body coil and receiving with the 7.5-cm surface coil. The localized ~H images and 31p MRS spectra are from approximately 15 to 20 cm 3 of the dorsal prefrontal cortex. A small contribution from the underlying white matter cannot be ruled out. Suppression of signal from extracranial skin and muscle is accomplished by using an approximately 180~ pulse width at the plane of the surface coil. 31p MRS acquisition standards employed were 800-/xsec pulse width, 2-kHz spectral width, 2048 data points, 0.512-second acquisition time, a 2.0second recycle delay for a total interpulse interval of 1.512 seconds, and 300 acquisitions.

In Vivo 31p MRS Using Spin-Echo Localization Technique Spectroscopy and imaging are performed with a General Electric 1.5-T Signa System. A custom-designed (Robert Lenkinski, University of Pennsylvania, Philadelphia, PA) ~H/3~P head coil, quadrature for both 31p and ~H, is used. This coil fits in the same location as the GE quadrature imaging coil and has the same external and internal dimensions as the GE coil. The use of this dual-tuned coil allows us to perform the entire protocol without repositioning of the coil or the subject. An ~H MRI image is obtained to guide the selection of an axial MRS spectroscopic slice. Automatic ~H shimming is carried out on the selected axial siice. The hardware leads to the head coil are switched from the ~H to the 31p mode. The 90 ~ 31p pulse width is determined on the selected axial slice. The data for the 31p MRS spectra are collected using a slice thickness of 35 mm, a spectral width of 2500 Hz, 2048 data points (without zero filling), a TE of 2.5 msec, a repetition time of 4 seconds, and the SPINECHO pulse sequence supplied by GE and developed by Lim et al. (8). The 31p spectra obtained from a human volunteer (Fig. 8) using this procedure are plotted in voxels overlaying the corresponding MRI axial slice of the brain using the GE Sage/IDL program. These spectra are plotted at a very low digital resolution and are meant to very roughly represent total area to compare with brain volume. The spectrum from

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FIG. 8

31p spectroscopic image of a human head using the spin-echo pulse sequence.

a central voxel is plotted at higher resolution in Fig. 9 to illustrate the individual components of the spectrum.

Applications of Magnetic Resonance Spectroscopy for Examining Neural Tissue The examples of applications of MRS given in the following sections have been taken from studies done in this laboratory.

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1. Freeze-Clamped Fischer 344 Rat Brain 3 1 p MRS studies have been conducted in Fischer 344 rats ranging in age from newborn (12 hours old) to aged (24 months old) (12). The brains used in this study were freeze clamped to preserve high-energy metabolites such as PCr, ATP, and ADP. The frozen rat brains were extracted with PCA as described above and the extracts were analyzed by 3 1 p MRS to obtain mole% values for PCr, P~, ATP, a-glycerol phosphate (c~GP), phosphocholine (PC), phosphoethanolamine (PE), GPC, and glycerophosphoethanolamine. These studies demonstrate a marked influence on high-energy phosphate and membrane phospholipid metabolism during brain development and, to a lesser degree, aging. PME levels are high in the newborn rat brain and rapidly

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[12] MRS OF NEURAL TISSUE

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The PCr/Pi ratio provides a measure of the energy status of the brain. The PCr/Pi ratio is low in the newborn period until 5 days of age, after which time the PCr/Pi ratio rapidly increases up to 3 months of age. The PCr/Pi ratio remains relatively constant until 12 months of age, when the ratio again slowly increases up to 24 months of age. This may indicate either that the aging brain synthesizes PCr at an increased rate or that there is a decreased utilization of PCr.

Alzheimer's Disease Brain In Vitro 31p Studies The 31p MRS spectra of PCA extracts prepared from various regions of AD, non-AD, and control brain were examined. Elevated PME and PDE were observed in the AD versus control samples (27). This increase in acknowledged phospholipid precursors (PME) and catabolites (PDE) may be an indication of accelerated membrane phospholipid metabolism in AD brain. The postmortem diagnosis of AD is neuropathologically based on the abundance of neurofibrillary tangles (NFTs) and senile plaques (SPs). These structures were quantitated in slices adjacent to those taken for MRS analysis and correlated with the mole percentages of PME and PDE. There was a significant (p - 0.05; r - 0.76) negative correlation between the PME and number of SPs; in contrast, there was a significant (p = 0.01; r - 0.89) positive correlation between the PDE and SPs (Fig. 11) (28).

In Vitro 1H Studies The ~H MRS spectra of PCA extracts of 12 AD and 5 control brain samples were examined (13). The relative concentrations of taurine, aspartate, glutamine, glutamate, y-aminobutyric acid (GABA), and the putative neuronal marker NAA were measured. NAA levels were found to be lower in the AD brains compared to controls. Comparison of the number of SPs in tissue sections adjacent to those analyzed by MRS with NAA levels demonstrated a decrease in NAA with increased numbers of SPs. Glutamate levels were greater in AD than controls; GABA levels were decreased. No significant changes were found in the levels of taurine, aspartate, or glutamine in the AD brains compared to controls. These findings suggest that the decrease in NAA reflects neuronal loss and that remaining neurons could be exposed to a relative excess of glutamate and a relative lack of GABA.

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In Vivo 3]p M R S Studies o f AD Brain

The initial molecular events in AD that culminate in neuronal cell death have not been elucidated and could start decades before the onset of symptoms. Widespread and severe cellular and synaptic membrane damage is evident by the time symptoms become manifest (29-31). The alterations in phospholipid metabolism found in the postmortem in vitro 31p MRS studies also are detectable by in vivo 3~p MRS studies (18, 19). An early cross-sectional 3~p MRS study found significant (p -< 0.05) elevations of PME levels in the temporoparietal region of AD brain compared to either controls or subjects with multiple subcortical infarct dementia (18). In a recent longitudinal study in our laboratory (19), the in vivo 3~p MRS spectra from the prefrontal cortex of 12 probable AD (5 males; 7 females) and 21 control subjects (11 males; 10 females) 63 years of age or older were examined. Mattis test scores, a neuropsychologic measure of clinical severity of AD, were used to classify the AD patients as mild (> 120) or moderate (60-119). The results of this study demonstrate that PME levels of the mildly demented group are increased compared to the moderately demented group. Also, PME levels correlate negatively with the clinical rating, the Mattis

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Fro. 12 Distribution of phosphomonoester (PME) levels (mole%) by Mattis score for Alzheimer's disease patients. The p and r values represent significance level and goodness of fit, respectively. Reprinted from Neurobiol. Aging 15, Pettegrew, Panchalingam, Klunk, McClure, and Muenz. Alterations of cerebral metabolism in probable Alzheimer's Disease: A preliminary study, 117-132, Copyright 1994, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK. score (Fig. 12). Taken together, these results indicate that the milder the dementia the greater the PME levels. This suggests that alterations in membrane phospholipid metabolism could be an early "molecular trigger" in AD, perhaps resulting in alterations to mitochondrial membranes as well as to the plasma membrane. Levels of PCr were decreased in the mildly demented AD subjects and increased as the dementia worsened. The data indicate that both of the immediate precursors of ATP (PCr and ADP) are diminished early in AD. This decrease in energy availability and reserve may lead directly to neuronal dysfunction and possibly place neurons at more risk of neurotoxic insult from glutamate (32).

In Vivo 31p M R S Studies of Acetyl-L-Carnitine Treatment of AD Brain In vivo MRS is a technique well suited to monitor the course of treatment of AD brain. We have monitored the effect of the administration of acetyl-Lcarnitine to AD patients by in vivo 31p MRS using the depth-pulse localization technique. In a double-blind, placebo study, acetyl-L-carnitine was administered to 7 probable Alzheimer's disease patients who were then compared by clinical and 3~p magnetic resonance spectroscopic measures to 5 placebotreated probable AD patients and 21 age-matched healthy controls over the course of 1 year (7). Compared to AD patients on placebo, acetyl-L-carnitinetreated patients showed significantly less deterioration in their Mini-Mental

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Status (MMS) and Alzheimer's Disease Assessment Scale test scores. Furthermore, the decrease in phosphomonoester levels observed in both the acetyl-L-carnitine and placebo AD groups at entry was normalized in the acetyl-L-carnitine-treated but not the placebo group. Similar normalization of high-energy phosphate levels were observed in the acetyl-L-carnitinetreated but not placebo-treated patients. This is the first direct in vivo demonstration of a beneficial effect of a drug on both clinical and CNS neurochemical parameters in AD. Acetyl-L-carnitine-treated patients showed significantly less deterioration in their MMS scores compared to AD patients on placebo. Although MMS scores were equivalent at entry in the two AD groups (p = 0.97), acetyl-Lcarnitine-treated patients had significantly higher scores than placebo-treated patients at 6 (p = 0.01) and 12 months (p = 0.01). This is the first direct in vivo demonstration of a beneficial effect of a drug on both clinical and CNS neurochemical parameters in AD.

Conclusion MRS spectroscopy provides useful information about the status of highenergy and phospholipid metabolism from observations of characteristic metabolites. MRS is useful for studying AD brain, both in vivo and in vitro. MRS findings support the suggestion that there is derangement of both the membrane structure and the metabolism of characteristic membrane lipids, which would contribute to deranged cellular functions. In vivo MRS is uniquely suited to identify the molecular underpinnings of neural injury and to monitor the efficacy of treatment protocols.

Acknowledgments This work was supported in part by NIA Grants AG08371, AG08974, AG50133, and AG9017.

References 1. J. W. Pettegrew, in "Handbook of Neuropsychology" (F. Boller and J. Grafman, eds.), pp. 39-56. Elsevier, Amsterdam, 1991. 2. "NMR: Principles and Applications to Biomedical Research" (J. W. Pettegrew, ed.) Springer-Verlag, Berlin and New York, 1990.

[12] MRS OF NEURAL TISSUE

,

10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22.

23. 24.

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"Spectrometric Identification of Organic Compounds." (R. M. Silverstein, G. C. Bassler, and T. C. Morril, eds.), 5th Ed. Wiley, New York, 1991. K. Panchalingam, S. Sachedina, J. W. Pettegrew, and T. Glonek, Int. J. Biochem. 23, 1453-1469 (1991). R. Vink, Mol. Chem. Neuropathol. 18, 279-297 (1993). M. R. Bendall, Bull. Magn. Reson. 8, 17-44 (1986). A. A. Maudsley, D. B. Tweig, D. Sappey-Marinier, B. Hubesch, J. W. Hugg, G. B. Matson, and M. W. Weiner, Magn. Reson. Med. 14, 415-422 (1990). K. O. Lim, J. Pauly, P. Webb, R. Hurd, and A. Macovski, Magn. Reson. Med. 32, 98-103 (1994). W. P. Aue, Magn. Reson. Med. 1, 21-72 (1986). L. Bolinger and R. E. Lenkinski, in "Biological Magnetic Resonance 11, In Vivo Spectroscopy" (L. J. Berliner and J. Reuben, eds.), pp. 1-53. Plenum, New York, 1992. T. Glonek, S. J. Kopp, E. Kot, J. W. Pettegrew, W. H. Harrison, and M. M. Cohen, J. Neurochem. 39, 1210-1219 (1982). J. W. Pettegrew, K. Panchalingam, G. Withers, D. McKeag, and S. Strychor, J. Neuropathol. Exp. Neurol. 49, 237-249 (1990). W. E. Klunk, K. Panchalingam, J. Moossy, R. J. McClure, and J. W. Pettegrew, Neurology 42, 1578-1585 (1992). J. W. Pettegrew, G. Withers, K. Panchalingam, and J. F. Post, Magn. Reson. Imaging 6, 135-142 (1988). J. H. Ackerman, T. H. Grove, G. G. Wong, D. G. Gadian, and G. K. Radda, Nature (London) 283, 167-170 (1980). H. Bruhn, J. Frahm, M. L. Gyngell, K. D. Merboldt, W. Hanicke, and R. Sauter, Magn. Reson. Med. 9, 126-131 (1989). J. W. Pettegrew, M. S. Keshavan, K. Panchalingam, S. Strychor, D. B. Kaplan, M. G. Tretta, and M. Allen, Arch. Gen. Psychiatry 48, 563-568 (1991). G. G. Brown, S. R. Levine, J. M. Gorell, J. W. Pettegrew, J. W. Gdowski, J. A. Bueri, J. A. Helpern, and K. M. Welch, Neurology 39, 1423-1427 (1989). J. W. Pettegrew, K. Panchalingam, W. E. Klunk, R. J. McClure, and L. R. Muenz, Neurobiol. Aging 15, 117-132 (1994). W. E. Klunk, M. Keshavan, K. Panchalingam, and J. W. Pettegrew, in "American Psychiatric Press Review of Psychiatry" (J. M. Oldham, M. B. Riba, and A. Tasman, eds.), Vol. 12, pp. 383-419. American Psychiatric Press, Washington, D.C., 1993. W. E. Klunk, C. J. Xu, K. Panchalingam, R. J. McClure, and J. W. Pettegrew, Neurobiol. Aging 15, 133-140 (1994). M. Baramy and T. Glonek, in "Methods of Enzymology" (D. L. Frederiksen and L. W. Cunningham, eds.), Part B, Vol. 85, pp. 624-676. Academic Press, New York, 1982. M. M. Cohen, J. W. Pettegrew, S. J. Kopp, N. Minshew, and T. Glonek, Neurochem. Res. 9, 785-801 (1984). P. A. Bottomley, J. P. Cousins, D. L. Pendrey, W. A. Wagle, C. J. Hardy, F. A. Eames, R. J. McCaffrey, and D. A. Thompson, Radiology 183, 695-699 (1992).

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PARADIGMS OF NEURAL INJURY 25. R. S. Lara, G. B. Matson, J. W. Hugg, A. A. Maudsley, and M. W. Weiner, Magn. Reson. Imaging 11, 273-278 (1993). 26. O. A. C. Petroff, J. W. Prichard, K. L. Behar, J. R. Alger, J. A. den Hollander, and R. G. Shulman, Neurology 35, 781-788 (1985). 27. J. W. Pettegrew, J. Moossy, G. Withers, D. McKeag, and K. Panchalingam, J. Neuropathol. Exp. Neurol. 47, 235-248 (1988). 28. J. W. Pettegrew, K. Panchalingam, J. Moossy, J. Martinez, G. Rao, and F. Boller, Arch. Neurol. 45, 1093-1096 (1988). 29. D. M. A. Mann, Mech. Ageing Dev. 31, 213-255 (1985). 30. E. Masliah, M. Ellisman, B. Carragher, M. Mallory, S. Young, L. Hansen, R. DeTeresa, and R. D. Terry, J. Neuropathol. Exp. Neurol. 51, 404-414 (1992). 31. E. Masliah, A. Miller, and R. D. Terry, Med. Hypotheses 41, 334-340 (1993). 32. R. C. Henneberry, Neurobiol. Aging 10, 611-613 (1989). 33. E. L. Harris, "NMR and Periodic Table." Academic Press, New York, 1978. 34. R. J. McClure, J. N. Kanfer, K. Panchalingam, W. E. Klunk, and J. W. Pettegrew, Neuroprotocols 5, 80-90 (1994).

[13]

Acute Stroke Diagnosis with Magnetic Resonance Imaging S t e p h e n C. J o n e s , N e n g C. H u a n g , M i c h a e l J. Q u a s t , A l e j a n d r o D. P e r e z - T r e p e c h i o , G i l b e r t R. H i l l m a n , a n d T h o m a s A. K e n t

Introduction The diagnosis of early ischemic stroke is primarily clinical within the first 8 hours, the time period during which therapeutic measures should be administered in order to be maximally successful. After 6 hours, the damage to the brain and the blood-brain barrier is generally throught to be irreversible (1) and the most recent evidence indicates that therapy must start before 4 hours to be effective (2). Thus we are left with a dilemma: How can early therapy be administered if early diagnosis cannot be made? We will review the use of three new magnetic resonance imaging (MRI) techniques that might alleviate, if not solve, the dilemma: first, diffusion-weighted imaging (DWI) is sensitive to proton diffusion and to the very early stages of ischemia; second, magnetic resonance spectroscopy (MRS) produces lactate and Nacetylaspartate (NAA) images using chemical shift imaging (CSI); and third, MR perfusion imaging yields on-line circulatory status. This review is focused on the immediate clinical application of these new MR techniques by the cerebrovascular community. The question that this review poses is whether the ischemic penumbra, that region that is salvageable in the early stages of ischemic stroke, can be identified by these new MR imaging methodologies.

E a r l y D i a g n o s i s of S t r o k e The early diagnosis and recognition of ischemic stroke is complicated by its symptomatology and pathophysiology. The changing pathophysiological situation during the early stages of ischemia has made predictions concerning ultimate outcome based on clinical findings alone difficult. Angiography, computed tomography (CT) (3), single-photon emission computed tomography (SPECT), positron emission tomography (PET), or conventional MRI Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(4, 5) all have limited utility or applicability for the very early diagnosis of ischemic stroke. The early diagnosis of arterial occlusion that precedes infarction is possible using angiography, an invasive and risky procedure. However, early angiography has been used safely in one tissue plasminogen activator (tPA) trial to confirm clot position and lysis (6). Cerebral blood flow (CBF) techniques such as SPECT (7), PET, or stable xenon CT are neither highly available, accepted, nor proved. Noninvasive vascular imaging techniques such as transcranial Doppler ultrasound or MR angiography have not yet been utilized for clinical trials. Conventional CT can be used to exclude intracranial hemorrhage, and has been used for this purpose in another tPA trial for the early treatment of stroke (8), but is typically normal during the first 24 hours and cannot provide a diagnosis of early stroke. Currently available MRI techniques are not sensitive to early stroke. Tz-weighted MRI may be abnormal 12 hours after stroke onset, but is not useful for the very early diagnosis of stroke (9).

Early Treatment of Stroke: Entry Time, Therapeutic Window, and Ischemic Penumbra Much evidence exists that early treatment is important for stroke. Early entry times (or pretreatment) in experimental studies are associated with successful outcomes (10-13). Clinical studies that have been relatively successful tend to have earlier entry times (14-16) than those that were unsuccessful (17-20). Entry times of 3-4 hours after stroke have been suggested as necessary for clinical trials to take advantage of the therapeutic window (2, 21), that period during which some cells are at risk of death and can be saved by the restoration of blood flow. The limit of entry time is characterized by the progression of cerebral ischemic edema (22) from cytotoxic edema to vasogenic edema (23), the subsequent leakage of the blood-brain barrier to macromolecules in plasma, and the permanent disruption of cellular integrity 6-24 hours after stroke onset. Evidence from the comparison of H 2 clearance CBF and neuropathology suggests that 3 hours is the longest time that ischemia can be tolerated during which the "therapeutic window" still exists (1). Using a rat model of ischemic stroke, Kaplan et al. (2) have come to the same conclusion and defined the therapeutic window in terms of the ischemic penumbra. Clinical trials have disregarded this time factor until now, because of results of trials of the thrombolytic agent, tPA; treatment within 90 minutes has been achieved using emergency medical personnel to recognize stroke (8, 24). Another tPA trial that uses the much lengthier process of angiography

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for screening has reduced the entry time to 6 hours (6). However, one of the weak links in clinical trials is the lack of quick and noninvasive diagnostic techniques that are specific for very early stroke and that can identify and characterize evolving brain infarction, because treatment based solely on neurologic findings and time parameters is clearly suboptimal.

Diffusion-Weighted Imaging Diffusion-weighted imaging is a relatively new MR modality that is sensitive to the translational motion of water protons and was first introduced, implemented, and extended by Le Bihan et al. (25-27). Its role in ischemia is based on the observation that the diffusional rate of water in ischemia is much lower than that of normal brain. For diffusion-weighted imaging, gradient coils capable of generating high magnetic field gradients are required. The diffusion-weighted image is acquired by introducing a pair of gradient pulses with respect to the refocus 180~radio frequency pulse in the spin-echo sequence to increase the effect of motion on the echo signal (25). The resulting echo signal is dependent on proton motion (26). Therefore only the a p p a r e n t diffusion coefficient (ADC) can be obtained. The ADC image is a quantitative measure of the amount of diffusion and is inversely proportional to the DW image. It is calculated, on a pixel-by-pixel basis, from two or more images with different diffusion weighting (26). Values in normal and ischemic brain have been estimated to be 700-800 ~m2/sec and 200-400/xmZ/sec, respectively (28). Several theories have been advanced to explain the sensitivity of DWI to cerebral ischemia. Although it is reasonable that the increased DWI is not due to the magnitude of total water increase in cerebral ischemia, shown to be less than 5% (29), it is possible that the shift of water into the intracellular compartment (30), where its diffusion is restricted, or the associated decrease in membrane permeability due to the inactivation of the Na+/K + pump, could well be the cause of the decreased ADC in early ischemia (31). In either case, the decrease in ADC during early cerebral ischemia is due to the presence of cytotoxic edema. In normal brain, the Na+/K + pump maintains a large space of extracellular water, which has a higher diffusional constant than intracellular water. In ischemia, the pump is disabled and the extracellular space is decreased (22). The evidence supporting this conjecture is based on an experiment in which ouabain, which disables the Na+/K + pump, was administered intraparenchymally, decreasing the ADC from 840 to 460/xm2/ sec (30). The initial decrease of ADC after ischemia (28, 32) certainly is consistent with the initial decrease in extracellular space after arterial occlu-

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sion (22). Thus the decrease in ADC during early cerebral ischemia is most probably due to some characteristic of cytotoxic edema. In the late phase of cerebral ischemic edema characterized by vasogenic edema, in which the blood-brain barrier becomes dysfunctional, evidence concerning DWI is not conclusive. Using a model ofphotochemically induced intracerebral thrombosis (33), which has an uncertain relationship to classical vasogenic edema, DWI was increased in the periphery of the lesion, but not in the central area that suffered immediate endothelial barrier dysfunction (34). In several other reports, high DWI intensities at 12 hours (35) or low ADC values up to 24 hours (32) were noted after permanent occlusion, indicating that DWI is sensitive to vasogenic edema, as well as cytotoxic edema. Although DWI changes after middle cerebral artery (MCA) occlusion in the rat have been described by Mintorovitch et al. (28), the data lack certain features that limit our understanding of DWI for the diagnosis of ischemic stroke. First, the data are reported in terms of the ratio to contralateral cortex. Second, DW intensity is used, not ADC values. Third, the data are collected at only one gradient factor (b = 1413 sec/mm2). Fourth, ischemia was produced for only 33 minutes, so no information beyond this period is available. Finally, quantitative regional comparison to CBF or histology was not performed, although infarct was confirmed histologically at the end of the experiment. Although this study does not provide necessary CBF or histology data, it is extremely interesting because the initial DWI intensity at approximately 20 minutes after occlusion is 1.38 times the preocclusion control. It should be noted that this work used the intraluminal suture model of focal cerebral ischemia of Zea-Longa et al. (36), which produces large ischemic regions. Thus the sensitivity reported is based on only large ischemic regions and does not provide knowledge on the detectability of small ischemic regions. The time course of DWI changes after MCA occlusion in the rat has been described by Knight et al. (32) at 1.5-4, 4-8, 18-24, and 48-72 hours, in addition to longer periods. ADC remained at 50% of control values until the 18- to 24-hour time point, then increased to control thereafter. The time resolution is coarse, especially for the early and more interesting times, and although regions of interest were placed over "ischemic" cortex, no confirmation of ischemia was provided. In a further publication in which only ipsilateral/contralateral differences are given, Knight et al. (37) showed ADC changes until 24 hours in core and bordering regions. DWI has been proposed as a neuropathological marker at 30 minutes after ischemic onset using the suture model of MCA occlusion (38), but interestingly a subsequent work from the same group suggests that the ADC threshold changes during the first 2 hours (39). Both of these studies use

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neuropathology at 24 hours to assess ADC changes, without regard to the changing pathophysiology of CBF during the first hours after ischemia. Although ADC alone will undoubtedly have a role in stroke detection, we are unsure of exactly how ADC will be used for penumbra localization. Most probably, the addition either of an MR perfusion technique (40) or of MRS with NAA and lactate (41) as additional pointers will be necessary for clinical utility.

Chemical Shift Imaging Spectroscopy and Lactate Spectroscopy and Imaging MR proton spectroscopy was first performed with a surface coil placed over a region from which the spectra were to be obtained. This limitation made it difficult, if not impossible, to use the technique in either experimental or human focal cerebral ischemia, because the location of the ischemic core is often unknown, making the placement of the surface coil problematic. The combination of diffusion-weighted imaging and surface coil lactate spectroscopy has been applied to experimental stroke (42, 43). Recently, spectra have been obtained in a specified position by choosing a volume of interest using a saddle or slotted tube resonator (44). Localized proton spectroscopy with a vowel size of 2 x 2 x 2 cm has been used in normal humans (45) and a stroke patient (44). A technique to obtain a voxel size of 12/xl (3 x 2 x 2 mm) has been described, making this method useful for the study of experimental stroke in rats (41).

Cerebral Tissue Lactate in Stroke: Time Course and Levels Lactate plays a central role in ischemia. Figure 1 shows the results of combining cerebral tissue lactate determinations from different ischemia models and species (46-55). These data from different sources agree well, showing an immediate elevation of brain tissue lactate to 10 mM within minutes of ischemia, with a linear rise to 20 mM at 6 hours, dropping to 10 mM at 24 hours. The increasing lactate up until 6 hours, and perhaps beyond, could be a factor in the loss of tissue vitality that occurs when the last stages of cytotoxic edema end at 6-10 hours after onset. The only points that are not consistent with this pattern are those above 30 mM, after complete (53) or focal (54) ischemia in the cat. These values are two to three times the other values, because in the global ischemia model (53) there presumably was just enough residual flow to supply glucose for the anaerobic production of lac-

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FIG. 1 Data from various ischemic models (O, focal ischemia; IS],directed sampling in focal ischemia or global ischemia) and species showing the immediate increase and the eventual decrease of cerebral lactate after ischemia. The curve is arbitrarily fitted.

tate, yielding very high lactate values. In the focal ischemic model (54), tissue sampling was directed to areas of very high 2-deoxy-D-[14C]glucose accumulation, where high lactate production would occur. In studies without this directed sampling, this tissue with high lactate would be mixed with more normal tissue, yielding lower lactate values. If the spatial resolution of the lactate image were high enough to resolve these heterogeneities in lactate, they could be used as a marker for areas that have a dribble of flow and are potentially viable if flow is restored. Barker et al. (56) and Monsein et al. (57) have observed a similar time course of lactate accumulation, with a continued elevation until 12 hours after arterial occlusion in the baboon using localized 1H magnetic resonance spectroscopy for lactate. However, the lactate concentrations do not agree with the biochemical results summarized in Fig. 1, most probably because lactate concentration was referenced to NAA as a standard. NAA concentration has been shown to vary during ischemia (58, 59), and therefore is not an acceptable or accurate standard for ~H spectroscopy. What will be the relation between lactate, NAA, and ADC in early stroke? Can these three quickly responding entities help in the regional identification of the evolution of ischemia to infarction? We know lactate rises quickly, within minutes, in the ischemic core, but we have little information between 6 and 24 hours for lactate, and little information about NAA in the first 6 hours. The early rise of ADC in the first 2 hours has been characterized (60): there is a rapid rise of ADC starting at 5 minutes, but this study does not

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report the progression of ADC values, but rather the evolution of brain volume below the ADC threshold of 550/zmZ/sec.

N - A c e t y l a s p a r t a t e in N o r m a l Brain and Cerebral I s c h e m i a N-Acetylaspartic acid, discovered in 1956 by Tallan (61), is the major peak seen in water-suppressed NMR proton (hydrogen) spectroscopy at a resonance of 2.02 ppm, although N-acetylaspartylglutamate also contributes to this resonance. NAA makes up about one-thousandth of the wet weight of human brain at about 10 mM. This compound has been shown to be relatively stable for a period of 24 hours postmortem (62). NAA is a major source of acetyl groups for lipid synthesis during brain development in rats (63) and appears to be limited solely to neurons. Immunohistochemical localization of NAA has demonstrated that it is discretely localized in many neurons throughout the rat CNS (64), but is undetectable is nonneuronal tissue (65). Although it is now commonly accepted that NAA is a neuronal marker, NAA was found in high concentrations in oligodendrocyte type 2 astrocyte progenitor cells grown in vitro (66). This finding suggests that alterations in NAA in specific brain disorders might not be due solely to neuronal factors. The time course of NAA has been determined in human stroke starting at 6 hours after stroke onset (67) and compared with SPECT CBF maps (68). A normal level was determined at the earliest data point at 6 hours, and then decreased at later times. The decrease in NAA correlated with the severity of ischemia. In the rat MCA occlusion model, NAA decreased in the histological location of the infarct (69). Several studies have measured NAA after 1 hour of ischemia, then recirculation, in gerbils (70, 71), but did not include permanent ischemia. Evidence that NAA levels decrease without neuronal degradation during acute hypoxia indicates that we must be careful when proposing that NAA is a marker of neuronal counts (72).

Ischemic Penumbra Hypotheses concerning ADC, CBF, NAA, and lactate in the penumbra are complex and can be easily constructed to be circular. The penumbra, during the initial 4 hours, is a moving target. At these early times, the penumbra, when it is defined as that region which can be salvaged, is dependent on what is doing the salvaging. The volume of tissue under the ischemic threshold can change. One problem with defining the penumbra for stroke therapeutics is that its definition is dependent on how well the therapy works. The classical definition of the penumbra by Strong et al. (73) is low EEG amplitude and

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TABLE I Observable Parameters before and after 4-Hour Postonset in Ischemia a Cortical region Ischemic core

Penumbra

Normal

Variable

<4 hr

>4 hr

<4 hr

>4 hr

<4 hr

>4 hr

CBF ADC [Na+] Specific gravity Lactate NAA

~ $ 1' 1' 1' 1'

$ $ 1' 1' 1' {

~ -~ ~ $ 1' 1'

DNE DNE DNE DNE DNE DNE

1' 1' $ ~ $ 1'

1' 1' $ $ 1'

a ~, Low value; 1', high value; ---~, near-threshold value; DNE, does not exist.

normal extracellular K +, but when dealing with therapeutic measures a definition based on noninvasive methods that include time and various modes of salvage is needed. Knowledge of the location and size of the ischemic penumbra would permit an answer to the most pertinent diagnostic question: "Is it too late to treat?" Concrete evidence of the ischemic penumbra would be useful in making clinical decisions. In previous work, the ischemic penumbra was related to the slightly reduced p a C O 2 reactivity in the area between the ischemic core and normal cortex (74). This area is no longer viable by 24 hours, but hypothetically is still viable during the first 3 hours of ischemic stroke. Possibilities for the relationship between cerebral perfusion, lactate, NAA, and ADC in normal, penumbral, and ischemic cortex before the therapeutic window expires, at 4 hours, are presented in Table I. ADC will be higher than threshold in normal cortex ( 1', Table I). Because the penumbra does not exist after the therapeutic window, no entries can be made for penumbral parameters after 4 hours. After 4 hours, tissue that was previously penumbra will either be ischemic core or normal. NAA will remain normal ( 1' ) in all cases in which neurons are alive and viable. Only in the ischemic core after 4 hours will NAA be depressed ( + ). In these areas at risk before 4 hours, both ADC and CBF should decrease to their respective near-threshold values of 460/~mZ/sec (75) and 35 ml 100 g-1 min -~ (76). Lactate will be abnormally high in both the core and the penumbra, both before and after 4 hours. CBF will serve as an early indicator of later damage, as determined by the ischemic threshold. If these quantitative relations exist between regional ADC, lactate, NAA, and MR perfusion during early stroke, then they should help in the location of the ischemic penumbra. Even in the early

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stages of DWI research, this goal has been expressed (77). It is possible that the combination of near-threshold ADC, near-threshold perfusion, normal NAA, and high lactate will provide this identification. The utility of DW, lactate, NAA, and MR perfusion images for penumbral location could require the careful correlation in time and space between these new MR modalities.

Comparison of Apparent Diffusion Coefficient with Cerebral Blood Flow Autoradiography for Assessing Early Stroke Although initial studies have attempted to define the utility of DWI and 1H MR spectroscopy for lactate and NAA in ischemic stroke, none has compared DWI, lactate, and NAA to CBF. Instead, either no correlation to physiology or morphology was attempted (32) or correlation was made to neuropathology at the end of the experiment (28, 35, 42, 43, 78, 79). Comparison of ADC with a magnetic resonance index of cerebral blood volume in a cat model of partial MCA stenosis has been performed (80). At 7 hours after MCA occlusion, Back et al. (8 l) compared ADC with pH, lactate, and ATP distributions, with the conclusion that all of the parameters are highly correlated at this time. Bradley et al. (82) have used CBF autoradiography as a standard when evaluating a new cerebral perfusion technique using MR with a superparamagnetic agent.

Methods The distribution of DWI was compared with CBF autoradiography in an experimental model of focal ischemia in the rat to define the time course of DWI intensity over the evolution of stroke and the sensitivity of stroke detection (75). Sprague-Dawley rats were embolized with a single silicone cylinder introduced into the internal carotid artery (ICA) with both common carotids ligated (13). The animals were paralyzed (gallamine), anesthetized (isoflurane in O2), and MABP was monitored during the MR procedure. MR-DWI were obtained with a General Electric (GE) 4.7-T small-bore magnet. DWI was performed within 40 _+ 4 minutes (mean _+ SEM) after stroke and repeated at 166 + 4 minutes. A T2-weighted image was obtained at 72 _+ 2 minutes, followed by CBF determination at 232 -+ 4 minutes. Four DWI, their corresponding ADC images, and four anatomically matching CBF images from each animal were compared.

218

PARADIGMS OF N E U R A L INJURY TABLE II

S e n s i t i v i t y a n d S p e c i f i c i t y o f 1- a n d 3 - H o u r D W I f o r I s c h e m i c Localization a

Technique

Sensitivity

SE

95% CI

Specificity

SE

95% CI

DWI (1 hr) T2 weighted D W I (3 hr) CBF

56 2 94 99

6 1 2 1

44-67 0-3 89-98 96-100

95 100 94 71

2 __ 1 4

90-99 m 92-96 63-80

Abbreviations: CI, confidence interval; SE, standard error of the estimate; DWI, diffusion-weighted imaging; and CBF, cerebral blood flow. a Values expressed as percent; n = 10. From Ref. 75 with permission.

Sensitivity and Specificity As shown in Table II (75), the sensitivity analysis indicates that early DWI is not as sensitive as late DWI or CBF. Using a latent class model for statistical comparison (83), the sensitivities for 3-hours DWI and CBF are 94 and 96%, respectively, and agree because their 95% confidence intervals overlap, whereas the 56% sensitivity of 1-hour DWI differs from both sensitivities for the 3-hour DWI and CBF. The Tz-weighted images failed to show alterations in any animal. Thus DWI might not be completely sensitive in the very early stages of ischemic stroke. According to the initial reports from other workers, DWI imaging is extremely sensitive for early stroke, although sensitivity was not measured (35, 43, 77, 84). This result of low early sensitivity could be due to small infarct volumes in several animals (750. Animals in whom a DWI hyperintensity was detected in the first DWI had larger ischemic areas (35 -+ 9 mm 2, n = 5) than those detected in the second DWI (7.9 +_ 2 mm 2, n = 3; p = 0.06). Thus DWI has a high degree of sensitivity in the detection of cerebral ischemia, with the caveat that a certain critical volume of affected tissue appear to be necessary for early detectability.

Determination of ADC Threshold Profiles through the most hypointense area in the left, ischemic hemisphere and through a homologous area in the opposite, normal hemisphere were extracted from both the first and second ADC images (Fig. 2) (75). These profiles consist of a plot of the ADC signal intensity versus length along a 1-mm-wide curvilinear segment drawn through the right and left hemispheres. As presented in Fig. 2, for the profile on the ischemic side, contiguous ADC values within 1.1 times the minimum ADC were averaged, along with the

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FIG. 2 (A) Transect strips (1 mm wide) drawn, starting at point b, through the ischemic region, represented as the cross-hatched area in the left cortex, and through the nonischemic, contralateral region, starting at point a. (B) The profiles from these ADC transects through the normal (a) and ischemic (b) hemispheres are plotted from the 1-mm-wide transect strips that are shown in A. The ADC threshold is chosen at 45% of the difference between the normal and ischemic ADC plateaus. Here, the threshold is 309 p~mZ/sec. The mean (_+ SD) threshold was determined to be 460 + 95/~mZ/sec from 13 determinations. From Ref. 75, with permission.

same number of ADCs from the corresponding contralateral profile. The ADC value corresponding to 45% of the difference between the average ADCs of the ischemic and contralateral profiles was obtained for each animal. These ADC thresholds were averaged to produce a mean ADC threshold. The mean ADC threshold was 460 _+ 95 /xm2/sec (75). Values below this value were used to establish the cross-sectional area of abnormality in the ADC images and to classify regions of interest (ROIs) as abnormal.

CBF versus A D C in Ischemic and N o r m a l Cortex ADC and C B F values from nonischemic gray matter from the second ADC images (Fig. 3A) were linearly correlated. Thus the p e n u m b r a is characterized by ADCs and CBFs that are near their thresholds, 460/xm2/sec and 35 ml

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FIG. 3 (A) CBF vs. 3-hour ADC values (A) in ROIs representing nonischemic gray matter. A significant inverse correlation between CBF and the 3-hour ADC was observed. There are two data points plotted at the asterisk. (B) CBF vs. the 1-hour (O) and 3-hour ADC values (A) in ROIs representing only ischemic gray matter (CBF < 35 ml 100 g-] min-1). Both coefficients of determination, r 2, for the 1- and 3-hour ADCs are significant (p < 0.05). (C) The ipsilateral/contralateral CBF ratio vs. the ADC ratio in ischemic (ipsilateral CBF < 35 ml 100 g-i min-l) and nonischemic cortex at 1 and 3 hours. The coefficient of determination, r 2, for the 3-hour ADC ratio in ischemic cortex (A, solid line) is 0.48 (p < 0.05), whereas the other ADC ratios are not linearly related to CBF ratio, indicating that relationship between ADC and CBF ratio has changed from 1 to 3 hours. The mean ischemic ADC ratio at 1 hour (O) is different from both the 1-hour (e) and the 3-hour (A) ratios (segmented lines) in nonischemic cortex.

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ACUTE STROKE DIAGNOSIS WITH MRI

221

100 g-1 min-~, respectively, at < 4 hours. This supposition reflects the fact that in the CBF versus ADC plot for ischemic cortex (Fig. 3B) the ADCs from the points with the higher, near-threshold CBFs do not drop from 1 to 3 hours. In contrast, the ADCs of those five points with severely depressed CBF, far under the ischemic threshold, but with near-threshold ADC at 1 hour, drop to under 200 ~mZ/sec at 3 hours. When the ipsilateral/contralateral CBF and ADC ratios were compared, only the 3-hour ADC ratio from ischemic areas correlated with the CBF ratio (Fig. 3C; r 2 -- 0.48, n = 15, p < 0.05). The 1-hour ADC ratio from ischemic cortex and both the 1- and 3-hour ratios from nonischemic cortex showed no relation to the CBF ratio. However, the ischemic cortex 1-hour ADC ratio, 0.74 + 0.14, n = 18, differed (p < 0.01) from both the 1- and 3-hour ratios from nonischemic cortex (0.99 -+ 0.08, n = 10; 0.97 +_ 0.04, n - 9). Although there is no difference between the slopes of the ADC and CBF for ischemic cortex from 1 to 3 hours (Fig. 3B), there is a striking change when the data are normalized using the ipsilateral/contralateral ratio, as shown in Fig. 3C. This is because the five ROIs with 3-hour ADCs less than 200 mZ/sec in Fig. 3B showed a dramatic drop in ADC from 1 to 3 hours, and in addition had low ipsilateral, in relation to contralateral, CBF. Whereas the other ROIs classed as ischemic did not show a drop in ADC from 1 to 3 hours, these five points showed a mean drop of 45%, suggesting a more rapid progression than the other below-threshold ROIs. The comparison of CBF and ADC ratios in Fig. 3C shows several aspects of the progression of ADC over time and from normal to ischemic cortex. There is no relation between CBF ratio and ADC ratio for nonischemic cortex at both 1 and 3 hours after ischemic onset and for ischemic cortex at 1 hour. The mean ADC ratio in ischemic cortex at 1 hour has fallen about 25%, from 1 to 0.74 (p < 0.01). At 3 hours, the relation has become linear, driven by the further progression of cytotoxic edema or the initial stages of vasogenic edema. This study using autoradiographic CBF as close in time to the MR imaging sequence as possible (75) used the same approach that showed the lack of correspondence between T2 imaging and early stroke (9). This previous T2 study (i) documented the time course and location of Tz-weighted nuclear magnetic resonance imaging changes in stroke from 2 to 12 hours in the MCA occlusion model in cats, (ii) showed that edema-sensitive T2-weighted images only correspond with the ischemic area after 12 hours, and (iii) showed that Tz-weighted imaging is not representative of ischemia during the first 6 hours.

222

PARADIGMS OF N E U R A L I N J U R Y

FIG. 4 Coronal images of a rat with an MCA occlusion. The DWI at 3 hours (left) and the autoradiographic CBF image at 3.7 hours (right) do not spatially correlate. Note the area of high intensity in the DWI image that corresponds to the ventral border of the ischemic region in the autoradiograph. From Ref. 85, with permission.

M i s m a t c h between D W I and C B F In several animals, there was a region of very high DW intensity at the border of the ischemic area. Figure 4 shows an example of a 3-hour DWI image compared to a 3.7-hour CBF autoradiographic image. This figure (85) shows coronal images of a rat with an MCA occlusion. The DWI at 3 hours (left) and the autoradiographic CBF image at 3.7 hours (right) do not spatially correlate. Note the area of high intensity in the DWI image that corresponds to the ventral border of the ischemic region in the autoradiograph. This region leads us to speculate that low ADC, high lactate, and normal NAA with CBF greater than the ischemic threshold in the first 3 hours of ischemia will correlate with eventual viability. Even though we do not have histological analysis, we can make the presumption that the region with CBF less than the ischemic threshold of 35 ml 100 g-1 min-1 defines the eventual infarct. This region is shown as the black area in Fig. 4 and is clearly not associated with the high intensity in the DW image at the ventral border of the ischemic region. Thus we must assume that this region of high DWI intensity will not become infarcted.

[13] ACUTE STROKE DIAGNOSIS WITH MRI

223

FIG. 5 Coronal images of a rat with a large stroke. Top:2,3,5-Triphenyltetrazolium chloride staining at 24 hours showing an infarct in the left cortex. Bottom: DW image of same section at 4 hours (above) and at 24 hours (below).

Apparent Diffusion Coefficient Changes from 4 to 24 Hours after Ischemia Figure 5 presents DWI images from which ADC was calculated in various regions at 4 and 24 hours after stroke onset. The plots of D W intensity versus b are shown in Fig. 6. All the plots are monoexponential, and the ADC decreases from 294 to 186/~m2/sec in ischemic cortex, but remains constant

224

PARADIGMS OF N E U R A L INJURY

/~

ICO

ICT

9 ICO F-I 24hr 514pm21s 186pm~ls

+

4hr

ICT 24hr 522 pm21s

4hr

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1.01Z 0.9 0.8 0.7 o 0.6

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GRADIENT, b (slmm a) FIG. 6 Diffusion-weighted signal intensity, Si/So, v s . gradient factor, b i , for normal (ICT) and ischemic (ICO) cortex 4 and 24 hours after stroke onset. The ADCs are the slope of the monoexponential nonlinear fits obtained using the LevenbergMarquardt algorithm.

in contralateral normal cortex. The initial exponential component described by Le Bihan et al. (27) is probably very small and does not contribute to our data. Thus the ADC decrease will continue into the period of vasogenie edema.

A D C in V a s o g e n i c E d e m a By piecing together two sets of data, we suggest that ADC continues to fall during the first 24 hours. Evidence presented by Perez-Trepichio et al. (75) suggests that ADC falls from 1 to 3 hours in severely ischemic regions. From data shown in Fig. 6, ADC falls from 4 to 24 hours. Combining these studies suggests that ADC continues to drop from soon after stroke onset until 24 hours. Thus it appears that over the time course of the transition from cytotoxic to vasogenic edema that is characteristic of ischemic brain edema, ADC continuously falls. MRI Blood Flow Techniques This section contains more detail about detection of CBF using MR techniques, with discussion of the potential of performing experiments as described previously using solely MR techniques.

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Magnetic Resonance Imaging Several proton MRI techniques delineate brain perfusion, edema, and brain injury with high spatial and temporal resolution. Techniques developed at high field strengths have already been applied in humans at 1.5 T (86). Because MRI is noninvasive, sequential data may be obtained to trace the temporal evolution of these abnormalities in a single experimental animal. Advances in tomographic imaging techniques such as PET and MRI have provided powerful tools to study cerebral ischemia (77, 87, 88). These techniques provide pictorial and often quantitative representation of metabolic and hemodynamic variables in the brain during cerebral ischemia.

Cerebral Perfusion Several specialized magnetic resonance imaging procedures have been developed and are very useful for studying cerebral ischemia. Techniques have been developed to image cerebral perfusion using MR contrast agents (8992). These techniques are very sensitive to perturbations in tissue perfusion and yield semiquantitative cerebral plasma volume (CPV) and cerebral mean transit time (MTT) and CBF images (40), using exogenously administered intravascular contrast agents.

Animal Model Male Sprague-Dawley rats, weighing 300-350 g, are fasted overnight. After initial induction of anesthesia with 3.0% halothane in balanced breathing air (IWECO, Inc., Houston, TX), the rat is intubated with a 14-0 i.v. catheter and artificially ventilated with halothane maintained at 0.5-1.0% during the surgery and MRI procedure. The respirator used is a modified, compressed air-powered clinical pressure ventilator (Monaghanin, Littleton, Colorado). The ventilating pressure is maintained between 20-25 cm H20 and the respiration rate at 25-30 per minute. At these ranges, the rat paCO2 can be maintained at 35-40 mm Hg. The rat tail artery is cannulated for monitoring the blood pressure, paCO2, paO2, and pH. Rectal temperature is maintained at ---37.0-37.8~ with a heating pad under the rat during the surgery. A tail vein cannula is placed to deliver the MR contrast agent. During MR imaging, the rat core temperature is maintained by blowing warm air into the magnet bore. Blood pressure and rectal temperature are recorded when imaging procedures are finished and the rat is removed from the magnet. Cerebral ischemia is induced by occluding the right MCA using the intraluminal suture insertion method (36, 93) with some modifications. A 1-cm-

226

PARADIGMS OF NEURAL INJURY

long oblique incision is made on the right ventral lateral side of the neck. The ICA is isolated and ligated at the base of the common carotic artery bifurcation. The pterygopalatine artery, which is the only branch of the extracranial ICA, is then isolated and also ligated. An incision is made in the ICA, and a 3-0 monofilament nylon suture with the insertion end coated with silicone is introduced into the lumen of the ICA. Exactly 15 mm of suture is advanced into the ICA from the base of the bifurcation of the ICA. The suture passes the origin of the ipsilateral MCA, blocking the blood supply to it.

NMR Techniques Proton MRI is performed with a 4.7-T imaging system (SISCO, Fremont, CA). The imaging coil is a home-built 2-inch saddle-style radio frequency coil tuned to 200 MHz, mounted on a plastic cradle. The rat is placed in a supine position on the cradle with its head positioned inside the imaging coil. After the cradle is placed inside the magnet, a sagittal scan of the rat brain using a fast low-angle shot gradient echo-imaging sequence, or FLASH (94), is acquired to define the relative position of the rat inside the magnet. The MRI protocol consists of the following imaging regimens: (1) DWI spin-echo; (2) relative MTT and relative CBF image. All the images are acquired with a field of view (FOV) of 80 mm, 128 phase-encode (PE) steps, and reconstructed using a 256 x 256 matrix, which translates to an in-plane resolution of 0.31 mm/pixel. DWI is acquired using a multislice mode. Twelve consecutive transverse slices cover a 2-cm length from the cerebellum to the olfactory lobe, with a slice thickness of 1.7 mm. Due to hardware limitations of the MR imager, only one slice is acquired at the level of the caudoputamen for the MTT and CBF images.

Diffusion-Weighted Spin-Echo Image DWI shows the contrast due to differences in tissue water diffusion (25). DWI is implemented by modifying the standard spin-echo sequence; T is 3200 msec and TE is 90 msec. Two 18-msec rectangular-shape, diffusion gradient pulses are added symmetrically on either side of the refocusing 180 radio frequency pulse. The diffusion gradient separation time is 29 msec. The diffusion gradient is set at 4.0 g/cm, which results in an attenuation factor (b value) of 1500 mm/sec 2 (25).

Relative MTT and CBF Images Theory Measurement of MTT (of blood) through the brain is based on the principle of the indicator-dilution method (95). Originally, the indicator dilution method was used to estimate the total blood flow of an organ, such as cardiac output

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or renal blood flow. To implement this principle, an intravascular indicator is injected into the blood supply of an organ of interest. The indicator is then monitored at the outlet of the organ, and a concentration-time curve is constructed to describe the passage of the indicator through the organ. From this concentration-time curve, a series of parameters such as MTT, blood volume, and flow are derived. More recently, the indicator-dilution method has been adapted in topographic imaging techniques, such as CT (96, 97) and MRI (90, 98). In the case of CT or MRI, a time-concentration curve of the indicator is needed for each pixel in order to obtain a map of the perfusion parameter of interest for the entire image. This time-concentration curve can be obtained by repeated dynamic imaging of the slice(s) of interest while the indicator is passing. Intensity change at each pixel over the series of scans can be used to derive the time-concentration curve of that pixel. MR Contrast Agent Measurement of MTT by MRI requires the use of an MR contrast agent, an indicator that can be detected by MRI. One type of MR contrast agent, called superparamagnetic iron oxide (SPIO), affects the MR signal by altering the magnetic susceptibility of the plasma when it is given intravenously, thereby enhancing the spin-spin relaxation processes (T2) of the protons in the plasma. The contrast agent used in the present study is an intravascular SPIO tracer (AMI-227, Advanced Magnetics Inc. (Cambridge, MA)/Squibb Diagnostic). The vascular half-life of AMI-227 is about 5.5 hours. Its effect on MR signal attenuation has been shown to be proportional to its concentration in the vasculature (40). k-Space Substitution Because cerebral transit times are on the order of seconds, a high-speed imaging technique is needed in order to obtain enough scans to resolve temporally the passage of contrast agent through the brain. The FLASH imaging sequence used in the present study requires 2 seconds per scan with an imaging acquisition matrix of 128 x 128 and signal average of 2. This temporal resolution is not sufficient for proper digitization of the bolus transit through the rat brain. To improve the temporal resolution, the k-space substitution acquisition method is used (99). This method is based on the premise that, of all the phase-encoding steps needed for reconstructing an image, the high phase-encoding steps (high frequencies) primarily contribute to the fineness of the spatial resolution, and thus appearance of the image, whereas the image intensity, and therefore image contrast, is primarily determined by low phase-encoding steps (low frequencies). These characteristics of kspace allow the spatial resolution and contrast in an image to be extracted from different phase-encoding steps. Using this concept, a reference image

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PARADIGMS OF N E U R A L INJURY

prior to the injection of the MR contrast agent is acquired, in which all 128 phase-encoding steps ( - 6 4 , . . . , - 1,0, 1 , . . . , 63) are collected. In dynamic imaging, during which the contrast agent is introduced, only the middle 32 low phase-encoding steps ( - 1 6 , . . . , - 1 , 0, 1. . . . ,15) are collected. This low phase-encoding data set is then used to replace the corresponding low phase-encoding data in the reference image. The reconstructed images preserve both the contrast from the fast dynamic scanning images and the spatial resolution from the reference image. The signal-to-noise ratio of the reconstructed images is similar to the reference image but with only a quarter of the scanning time (32 phase-encoding steps instead of 128 phase-encoding steps). With T at 7.8 msec and TE at 2.5 msec, and signal averaging of 2, the acquisition time is 0.5 second per scan. Due to a computer reset delay, there is a 0.2-second pause between subsequent images, resulting in an effective image repetition time of 0.7 second. A total of 30 scans over the same slice is acquired, with a total scanning time of 21 seconds. Calculation of Flow Indices The intensity at each pixel in each of the images from the dynamic imaging sequence is converted into the contrast agent concentration using the following formula (98): C(t) = [ 1 / - T E AR2)] ln[I(t)/I(O)],

where I(0) is the pixel intensity in the absence of contrast agent, I(t) is the pixel intensity at time t, and Z ~ 2 is the molar relaxivity of the contrast agent, which was set at 484 sec-~mmol/liter (40). From the 30 images, a time-concentration curve representing the concentration change over a 21second period after the injection of the contrast agent is built for each pixel. The time-concentration curve is then fitted with a gamma-variate function to eliminate the contamination of recirculation of contrast agent to the original first pass trace (100): C(t) = Cp(elafl)~(t - Ta)~ e -(t-ra)/~,

where Cp is peak concentration of the passing contrast agent, a and/3 are two parameters determining the shape of the curve, and Ta is the time between the bolus injection and bolus appearance at the pixel of interest. The Simplex algorithm (101) is used to fit parameters Cp, a,/3, and Ta. From the fitted parameters, the following functional perfusion indices are calculated:

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A C U T E S T R O K E D I A G N O S I S WITH MRI

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Time after Injection FIG. 7 Time-concentration curve of a bolus of the MR contrast agent AMI-227 passing through a representative pixel in the cerebral cortex. The curve is fitted to a gamma-variate function: C(t) = Cp(e/c~B)~(t - Ta) ~ e - ( t - ~ )/~. Cp, Peak concentration of the passing contrast agent; Ta, time of arrival, the time when the contrast agent first reaches the pixel after injection; c~,/~, parameters determining the shape of the curve; t, time after the injection of the contrast agent.

Mean transit time (MTT) =/~(a + 1) Area under curve (V) = C p ( e / a ~ ) ~ ( ~ + l ) F ( a Cerebral blood flow ( F ) = V/MTT

+ 1)

Quantitation of CBF using the method of transit time requires an arterial input function to correct for the bolus dispersion (102, 103). This arterial input function can be obtained by simultaneous MR acquisition of both arterial and brain tissue time-concentration curves (104). Limits in our hardware restrict the current study to acquire only the brain time-concentration curve. Therefore, the CBF obtained in the current study is a relative cerebral flow index, proportional to the nonischemic hemisphere. Figure 7 illustrates the time-concentration curve derived from a set of MR scans tracing a bolus of contrast agent passing through a pixel in the brain. The MR contrast agent is given via the tail vein cannula at a dose of 3 mg/kg. A special computer program was developed to calculate the MTT image and blood flow image from the time-concentration curve.

230

PARADIGMS OF N E U R A L I N J U R Y

.

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C FIG. 8 Diagram illustrating the steps for automatic region of interest analysis. (a) The original spin-echo image is rotated so that the vertical principal axis of the image is perpendicular. (b) The rotated image is then standardized into a 46 x 32-pixel rectangle. A standard grid mask (c) with six regions of interest for each hemisphere is then applied to b to give the final image in d. The six regions of interest are (1) frontal and cingulate cortex, hindlimb and forelimb area of cortex, (2) parietal cortex, (3) piriform and insular cortex, (4) preoptic area, (5) caudate putamen, and (6) thalamus.

Region of Interest Analysis Regional analysis of CPV level and relative flow indices in ischemic lesions is applied to the corresponding MR images at the level of caudoputamen (one slice per animal). The regions of interest studied include six structures as shown in Fig. 8. Because manually outlining each structure is very subjective, and very time-consuming considering the number of images needed to be analyzed, a computer program was written to assist the analysis. The program incorporated the following analytical steps. (a) The brain MR image at the level of caudoputamen is selected. (b) The brain image is appropriately aligned by rotating the image according to the principal axes of the images. The position of the brain in the image is then standardized within a 46 x 32pixel rectangle. (c) A standard structure mask including the six brain regions

[13]

A C U T E STROKE DIAGNOSIS WITH MRI

231

is made for all the images. (d) The mask is applied to the standardized image, and various indices are calculated for each structure. The following indices are calculated for each of the six structures in both the stroke hemisphere and the contralateral normal hemisphere: (a) the average value of MTT from MTT images, and the MTT ratio of the structure in the ischemic hemispheres to that of normal hemisphere; (b) the value of volume/transit time (calculated flow) relative flow images, and the blood flow ratio of the structure in the ischemic hemispheres to that of normal hemisphere; and (c) the area of hyperintensity from DWI. Correlation o f CBF with D WI Hyperintensity

A mathematical model based on the logistic function is used to establish the relationship between CBF and DWI hyperintensity in the same animal. The purpose of this correlation is to predict the hyperintense DWI regions, presumably representative of cytotoxic edema, based on early measurement of blood flow index. The following steps are employed to establish the correlation: 1. The 4-hour relative CBF map and its corresponding 4- or 24-hour DWI at the same anatomical position (approximately the same slice acquired at different times can be obtained by a sagittal line-up scan in MRI) are selected. The pair images are rotated and aligned according to their principal axis. The aligned images are then standardized into a 46 x 32-pixel rectangle as described previously. The corresponding anatomical structures in the standardized pair images can now be addressed by the same pixel coordinates. 2. A total of 20 ROIs, each consisting of a 2 x 2-pixel rectangular box, are applied to the ischemic hemispheres in various representative locations in the standardized DWI image. The ROIs located at the region of hyperintense DWI are assigned a value of 1 and those in the normal intensity region are assigned a value of 0. The locations of these ROIs and their corresponding value (1 or 0) are then recorded. Next, these 20 ROIs are applied to the corresponding 4-hour CBF flow map in the same pixel locations. The mean value of the CBF at each ROI is calculated and normalized to the corresponding structures in the contralateral hemisphere, so that the CBF level in that ROI is now expressed as a percent reduction of the normal hemisphere CBF level. For example, a ROI with CBF value of 10% would mean the blood flow level of that region is 90% of the corresponding region in the contralateral normal hemisphere. An example of such a measurement is given in Table III. 3. The data set is then fitted with the logistic function (105): R = DP/(D e + K P)

232

PARADIGMS OF NEURAL

TABLE III

INJURY

Regional CBF Levels and Development of Hyperintense DWI Indicative of Cytotoxic Edema at Representative Voxels in Ischemic Hemisphere a

% C B F level b

Hyperintense DWI c

0.33 0.31 0.35 0.45 0.50 0.56 0.61 0.61 0.65 0.70 0.78 0.83 0.73 0.62 0.85 0.87 0.76 0.92 0.93

0 0 0 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 1

a Data represent 20 voxels sampled from the stroke hemisphere in a rat with permanent MCA occlusion. b % CBF level: the percent reduction of the mean CBF level of that voxel to the mean CBF level of the corresponding voxel located in the contralateral hemisphere. c Hyperintense DWI: 0 indicates normal intensity and 1 indicates the presence of hyperintensity.

where R represents the response with quanta value of either 1 (presence of hyperintense DWI in a ROI) or 0 (normal DWI intensity in a ROI), and D is the "dose" used, which in our case would be the percent flow reduction of an individual ROI. K and P are measurements of EDs0 and slope, respectively. A logistic modeling of the data in Table III is illustrated in Fig. 9. From the curve, the EDs0 and ED95 can be calculated. ED95 represents the dose causing 95% of the sample population to have a response. In our case, ED95 would be the flow level leading to 95% of the ROIs having hyperintense DWI. We propose reporting the value of the ED95 as the flow threshold for ischemic penumbra.

[13]

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All the image analysis procedures were performed by our own programs written in C language in the Sunview windowing environment for Sun computers. Dynamic imaging of the first passage of a bolus injection of the intravascular contrast agent demonstrated prolonged vascular MTT in certain regions with relatively normal CPV levels (Fig. 10). For example, the cortical region of Fig. 10 that shows relatively normal CPV level (as compared to the contralateral cortex) had a longer MTT than that of the contralateral hemisphere. The relative CBF index derived from the vascular MTT measurement decreased by more than 50% in the MCA-occluded cortex compared to its corresponding cortex in the contralateral hemisphere (Fig. 11). An elevation plot of the calculated relative cerebral flow map reveals a topographical gradient of ischemia in the MCA-occluded hemisphere, with a core of absent flow in the center and increasing flow toward the periphery, where the flow level is still lower than that in the contralateral hemisphere (Fig. 12). Correlation of the 4-hour relative cerebral flow index with the 4- and 24-hour DWI hyperintensity using the logistic model indicates that a flow reduction of

234

PARADIGMS OF NEURAL INJURY 4 hr D W I

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FIG. 10 Coronal forebrain images showing spatial correlation between the severity of cerebral perfusion deficit and ischemic lesion. The images are from a rat with the right MCA permanently occluded. Relative MTT and CBF images were taken 4 hours after MCAO from the fast dynamic images, and the CPV index was taken from the contrast-enhanced spin-echo difference image. DWI was taken at both 4 and 24 hours post-MCA occlusion, and TTC staining was performed 1 week after the MCA occlusion. The region of hyperintensity in the 4-hour DWI corresponds well with the area without plasma volume in the CPV index, whereas the high-intensity region in the 24-hour DWI or nonstaining region in the TTC slice corresponds better with the high-intensity area in the 4-hour MTT map and the low-intensity area in the 4-hour CBF map.

87.1% would result in DWI hyperintensity at 4 hours, and tissue with flow reduction by 67.6% or more would turn into DWI hyperintensity 24 hours after MCA occlusion (Fig. 13). Hence, the ischemic tissue with a residual flow level between 12.9 and 38.4% of the CBF level of the contralateral hemisphere represents a region of brain tissue that is not infarcted at 4 hours but is destined to develop DWI hyperintensity by 24 hours after MCA occlusion. These data produce a valid comparison for relative CBF values, but preferable would be an MR method that produces quantitative values of CBF, eliminating the need to rely on the contralateral hemisphere for relative CBF values. This goal is dependent on quantitating the arterial input function

[13]

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(106), and may be able to produce actual CBF values using a variety of models (107). Together these techniques show promise for quantifying events occurring within the ischemic penumbra and elucidating the mechanisms of neuroprotection.

Conclusion Agents for the early treatment of ischemic stroke, within 1 to 2 hours of onset, are now available and being evaluated in clinical trials. Because of the wide variability in size, location, and etiology of stroke, very early characterization of the ischemic process, especially distinguishing infarcted from salvageable brain tissue, will aid in optimizing early therapeutic intervention. However, there is no established diagnostic technique for identifying and localizing evolving ischemic stroke within this early time period. Three magnetic resonance techniques have been introduced that can image processes detectable in the early stages of brain injury that are relevant to ultimate outcome: (1) DWI, sensitized to the diffusion of protons in water,

236

PARADIGMS OF N E U R A L INJURY

FIG. 12 Elevation plot of the relative CBF map measured at 4 hours post-MCAO from a rat. Signal intensity in the flow map is converted into the height of the plot. The map reveals a core of absent flow in the subcortical region of the stroke hemisphere, with a gradual increase in the flow toward the periphery in the cerebral cortex. Reprinted by permission of the publisher from the evolution of acute stroke recorded by magnetic resonance imaging, M. J. Quast, N. C. Huang, G. R. Hillman, and T. A. Kent, Mag. Res. Imaging 11, 465-471. Copyright 1993 by Elsevier Science Inc. has been used to visualize parenchymal changes in ischemic brain within 1 hour from vascular occlusion and provides an image of the ADC. In ischemic regions, the DWI is more intense and the ADC is less intense. (2) It is now possible, although with limited spatial detail, to produce brain lactate or NAA images using MR proton spectroscopy. Lactate has a central role in the bioenergetics of ischemia, whereas NAA is a putative neuron-specific metabolite. (3) Several MRI techniques are being developed that can detect parameters of brain perfusion. The potential of these techniques in brain ischemia and injury is discussed here. What is the relationship between brain perfusion detected by MRI techniques and the thresholds of ischemia that produce brain damage? How best can MRI techniques be used for their utility and effectiveness for very early stroke diagnosis and intervention? Can the combination of ADC, lactate, NAA, and MR perfusion provide specificity for the localization of the penumbra? The question of paramount

237

[13] ACUTE STROKE DIAGNOSIS WITH MRI

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importance in ischemic stroke underlies these questions: Is it possible to identify salvageable ischemic tissue before it progresses to infarction? Do DWI and its quantitative counterpart, ADC mapping, MRS, and MR perfusion have potential for the early evaluation of stroke and stroke therapy in humans by providing delineation of pathophysiological heterogeneity despite similar clinical symptoms in the very early hours of ischemia? Are these predictions just a product of the hope that any new measure that is sensitive to cerebral ischemia can be used to identify the ischemic penumbra, that region which is salvageable in the early stages of ischemic stroke? Although it is not clear that the penumbra may be found by these new techniques, it is clear at this point in time that DWI will have a major role in the diagnosis of stroke in the first 24 hours.

238

PARADIGMS OF NEURAL INJURY

Acknowledgments This work was supported in part by an American Heart Association (National) Grantin-Aid 91-1315 (S.C.J.), an American Heart Association (Texas Affiliate) Grant-inAid 91D-633 (M.J.Q.), a Fellowship from the American Heart Association (Northeast Ohio Affiliate) F244 (A.D.P.-T.), National Institutes of Health Grant NS 30839 (S.C.J.), and National Institutes of Health Grant NS 33575 (M.J.Q.). The image analysis software was supported in part by National Institutes of Health Grant AR03AA07689. Ultrasmall superparamagnetic iron oxide was supplied by Advanced Magnetics, Inc. (Cambridge, MA) and Squibb Diagnostics.

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[13] ACUTE STROKE DIAGNOSIS WITH MRI

70.

71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84.

85. 86. 87. 88. 89.

90. 91.

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Barrere, M. D6corps, E. Pinard, D. Riche, A. L. Benabid, and J. Seylaz, N M R Biomed. 5, 11 (1992). K. Allen, A. L. Busza, H. A. Crockard, R. S. Frackowiak, D. G. Gadian, E. Proctor, R. W. Russell, and S. R. Williams, J. Cereb. Blood Flow Metab. 8, 816 (1988). H. A. Crockard, D. G. Gadian, R. S. J. Frackowiak, E. Proctor, K. Allen, S. R. Williams, and R. W. R. Russell, J. Cereb. Blood Flow Metab. 7, 394 (1987). G. A. Rosenberg, J. White, C. Gasparovic, E. A. Crisostomo, and R. H. Griffey, Stroke 22, 73 (1991). A. J. Strong, G. S. Venables, and G. Gibson, J. Cereb. Blood Flow Metab. 3, 86 (1983). S. C. Jones, B. Bose, A. J. Furlan, H. T. Friel, K. A. Easley, M. P. Meredith, and J. R. Little, Am. J. Physiol. 257, H473 (1989). A. D. Perez-Trepichio, M. Xue, T. C. Ng, A. W. Majors, A. J. Furlan, I. A. Awad, and S. C. Jones, Stroke 26, 667 (1995). G. W. Tyson, G. M. Teasdale, D. I. Graham, and J. McCulloch, Ann. Neurol. 15, 559 (1984). L. L. Baker, J. Kucharczyk, R. J. Sevick, J. Mintorovitch, and M. E. Moseley, A JR 156, 1133 (1991 ). M. E. Moseley, R. Sevick, M. F. Wendland, D. L. White, J. Mintorovitch, H. S. Asgari, and J. Kucharczyk, Can. Assoc. Radiol. J. 42, 31 (1991). K. Houkin, I. L. Kwee, and T. Nakada, J. Neurosurg. 72, 763 (1990). T. P. L. Roberts, Z. Vexler, N. Derugin, M. E. Moseley, and J. Kucharczyk, J. Cereb. Blood Flow Metab. 13, 940 (1993). M. Eis and M. Hoehn-Berlage, J. Magn. Resort. 107, 222 (1995). R. H. Bradley, T. A. Kent, H. M. Eisenberg, M. J. Quast, G. A. Ward, G. A. Campbell, and G. Hillman, Stroke 20, 1032 (1989). D. Rindskopf and W. Rindskopf, Star. Med. 5, 21 (1986). J. Mintorovitch, Y. Cohen, L. Chileuitt, H. Shimizu, P. Weinstein, and M. E. Moseley, in "Works in Progress~Society of Magnetic Resonance in Medicine," p. 1002. Society of Magnetic Resonance in Medicine, Berkeley, 1989. S. C. Jones, A. D. Perez-Trepichio, M. Xue, A. J. Furlan, and I. A. Awad, Acta Neurochir. Suppl. 60, 207 (1994). J. W. Prichard and B. R. Rosen, J. Cereb. Blood Flow Metab. 14, 365 (1994). C. Fieschi, V. Di Piero, G. L. Lenzi, P. Pantano, F. Giubilei, C. Buttinelli, and A. Carolei, Stroke 21 (Supp. IV), IV9 (1990). A. L. Brownell, M. Kano, R. C. McKinstry, M. A. Moskowitz, B. R. Rosen, and G. L. Brownell, J. Comput. Assist. Tomogr. 15, 376 (1991). T. A. Kent, M. J. Quast, B. J. Kaplan, A. Najafi, E. G. Amparo, R. M. Gevedon, F. Salinas, A. D. Suttle, D. J. DiPette, and H. M. Eisenberg, A J N R 10, 335 (1989). R. R. Edelman, H. P. Mattle, D. J. Atkinson, T. Hill, J. P. Finn, C. Mayman, M. Ronthal, H. M. Hoogewoud, and J. Kleefield, Radiology 176, 211 (1990). M. E. Moseley, M. F. Wendland, and J. Kucharczyk, Top. Magn. Reson. Imaging 3, 50 (1991).

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PARADIGMS OF NEURAL INJURY 92. L. M. Hamberg, R. Macfarlane, E. Tasdemiroglu, P. Boccalini, G. J. Hunter, J. W. Belliveau, M. A. Moskowitz, and B. R. Rosen, Stroke 24, 444 (1993). 93. H. Nagasawa and K. Kogure, Stroke 20, 1037 (1989). 94. A. Haase, J. Magn. Reson. 67, 266 (1986). 95. K. L. Zierler, Circ. Res. 10, 393 (1962). 96. Z. Szabo and F. Ritzl, Eur. J. Nucl. Med. 8, 201 (1983). 97. W. H. Berninger, L. Axel, D. Norman, S. Napel, and R. W. Redington, Radiology 138, 711 (1981). 98. P. L. Davis, G. L. Wolf, and J. S. Gillen, Invest. Radiol. 24, 400 (1989). 99. R. A. Jones, O. Haraldseth, T. B. Muller, P. A. Rinck, and A. N. Oksendal, Magn. Reson. Med. 29, 830 (1993). 100. H. K. Thompson, C. F. Starmer, R. E. Whalen, and H. D. McIntosh, Circ. Res. 14, 515 (1964). 101. M. S. Caceci and W. P. Cacheris, Byte 5, 340 (1984). 102. O. Carlsen and O. Hedegaard, Phys. Med. Biol. 32, 1457 (1987). 103. R. M. Weisskoff, D. Chesler, J. L. Boxerman, and B. R. Rosen, Magn. Reson. Med. 4, 553 (1993). 104. W. H. Perman, M. H. Gado, K. B. Larson, and J. S. Perlmutter, Magn. Reson. Med. 28, 74 (1992). 105. J. A. Zivin and D. R. Waud, Stroke 23, 767 (1992). 106. T. A. Kent, D. S. DeWitt, M. J. Quast, D. S. Prough, L. W. Jenkins, R. H. Fabian, and G. R. Hillman, J. Cereb. Blood Flow Metab. 15 (Suppl. 1), $608 (1995). 107. K. B. Larson, W. H. Perman, J. S. Perlmutter, M. H. Gado, J. M. Ollinger, and K. Zierler, J. Theor. Biol. 170, 1 (1994).

[14]

Evaluation of Free Radical-Initiated Oxidant Events within the Nervous System Stephen C. Bondy

Introduction Free radicals are defined as any species with one or more unpaired electrons. Because oxygen is ubiquitous in aerobic organisms, oxygen-centered free radicals have been implicated in several physiological, toxicological, and pathological phenomena. However, although the superoxide anion and hydroxyl radical qualify as oxygen-centered radicals, hydrogen peroxide is a potent cellular toxicant that lacks unpaired electrons. The terms reactive oxygen species (ROS) and oxygen radicals have been used to describe all oxygen-centered radicals and nonradicals with oxidant properties. There has been an increasing accumulation of data suggesting that oxygen radicals are involved in a wide variety of disease processes. The works of Freeman and Crapo (31) and Halliwell and Gutteridge (38, 39) especially have addressed the ubiquitous role of free radicals in the biology of disease and tissue injury. Most of the issues considered originally emphasized the role of free radicals in the mechanisms of carcinogenesis, ischemia, and aging. In the past, free radical research has primarily focused on the area of pulmonary, cardiac, and hepatotoxicity. More recently, the brain, with its high lipid content, high rate of oxidative metabolism, and somewhat low levels of free radical-eliminating enzymes, has also been considered as a prime target of free radical-mediated damage. The localization of antioxidant systems primarily to glia rather than neurons (64, 74), although providing a first line of defense, may render the neurons especially susceptible to excess levels of free radicals. Any imbalance of cellular redox status in favor of greater oxidative activity can lead to several kinds of macromolecular damage, such as disruption of genomic function by alterations to DNA, or impairment of membrane properties by attack on proteins or lipids. Lipid peroxidative events are especially hazardous, because lipoperoxy radicals can initiate oxidative chain reactions (31). Thus, the high lipid content of myelin makes nervous tissue especially susceptible to oxidative stress. It has been known for several decades that mammalian brain contains large amounts of substrates that are susceptible to free radical attack, such Methods in Neurosciences, Volume 30

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PARADIGMS OF NEURAL INJURY as unsaturated lipids and catecholamines. Halliwell and Gutteridge (38) were among the first to discuss the potential role of oxygen radicals in the nervous system. Elevated rates of generation of reactive oxygen species have been associated with many neurological disorders, including various lipofuscinoses such as Batten's disease (36), Alzheimer's disease (22, 35), Parkinson's disease (28, 93), seizure disorders (77), stroke (37), ischemia (69), HallervordenSpatz disease (61), amyotrophic lateral sclerosis (27), and edema (42). Evidence exists that ethanol can lead to elevated levels of lipid peroxidation in liver and brain (10). This has been attributed, at least in part, to liberation of protein-bound iron by ethanol (12, 18, 70, 80). A concomitant of elevated levels of ROS generation is often the presence of excess levels of unsequestered iron, which have been reported for most of the neurological disorders listed above. Iron penetrance into the brain can also follow traumatic brain injury or hemorrhagic stroke when low molecular weight iron is liberated during degradation of free hemoglobin (72). The consequent elevation of ROS may in part account for the delayed appearance of seizure activity bleeding into brain tissue (90). The appearance of abnormally high levels of ROS may then constitute a final common pathway of a broad range of neurological diseases. Oxidative stress is to be regarded as a factor with the potential to contribute to and exacerbate such diseases rather than being the sole means by which damage is effected. The literature abounds with disparate results found after using various dyes as indices of oxidative stress. Some of this variance may be due to the fact the intracellular distribution of these dyes is related to their lipophilicity. Prooxidant status can vary within differing cell compartments and thus dyes report from various distinctive zones. In addition, the spectrum of sensitivity of each dye to oxidation by a precise type of ROS may vary. A difficulty is that little is known concerning the extremely short-lived and reactive species that actually perform the oxidations. The status of the hydroxyl radical is not fully established and there are several rivals for the role of the ultimate direct oxidant. A recent study concludes that distinction between the hydroxyl radical and a metal in a higher oxidation state, as the ultimate oxidant species, is "quite an impossible task" (33). The older literature has many descriptions of a selective assay for superoxide or hydrogen peroxide involving an oxidation, but these are relatively stable species and such assays generally can only implicate these species as precursors. In themselves, their oxidative capacity may be very limited (87). The precise nature of the oxygen radicals produced in the CNS is thus by no means clear. The difficulty of establishing this with certainty is due to the short half-life and rapid interconvertibility of many of the putative key species (50).

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Some of the mechanisms by which ROS are generated within cerebral tissues will be briefly delineated, because this basic information is important in allowing a choice of suitable analytical procedures. Endogenous

Sources of Generation of Reactive Oxygen Species

Oxidative Phosphorylation Around 2% of the oxygen consumed by mitochondria is incompletely reduced and appears as oxygen radicals (14). This proportion may be increased when the efficient functioning of mitochondrial electron transport systems is compromised. This could account for the increased lipid peroxidation found in the brains of mice exposed to nonlethal levels of cyanide (43).

Cytosolic Acidity Lowered pH, resulting from excess glycolytic activity, may not only accelerate the process of liberating protein-bound iron in organisms, but it may also lead to an impairment of oxidative ATP generation and to the appearance of the prooxidant protonated superoxide (8, 81). However, there is evidence that the reduction of pH during ATP depletion may be protective and enhance cell survival (44). Chlordecone, a neurotoxic insecticide, elevates pH within synaptosomes and depresses oxygen radical synthesis (11).

Presence of Metal Ions with Multivalence Potential Liberation of protein-bound iron can occur by enhanced degradation of important iron-binding proteins such as ferritin and transferrin. A small increase in levels of free iron within cells can dramatically accelerate rates of oxygen radical production (55). A key feature in establishing the rate of production of oxygen radicals by tissue is the cytosolic concentration of free metal ions possessing the capacity to change their valence state readily. Iron is considered the most important of these, but levels of free manganese and copper may also be significant factors (6).

Eicosanoid Production Enhanced phospholipase activity can lead to the release of arachidonic acid. This polyunsaturated fatty acid contains four ethylenic bonds and is readily autooxidizable. In fact, impure preparations of this chemical may explode

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spontaneously on exposure to air (38). The enzymatic conversion of this compound to many bioactive prostaglandins, leukotrienes, and thromboxanes by cyclooxygenases and lipoxygenases leads to considerable oxygen radical generation (31, 73). All major catabolic pathways of arachidonic acid involve the utilization of molecular oxygen and the formation of hydroperoxide or epoxide intermediates. Subsequent metabolism by peroxidases and hydrolases can lead to further formation of free radicals. The physiological relevance of this is illustrated by the finding that antioxidants can protect against arachidonic acid-induced cerebral edema (4). Increased levels of cytosolic free calcium may result either from breakdown of the steep concentration gradient of calcium across the plasma membrane, or by liberation of the large amounts of calcium bound intracellularly within mitochondria or endoplasmic reticulum. This elevation can activate phospholipases and thus stimulate oxygen radical production. In fact, the activation of phospholipase D has been functionally linked to superoxide anion production (13). A reciprocal relation exists because free radicals can enhance phospholipase A2 activity within cerebral capillaries (5). Conversely, phospholipase A2 may selectively induce oxidative changes to the y-aminobutyric acid (GABA)-regulated chloride channel, and thus increase cell excitability (78). Such bidirectional processes have the potential to reach a critical level and set in motion an ever increasing entropic cascade. Evidence is accumulating for a messenger role for reactive oxygen species within cells. Endothelium-derived relaxing factor has been identified as nitric oxide, a free radical (15).

Oxidases Chemical induction of cytochrome P-450-containing mixed-function monooxidases can increase the rate of phase I detoxification reactions. The oxidative metabolism of many lipophilic compounds, although necessary for their conjugation and excretion, often involves the transient formation of highly reactive oxidative intermediates such as epoxides (79). Mixed-function oxidases predominate in the liver, but they are also present in the nervous system, within both neurons and glia (40). At the intracellular level, most of these cerebral oxidases are mitochondrial rather than microsomal, and like the corresponding hepatic enzymes, they are inducible (60). Products of such enzymes may include epoxides in which the C - O - C ring strain often results in a potent oxidizing chemical. Xanthine oxidase is a prime generator of superoxide and may be a significant exacerbating factor in several pathological states.

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Phagocytosis Extracellular formation of superoxide anion by phagocytes has long been recognized as a bacteriocidal mechanism. Similar oxidative activity has been observed in cerebral microglia (39). Astroglial activation is a common event following neural trauma, and reactive astrocytes are active in clearance of cell debris and, ultimately, in the formation of glial scar tissue. Although the phenomenon of reactive oxygen species generation has not been documented in the injured brain during neuronophagia, the ROS-enhancing potential of such events is worthy of further study.

Evaluation of Cerebral Prooxidant Status A multitude of assays are available in the study of free radical-related events. Many of these assays are relatively simple to perform and have been welldescribed previously. When a range of indices is compared, it is clear that they are rarely consonant and that disparate kinds of free radical-initiated events exist. Several misinterpretations of data are possible if the basis of the assay is misjudged. For example, prevention of an adverse neurological event by superoxide dismutase does not demonstrate that the harmful oxidant

Short

ROS production Short lived OH Longer lived H202, 0 2 '

Macromolecular damaae Protein degradation Enzyme inactivation Lipid peroxidation Nucleic acid damage _

Intermediate

Antioxidant denletion GSH r Ascorbate _

Cellular resnonses Enzyme induction Altered neuronal excitability Cell death _

Long

FIG. 1 Time scale of quantifiable parameters relating to oxidative stress.

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species is superoxide. This oxidant is a relatively unreactive and long-lived species. Such data only demonstrate that superoxide is on the pathway leading to the production of a potent free radical species. In a parallel manner, the prevention by catalase of oxidative events does not imply a direct oxidant role for hydrogen peroxide. The selection of appropriate tests will vary with the hypothesis on which a study is based. The following discussions primarily focus on issues to consider when selecting indices of oxidative damage to be studied, rather than giving detailed methodological descriptions. Procedures that are used can be broadly divided into three kinds, relating to whether a primary, secondary, or more indirect event is under consideration. Assay of the properties of the free radicals themselves requires very short time frames, although evaluation of the damage effected by ROS occurs over a longer period. Finally, studies relating to adaptive response of the nervous system may require consideration of events over an even longer period (Fig. 1).

Direct Assay of Reactive Oxygen Species by Measurement of Formation of Oxidized Product from Exogenous Indicator Molecule The formation of a stable free radical with spin-trapping agents such as 5,5dimethyl-l-pyrroline 1-oxide (DMPO) and phenyl-N-tert-butylnitrone (PBN) can be determined by electron-probe or electron-spin resonance (ESR), and this permits direct quantitation of ROS (30). ESR has the advantage that specific free radical species can be detected. Furthermore, the procedure can be applied to more organized systems such as tissue slices, organs, or even small mammalian brains in vivo (95). These methods are subject to artifact but are continually being refined to give more unequivocal results (88). Direct ESR of free radicals in rapidly frozen tissue is also possible, but only when a stable radical such as superoxide is to be measured. The detection of products formed by oxidative modification of exogenous probes is another means of direct detection of ROS-induced changes. The generation of measurable fluorescent or colored products resulting from the oxidative conversion of nonsignaling precursors can be a very sensitive test in isolated systems. Using such procedures, data obtained by the direct addition of neurotoxic agents to tissue preparations are often in accord with parallel data obtained by animal dosing followed by tissue isolation and fluorescent assay for ROS (48, 51). Examples of fluorescent procedures that are readily adapted for CNS studies include the probes dihydrorhodamine and

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2',7'-dichlorofluorescin diacetate (47). In the latter case, the dye precursor, 2',7'-dichlorofluorescin (DCFH), is formed by intracellular esteratic hydrolysis. This allows the cytosolic accumulation of the precursor, in a manner analogous to that used in the assay of intracellular calcium with fura-2 or indo dyes. Such concentration serves to make the procedure more sensitive than the use of the similar dye precursor, dihydrorhodamine, which is not bioconcentrated (50). DCFH was formerly thought to be a selective assay for hydrogen peroxide, but the inhibition of such oxidation by the iron chelator, deferoxamine, indicates that a more reactive species, formed by the Haber-Weiss reaction, needs to be present. Another dye precursor, nitro blue tetrazolium, which can be oxidized to an insoluble blue formazan, is described as very sensitive (19). The superoxide selectivity claimed for this oxidation is questionable and the generation of a more active oxidant species by the Fenton reaction may be a prerequisite. Another analytic procedure that may be relatively selective for the hydroxyl radical involves the hydroxylation of benzoic acid to fluorescent 2and 3-hydroxybenzoate by the hydroxyl radical. This radical can also be selectively quantitated by chromatographic determination of the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (16). Such procedures can be utilized in the intact animal. The modification of deoxyribose by the hydroxyl radical to a compound forming a colored product with thiobarbituric acid is yet another means of determining hydroxyl radical synthesis (91). The dichlorofluorescein and benzoate hydroxylation methods are described below in more detail.

Assay of Reactive Oxygen with Dichlorofluorescein Cerebral morphological fractions in 40 mM Tris (pH 7.4) are loaded with 5 mM 2',7'-dichlorofluorescin diacetate (0.5 mM in ethanol; stored at -70~ for 15 minutes at 37~ to allow esteratic formation of the nonfluorescent compound DCFH. Following loading, the formation of dichlorofluorescein (DCF), the fluorescent oxidized derivative of DCFH, is recorded and incubation continued for an additional 60 minutes, when the fluorescence is again determined. For the in vitro studies, agents are added, at various concentrations to fractions after the initial fluorescence reading. Samples are then incubated for 60 minutes and the final fluorescence reading recorded. Fluorescence is monitored at excitation wavelength 488 nm (bandwidth 5 nm) and emission wavelength 525 nm (bandwidth 20 nm). The cuvette holder is maintained thermostatically at 37~ Corrections are made for any autofluorescence of fractions. This correction is always less than 6% of values in the presence of DCFH. DCF formation is quantified from a standard curve over the range of 0.05-1.0 mM.

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Benzoate Hydroxylation All reactions (final volume 2.0 ml) are performed in 25mM NaH2PO4, pH 7.4. The incubation mixtures contain benzoate (20/zl of a 5 mM stock solution), and reactions are started by the simultaneous addition of 10/zM H202 and 10/zM Fe 2§ Each mixture is incubated at 37~ for 5 minutes, and the reactions are terminated by the addition of 40/zl of 1 mM NaOH. Fluorescence of each solution, a measure of the formation of 2- and 3-hydroxybenzoate, is determined with excitation wavelength 300 nm (bandwidth 3 nm) and emission wavelength 390 nm (bandwidth 20 nm). Autofluorescence is allowed for by the inclusion of parallel blanks.

Estimation of ROS by Measuring Oxidation Products of Cellular Constituents The assay of chemically modified biological molecules has the potential to define the types of cellular deficit that are incurred by oxidative damage, rather than describing the nature of the oxidant species involved. All three cellular macromolecular species can reflect ROS-induced changes by forming adducts or showing oxidative degradation. In many tissues in which mutagenesis is a potential outcome of excess oxidant events, assay for damaged nucleotide sequences within DNA is important. In the CNS, oxidative events have the potential to disrupt phenotypic cell functioning rather than enabling transformation, and thus demonstration of alterations to constituents of proteins and lipids may be more relevant. Lipid peroxidation is a major means by which membranes can be oxidatively damaged. It is classically measured by the formation of colored products from reaction of thiobarbituric acid with malondialdehyde-like products of lipid breakdown. This procedure has been very widely used in the past, but its utility is limited by significant range of potential artifacts (7). It appears to reflect susceptibility of tissues to oxygen-induced lipid peroxidation rather than levels of lipid hydroperoxides in the intact animal (34). High-performance liquid chromatographic separation and quantitation of the malondialdehyde-thiobarbiturate derivative has been shown to improve the selectivity of this procedure (29). Another means of estimating oxidatively damaged lipids is by spectrophotometric assay of conjugated dienes (65) or by measuring exhaled ethane. This latter procedure, although noninvasive, only gives an index of overall rather than tissue-specific oxidative damage. The importance of free radical-induced degradation of proteins is becoming increasingly recognized (67, 82, 83). Although lipids have long been known for their ability to promote cell damage by catalyzing chain reactions of

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oxidant events, proteins have recently been also recognized as possessing this potential (56). Oxidative modification of proteins characteristically occurs predominantly within specific residues or sequences that are especially susceptible to damage by ROS. Analytical procedures are directed toward species that do not normally occur in intact proteins. These include tyrosine dimers, degraded tryptophan residues (23), and the appearance of carbonyl or excess free amino groups (24, 25, 57). Oxidatively modified proteins are also more hydrophobic and thus are susceptible to proteolytic dissolution. This is revealed by the presence of increasing numbers of free amino terminals (26). All of these changes can be exploited in the characterization of oxidative events. The procedures involved are either spectrophotometric or fluorometric and are relatively straightforward. We have found the spectrophotometric tracking of loss of protein tryptophan to be both uncomplicated and sensitive (49). Some enzymes are especially susceptible to oxidative denaturation by virtue of their tertiary structure or peptide sequences. Glutamine synthetase (glutamate-ammonia ligase), which occurs predominantly in astroglia and is readily assayed (71), is an enzyme that is readily oxidized and then preferentially degraded (68). This enzyme has been successfully used as an index of oxidative stress within the CNS (17, 58, 76). The oxidation of a critical lysine or proline residue within this enzyme to y-glutamyl semialdehyde appears to confer this susceptibility (52, 53). This can then be derivatized and quantitated (21). Creatine phosphokinase and several other enzymes are also selectively vulnerable to ROS (32). One advantage of assay for secondary events such as damaged proteins is that this gives a more historical perspective, describing oxidative events over a longer period. Gradual incremental accumulation of damage to proteins may allow detection of minor but persistent elevations of ROS production. In contrast, direct quantitation of very short-lived prooxidant species yields information of tissue status at a single moment in time. Other features characteristic of protein damage include the formation of lipofuscin (41), found in aging brain. This appears to be an accumulation of indissoluble proteinaceous material with lipid and carbohydrate constituents, and is not readily quantitated. Oxidative events can also cause quantitatable protein glycylation and protein cross-linking (92). Results obtained by use of secondary assays often reflect the consequences of more prolonged oxidative events and may appear to be in conflict with those from primary assays, which reflect "snapshots" of oxidative status at a single instant in time. Such apparent contradictions may serve to further illuminate underlying processes. For example, the rate of generation of ROS may be depressed in aging animals. This may reflect a slower metabolic rate in the elderly. However, greater levels of oxidative damage to proteins also

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exist within the same aged animals (49). This suggests that, despite a slower rate of ROS production, aged animals may show structural and enzymatic deficits resulting from cumulative oxidative damage being incurred over an extended period of time. Two detailed methods for some representative assays follow.

Degradation of Protein Tryptophan Residues Tissues are suspended in HEPES buffer, pH 7.4, and a sufficient volume of 10% sodium dodecyl sulfate (SDS; 10%, w/v) is added to bring the final concentration of SDS to 0.1%. The tryptophan content within solubilized proteins is fluorometrically determined using an excitation wavelength of 280 nm (bandwidth 5 nm) and emission wavelength of 345 nm (bandwidth 20 nm). More than 99% of this fluorescence is attributable to tryptophan residues.

Glutamine Synthetase This enzyme is assayed as y-glutamyltransferase activity by incubation (30 minutes, 37~ of 0.1 ml tissue preparation together with (mM) L-glutamine (50), hydroxylamine (75), NaADP (0.5), MnCI2 (0.2), imidazole hydrochloride (50), and sodium arsenate (25), in a final volume of 1 ml. 7-Glutamyl hydroxylamate formed can then be quantitated after centrifugation (5000 g, 5 minutes), by spectrophotometric assay of the colored product formed with acidified FeC13 (71). A standard curve is generated with 7-glutamyl hydroxylamate. The iron complex of 1 mmol/ml of this compound gives an absorbance of 0.340 at 535 nm. When the effect of various agents on enzyme activity is studied, a 30-minute preincubation of the tissue fraction with these chemicals can precede enzyme assay.

Determination of lntracellular Antioxidants and Enzymes Involved in Mitigation of Oxidative Damage This is a more indirect index of oxidative status and represents an integral of events occurring over an extended period. Tissue defense systems are capable of being depleted by prolonged oxidative stress, but homeostatic processes can also lead to enzyme induction and to elevated levels of antioxidant chemicals. Thus a biphasic response to elevated levels of ROS is sometimes observed. For example, glutathione levels may initially be depressed in response to an oxidative stressor and subsequently become significantly

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elevated above original basal levels (2). The assay of glutathione disulfide may provide a less ambiguous index of oxidative events (1). Key enzymes that may play a role in ROS detoxification include catalase (3) and glutathione peroxidase (89), both of which promote destruction of peroxides, and superoxide dismutase (59), which eliminates superoxide anion. Superoxide dismutase is clearly inducible and can become elevated in the CNS in the presence of oxidative stressors such as hyperbaric oxygen or following ischemia (85). However, bidirectional effects are also possible in this case, because oxidative damage can also inactivate superoxide dismutase and other enzymes that detoxify oxygen free radicals (75). Glutathione reductase is also critical in maintaining defense systems, because it is involved in the regeneration of reduced glutathione from the oxidized dimer, whereas glutathione transferase conjugates glutathione to electrophilic compounds. There is also evidence that glutathione transferase levels can be induced following chronic oxidative stress (20, 54), and that hydrogen peroxide can induce catalase within the CNS (62). Key low molecular weight antioxidant species include lipophilic a-tocopherol and/3-carotene and water-soluble elements such as ascorbic acid and glutathione. Water-soluble antioxidants that are presently less well defined include carnosine and anserine (45). Glutathione is present intracellularly at millimolar concentrations (38) and constitutes the main source of antioxidant potential within the cell. As mentioned above, both depression and elevation of the basal levels of glutathione have been regarded as evidence of oxidative stress. Such potentially biphasic changes are due to adaptive responses of tissues subjected to extended prooxidant events. The assay of glutathione can be performed by fluorometric or enzymatic procedures (1, 66). When establishing appropriate parameters to be assayed, it is important to recognize that the intracellular milieu is very heterogeneous and that the location of antioxidant enzymes and chemicals is critical. Although atocopherol is protective against lipid peroxidation and other events occurring predominantly in the lipid domain, it is not effective in protecting against oxidative damage to proteins taking place in the aqueous compartment (94). The hydroxyl radical appears to be formed largely in the hydrophilic areas of the cell (63). A significant concomitant of harmful oxidative events is the possibly mandatory requirement for the presence of low molecular weight iron compounds. Iron chelates have the potential to permit transitions from the ferrous to the ferric state and such valence cycling underlies the appearance of potent short-lived oxidant species. For this reason, it is thus often useful to be able to estimate levels of such incompletely sequestered forms of iron. Bathophenanthroline forms a strongly fluorescent complex with iron salts and this has

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been used to determine liberation of cytosolic iron from high molecular weight protein complexes (86). Resting levels of low molecular weight iron are in micromolar concentrations and can best be determined by ESR of paramagnetic iron complexes (46). Another important technology relates to the unequivocal induction of excess ROS within the nervous system. This can be a useful control in many studies, and is perhaps most readily achieved by depletion of glutathione levels using diethyl maleate or the more stable 2-cyclohexan-1-one (9). Buthionine sulfoxime is a good inhibitor of glutathione synthesis, and can more easily cross the blood-brain barrier when administered as the ethyl ester (84). Assays for glutathione and for the appearance of low molecular weight iron species are detailed in the following sections. Tissue Glutathione Glutathione (GSH) levels are determined using the method of Rice et al. (66). The principle behind the assay is that monochlorobimane (mBC1), a nonfluorescent compound, reacts with glutathione to form a fluorescent adduct. It has been shown that there is very little reaction between mBC1 and protein sulfhydryl groups, mBC1 is dissolved in ethanol to a concentration of 5 mM and stored at 0-5~ in the dark. mBC1 is added to 2 ml of a given tissue suspension to a final concentration of 20 mM, after which the suspension is incubated for 15 minutes at 37~ and then centrifuged for 10 minutes at 31,500 g. The fluorescence of the supernatant is read on an Aminco-Bowman spectrophotofluorometer at an excitation wavelength 395 nm and emission wavelength 470 nm. The tissue glutathione concentration is determined using a standard curve. Chelatable Iron Compounds The release of iron salts or low molecular weight organic complexes from the cerebral morphological fraction is followed by assay of the spectrophotometric absorbance of the bathophenanthroline complex. The tissue suspension (0.5 ml) is incubated for 20 hours at 37~ together with ferritin (250/~g/ ml), 1 mM succinate, 175/~M bathophenanthrolinesulfonic acid, and 50/~M flavin mononucleotide (FMN). Corn oil is layered over the incubation media in order to minimize aerobic respiration, because reduced FMN is necessary for mitochondrial mobilization of iron from ferritin. After the addition of 0.5 ml 2% (w/v) Triton X-100, and centrifugation (15,000 g, 15 minutes, 0~ the absorbance of the supernatant is determined at 530 nm. A parallel control incubation contained all agents in the absence of the tissue fraction. Iron release from ferritin is quantitated using a value of 22.1 for the molar absorption coefficient of the iron chelate.

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Conclusion Evaluation of the free radical-forming potential of neuropathological conditions and determination of whether oxygen radicals are common mediators of neurological disease should not be delayed until the identity of critical oxygen radicals is unequivocally clarified. However, the wide range of technologies available mandates a careful consideration of those to be selected, rather than applying a battery of unrelated tests. Obviously, apart from scientific considerations, the type of equipment available and the cost of various procedures will also be determinants. Although most techniques are ambiguous and subject to artifact, it is helpful to be aware of not only the limitations of a method but also what strengths it has and what it can determine. The approach used should reflect the hypothesis that is being tested. There are several issues that will help to pinpoint an appropriate test. 1. Does the study relate to rates of free radical production or rather to delineation of any resulting tissue damage? It is important not to confuse these related aspects. 2. Is the adaptive response the subject of study? Isolated and more defined systems will clarify mechanistic issues but many physiological responses can only be observed in whole animals. The time course of changes occurring as a consequence of oxidative stress can be quite complex and even biphasic. 3. The cell has lipophilic and aqueous domains and different methods focus predominantly on one of these. Lipid-soluble and water-soluble antioxidants certainly interact but have differing spheres of influence, and cytoplasmic heterogeneity must be taken into account. 4. If the protective potential of agents is to be studied, this will generally require a longer time frame, and tissue plasticity, including enzyme induction, will have to be taken into account. 5. The expense and complexity of a method may limit its utility. However, a relatively sophisticated method such as ESR can help to confirm results obtained with a simpler method. The use of differing types of assays that bear a relation to each other generally helps to validate a study. 6. A choice may have to be made between using an assay that is directed toward a very specific target (e.g., assay of glutamine synthetase), and one that is more general (e.g., detection of protein degradation). The relative sensitivity of such methods is currently unpredictable. This review has not dealt with molecular biological approaches to the study of the toxicity of reactive oxygen species. These include the use of genetically modified animal strains, wherein specific genes may be deleted

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PARADIGMSOF NEURAL INJURY (knockout mutations) or overexpressed. Such preparations will have great value toward understanding the role of enzymes critical for mitigation of prooxidant events.

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[15]

Exogenous Administration of Cytokines into the Central Nervous System" Analysis of Alterations in Cell Morphology and Molecular Expression M. A. Kahn and J. de Vellis

Introduction The effects of growth factors and cytokines on cell types derived from the central nervous system (CNS) have largely been studied in vitro. More recently, however, we have begun to examine the actions of these factors within an in vivo context. Unlike the peripheral nervous system (PNS), however, the CNS is protected by a blood-brain barrier (BBB). This barrier often complicates the experimental design; however, a number of different avenues do exist for the delivery of such exogenous growth factors. Information regarding growth factor function has been obtained using transgenic animals (MacMillan et al., 1993; D'Ercole et al., 1994; Timmusk et al., 1995) and through the intracerebral injection of cells engineered to express certain genes (Gage et al., 1990; Shimohama et al., 1993; Nikkhah et al., 1994). The last method, and probably the most simplistic, is to inject the cytokine or growth factor directly into the CNS parenchyma and/or ventricles (Giulian et al., 1988; Watts et al., 1989; Balasingham et al., 1994; Kahn et al., 1995). There are advantages and disadvantages to all of these methodologies. The method that is described in the following involves directly administering cytokines into the brain. It is well known that inserting a needle into the CNS creates a wound or lesion. In response to physical or chemical brain injury, the mammalian CNS often reacts by evoking astrogliosis. The most prominent feature describing this state is an up-regulation of glial fibrillary acidic protein (GFAP) (Eng et al., 1971; Chiu and Goldman, 1985). The agent(s) responsible for inducing astrogliosis remain unclear; however, recent observations have shown cytokines may play a pivotal role (Giulian et al., Balasingham et al., 1994; Kahn et al., 1995). During CNS trauma, macrophages and lymphocytes infiltrate the CNS, where they are thought to synthesize and secrete cytokines; moreover, activated microglia and reactive astrocytes are known to be capable of cytokine production (Poindron et al., 1981; Tedeschi et al., 1986; Giulian et al., 1989). Recent experimental evidence has shown that intracerebral injections of a pleiotropic cytokine, ciliary neurotrophic factor (CNTF), increase astrog260

Methods in Neurosciences, Volume 30

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liosis and the appearance of activated microglia in the neonatal rat, an induction that was comparable to the effect seen in animals receiving a well-known proinflammatory cytokine, tumor necrosis factor-a (TNF-a) (Kahn et al., 1995). The following protocols describe, in detail, the administration of cytokines and the techniques used in our laboratory to arrive at these conclusions. The descriptions of techniques are annoted with suggestions and hints to provide a researcher, having limited neurobiology experience, with a reliable reference to obtain high-quality, reproducible results.

Cytokine Preparation and Injection A fundamental concern in the injection of substances into the CNS is the ability to determine those changes in cell morphology and antigenic profile that result from the exogenous administration of such factors. A needle with a narrow diameter and injection of a minimal fluid volume are preferred to limit changes in the tissue resulting from the needle track or edema. For small rodents of approximately 15 g, we would recommend using a 22-gauge needle attached to a Hamilton syringe and a fluid volume that does not exceed 3/xl. Increasing the diameter of the needle or the amount of fluid may result in edema and severe disruption of the tissue.

Choosing Model System Although the precautions mentioned previously will help to limit some of the changes in the tissue resulting from a stab wound, it is well known that such a lesion does evoke a severe inflammatory response. The focal area surrounding the needle track often forms a glial scar that is composed of many cell types, including reactive astrocytes, endothelial cells, and cells of the monocyte/macrophage lineage (Fontana and Fierz, 1985; Perry et al., 1987; Giulian et al., 1989; David et al., 1990; Hatten et al., 1991). In further attempts to limit the changes resulting from the lesion, we chose to study the developing postnatal rat, prior to 1 week of age. The cerebral white matter tracts develop GFAP § cells early in embryonic development. In contrast, the cerebral cortical gray matter is devoid of this cell type until the second postnatal week of development. The cortical gray matter, therefore, provides for a low background level of GFAP; moreover, unlike the adult rat, the neonate experiences limited astrogliosis and microglia activation following injury to the CNS (Bignami and Dahl, 1974, 1976; Barrett et al., 1984). It is for these reasons that we have chosen to study the cerebral gray matter of early postnatal rat pups; this model system allows the changes in

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astrocytes and microglia to be observed easily. As will become evident, our results clearly show that early postnatal rats are capable of astrogliosis and activation of microglia if exposed to the appropriate stimuli; cytokines such as CNTF and TNF-c~ appear to be likely candidates.

Tracer Dye and Cytokine Preparation Prior to injecting cytokines, a substance should be used to locate the site of entry and to confirm delivery of the agent. Charcoal, colored beads, or dyes have been used with much success. The dye Evans blue (Sigma, St. Louis, MO), at a concentration of 0.1% v/v in phosphate-buffered saline (PBS), is a good choice as a colorimetric determinant of fluid location. Tissue at the injection site and in the path of the fluid will stain nonspecifically with the blue dye. Moreover, Evans blue displays fluorescence using the rhodamine filter, allowing for another method of analysis. A word of caution: autofluorescence of cells found within the ventricular system also occurs within this wavelength spectrum. Although some cells take in the Evans blue, for the most part, the dye leaves a diffuse fluorescence that can be observed in the surrounding tissue. The dye is often seen in regions distal to the injection site, but is usually limited to the ipsilateral hemisphere of the brain (Fig. 1). Cytokines should be resuspended in a recommended buffer. The cytokines used in our study were resuspended in PBS containing 0.1% w/v bovine serum albumin (BSA) to prevent nonspecific protein binding to plastic or glass surfaces. Postnatal day 5 (PN5), rat pups received 2/xl of PBS containing one of the following components: 0.5/xg CNTF, 0.5/xg TNF-a, or 0.5/xg BSA as a control (CNTF and TNF-a; Genzyme, Cambridge, MA). At least three animals should be analyzed per treatment.

Animals and Injection Procedure Sterilize surgery equipment by autoclaving. Weigh postnatal day 5 rat pups, anesthetize with an i.p. injection of Avertin at a dose of 60-70 mg/kg body weight, and place in a stereotaxic apparatus for surgery. Make a small incision in the skin of the left hemisphere to expose the scalp and coordinates, 1.5 mm lateral to the midline and 3 mm posterior to the bregma. Drill a 1-mm2 burr hole into the skull using ophthamology microsurgery equipment. Position a 5-/~1 Hamilton syringe attached to a 22-gauge needle above the burr hole and insert the needle 2.5 mm into the tissue; this allows for a reproducible needle track into the gray matter just above the lateral ventricle (Fig. 2). Inject the fluid slowly over a 5-minute interval. Leave the needle

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FIG. 1 Analysis of tracer dye at a site ventral to the corpus collosum in PN6 rat pups receiving 2-/21 injections at PN5 of PBS containing 0.1% Evans blue. (A) Light microscopy revealed some cells displaying selective uptake of the blue dye (arrows). (B) Fluorescence microscopy showed an absence of fluorescence in the contralateral hemisphere (open arrow). The ipsilateral hemisphere, however, displayed diffuse fluorescence of tissue within the path of the dye (solid arrow). Tissue counterstained with hematoxylin. Bar: 40 tzm.

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!

:

j

I

FIG. 2 Schematic representation of a coronal section illustrating the location of the injection site. A 22-gauge needle inserted into the ipsilateral hemisphere (coordinates, 1.5 mm lateral to the midline, 3 mm posterior to the bregma, and at a depth of 2.5 mm) allowed for a reproducible needle track into the cortical gray matter just above the lateral ventricle (arrow). Drawing is not to scale.

in place for 5 minutes and then retract slowly to prevent fluid from moving along the injection track. Cover the burr hole with bonewax and suture the area closed. Our experiences show a high survival rate, greater than 90%, using this procedure. If a lower survival rate is obtained, this is most likely due to an overdose of anesthesia. Wait until the animals fully recover from the surgery before returning them to their mother. The methods for animal sacrifice and preparation of the tissue for immunocytochemistry or Western analysis are described below.

Tissue Preparation Prior to immunostaining, the tissue must be adequately fixed. Proper fixation is necessary to preserve cellular morphology and to maintain antigens at their original locations; however, overfixation can disrupt antigenicity. The most likely factors attributing to a loss of antigenicity are delayed fixation, inadequate penetration, destruction of epitopes, overfixation, and fixation too quickly, which can result in disruption of the tissue and in edema.

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Fixation Fixation by perfusion is better than immersion fixation. Intracardiac perfusion of a 2.0% w/v paraformaldehyde solution proved to be an excellent choice for the preservation of cell morphology, immunocytochemical analysis, and combined immunocytochemistry with in situ hybridization. It is imperative to postfix the tissue and to cryoprotect with sucrose after the perfusion. These extra steps will provide proper fixation, the removal of fixative, and protection of the tissue from ice-related artifacts. The following protocols were used for the fixation of brains derived from postnatal day 7 rat pups.

Perfusion Fixation On the day of the perfusion, prepare a fresh 2.0% paraformaldehyde (Sigma, St. Louis, MO) solution in a fume hood. To make a l-liter solution of paraformaldehyde, add 20 g of paraformaldehyde to 250 ml of ddH20. While stirring, heat to 55-60~ and add 5 N NaOH dropwise until solution clears. Filter with Whatman (Clifton, NJ) # 1 paper and add 500 ml of ddH20. Bring volume to ! liter using 500 ml of 0.2 M NaHzPO4 buffer at pH 7.4, mix, and place on ice. The equipment needed to perfuse an animal is quite simple. A perfusion pump works best to set a constant flow rate, but is not essential. Obtain a piece of tubing and at one end attach a needle, while immersing the opposite end in the cold fixative. Place contents in a fume hood. Anesthetize animal with an i.p. injection of sodium pentobarbital (Abbott Laboratories, Chicago, IL) at a dose of 40-50 mg/kg and wait until nociceptive responses are absent. Tape animal to a perfusion tray and rapidly make an incision in the thorax to expose the heart. Insert the needle into the right ventricle, being careful not to tear the tissue. Perfuse at a rate of 15-20 ml/min until volume of perfusate reaches approximately four to five times the weight of the animal. Decapitate animal and excise the cerebral hemisphere from the cranium.

Postfixation, Cryoprotection, and Sectioning Postfix the brain in flesh, cold 2.0% paraformaldehyde for 4-5 days at 4~ Cryoprotect in a 20% w/v sucrose solution made in PBS containing 0.1% sodium azide for at least 24 hours. For the postfixation and cryoprotection, use a large volume of approximately 10 times the volume of the tissue. If antigens are susceptible to fixative, postfix brains for a shorter duration. Block tissue using optimal cutting temperature (OCT) medium (Miles Inc., Elkhart, IN) and dry ice. Although this procedure is often referred to as OCT embedding, this is a misnomer because the OCT compound does not

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penetrate the tissue. The purpose of the OCT is to create a hard block and to hold the specimen on the cryostat chuck. Be sure that no air bubbles surround the tissue while blocking. Blocked brains can be wrapped in plastic and stored at -70~ or immediately sectioned at an appropriate thickness. Examples given in this review were sectioned at 16/~m thickness; thinner sections are preferred for some antigens and for studies involving in situ hybridization. Place slides on a 37~ warm plate for 30 minutes and transfer to the -70~ freezer for storage or use immediately for immunocytochemistry.

I m m u n o c y toc he mis try The following protocols are examples of routine immunocytochemical procedures using the avidin-biotin complex (ABC) technique. To visualize antigens, commercial ABC kits are available (Vectastain; Vector Lab Inc., Burlingame, CA) and add an element of convenience for the researcher. Biotinylated secondary antibodies can be visualized with avidin-conjugated or avidin-biotin-conjugated complexes. These complexes can be tagged with enzymes (i.e., horseradish peroxidase), fluorochromes [i.e., fluorescein isothiocyanate (FITC) and electron-dense substances such as colloidal gold]. The major advantage in using avidin-biotin conjugates lies in the high affinity of avidin for biotin and in the multiple binding sites present for biotin on each avidin molecule, thus increasing the sensitivity of this method over other immunostaining techniques. Remove cryostat sections from the freezer and bring to room temperature. Place sections in phosphate- or Tris-buffered saline (TBS) for 15 minutes on a rotary shaker to rehydrate the tissue and to remove excess sucrose. Place tissue in 100% methanol in TBS containing 3.0% H202 for 15 minutes to permeabilize tissue and quench endogenous peroxidases. A lower concentration of methanol and/or the use of detergents such as Triton X-100 (Sigma, St. Louis, MO) will provide better results for cell surface antigens. Further permeabilize tissue using 0.5 mg/ml NaBH4 in TBS for 15 minutes and rinse 3 x for 5 minutes each in TBS. Apply primary antibodies at the appropriate dilution for 12-18 hours at 4~ Rinse tissue 3 • for 5 minutes each in TBS and add the appropriate biotinylated secondary antibody (Vector Lab Inc., Burlingame, CA) for 1 hour at room temperature. Rinse tissue 3• for 5 minutes each in TBS, followed by an avidin-biotinylated horseradish peroxidase (HRP) conjugate (ABC; Vector Lab Inc.) for 1 hour at room temperature. Rinse tissue 3• for 5 minutes each in TBS followed by 0.05% w/v diaminobenzidine (DAB; Vector Lab Inc.) treatment for 3 minutes. Remove the DAB and replace with 0.05% DAB containing 0.002% v/v H202; monitor the reaction under a microscope.

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The reaction product can be observed between seconds and minutes depending on the abundance of the signal and in the concentrations chosen for primary and secondary antibodies. A high background can be a problem, but is often improved by increasing the dilution of the antibodies, decreasing the H202 concentration and/or preincubating the tissue, before the addition of the primary antibody, in a 1.0% v/v preimmune serum in TBS. The serum should be derived from the same animal species as that of the secondary antibody. Stop the reaction by washing sections in TBS, 2x for 5 minutes each, and 1 x 5 minutes in doubly distilled H20. Allow slides to dry overnight, rehydrate in TBS, counterstain with hematoxylin or neutral red, rinse in doubly distilled H20, dehydrate in a descending ethanol series, clear in Histoclear, and mount in Permount. Visualize cells using phase-contrast optics (Zeiss, Axiovert 135M). Using the methodology described, we examined the appearance of reactive astrocytes and ameboid microglia in control and cytokine-treated animals. GFAP is considered a hallmark of astrogliosis, whereby reactive astrocytes often experience an increase in the expression of this intermediate filament protein. A polyclonal antibody for GFAP (1:3000, a gift from Dr. L. Eng, Stanford Univ., CA) revealed intensified GFAP immunoreactivity in the gray matter of control animals (Fig. 3A); the degree of GFAP up-regulation, however, is modest when compared to previously published results for the adult rat. CNTF-treated neonatal rats displayed more extensive astrogliosis that ranged from the pial surface, through the cortical gray matter (Fig. 3B), and down into the corpus callosum. In particular, the control animals had GFAP localized to a perinuclear network and within fine fibrils extending into processes. This distribution pattern is characteristic of immature glial cells. In contrast, CNTF-treated animals displayed both a mature and a reactive astrocyte phenotype. The reactive astrocytes exhibited an enlarged cell soma, thickened cell processes, and up-regulated GFAP immunoreactivity. This increase in astrogliosis was comparable to that observed for a well-known proinflammatory cytokine, TNF-c~ (Kahn et al., 1995). Examination of microglia cells was obtained by using the monoclonal antibody ED1 (1:2000; Harlan Bioproducts for Science, Indianapolis, IN), which recognizes a cytoplasmic and a membrane-associated macrophage antigen. Unlike the pattern for GFAP immunostaining, ED1 was restricted to the pial surface and corpus callosum in the control animal; rarely did we observe any microglia in the cortical gray matter of the controls (Fig. 3C). In CNTF-treated animals, ED1 revealed large, round microglia cells that contained prominent cytoplasmic vacuoles (Fig. 3D). ED1 + ameboid microglia were often found clustered in regions of severe astrogliosis (cf. 3B). No significant differences were observed in comparing CNTF-treated to

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FIG. 3 Immunocytochemical analysis of GFAP and ED 1 expression in the ipsilateral hemisphere of PN7 rat pups receiving 2-/~1 intracerebral injections at PN5 of PBS containing 0.5/zg BSA (control) or 0.5/zg CNTF. (A) A moderate increase in GFAP immunoreactivity was observed in the gray matter of control animals. (B) CNTFtreated neonates, however, displayed a significant increase in GFAP immunoreactivity. Many of the GFAP § cells in CNTF-treated animals displayed extensive hypertrophy (solid arrow), a characteristic not observed in the control astrocyte population

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TNF-a-treated animals; neonatal rats receiving T N F - a displayed a similar pattern of ED1 immunoreactivity (Kahn et al., 1995).

Tissue Preparation

for Western

Analysis

Immunocytochemistry is an invaluable technique that provides the researcher with the appropriate tools to examine changes in the CNS at the cellular level. This technique, however, may not be the fastest or most accurate way to determine quantitatively differences in the expression of specific genes resulting from cytokine injection. Proteins such as GFAP pose a particular problem in that an increase in immunoreactivity does not necessarily mean a concomitant increase in protein content (Amaducci et al., 1981; Smith et al., 1983). It is well known that inflammation of the CNS can cause the glial filaments to dissociate, thus allowing more epitopes to be accessible by the antibody. Western analysis provides a simplistic and reliable approach for quantitation. The following protocols are not intended to be an all-encompassing summary of Western analysis, rather they are intended to provide the researcher with our recommendations for examining alterations in specific proteins that arise from cytokine treatment.

P e r f u s i o n a n d Tissue H o m o g e n a t e The techinque here is similar to that used in the preparation of tissue for immunocytochemical analysis; however, fundamental differences do exist. After anesthetizing the animal, perfuse intracardially with ice-cold physiological saline (0.9% w/v NaC1). Dissect out the brain, and using a razor blade excise areas of interest (i.e., the ipsilateral or contralateral hemispheres

(cf. A). (C) The control animals showed no ED1 immunoreactivity in their cortical gray matter. (D) In animals receiving CNTF, however, a significant increase in ED1 immunoreactivity was observed. The microglia were large and ameboid in morphology; many contained prominent cytoplasmic vacuoles (solid arrow), a characteristic indicative of phagocytes. Occasionally ED 1§ microglia cells were observed enveloping pyknotic nuclei (open arrow). The distribution of the microglia cells mimicked the pattern observed for cytokine-induced astrogliosis; however, the number of activated microglia was far less than that observed for the reactive astrocytes (cf. B and D). Tissue counterstained with hematoxylin. Bar: 80/~m.

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surrounding the injection site and/or areas rostral and caudal to the injection). The Western results that follow were derived from a diced 2-mm 2 piece of tissue taken from the injection site and regions 2 mm rostral and caudal to the injection site. Using a mortar and pestle, homogenize brain pieces in icecold buffer containing 0.16 M Tris base, 1.0% sodium dodecyl sulfate (SDS), and 5.0% 2-mercaptoethanol (2-ME). Boil samples for 15 minutes, sonicate for 2 minutes, and determine total protein content by the Bradford method (Bradford, 1976). Store samples at -20~ until use.

Western Analysis Vacuum dry samples, resuspend in loading buffer (Promega, Madison, WI), boil for 5 minutes, place on ice, and load onto a 10-15% polyacrylamide gel. After appropriate separation, electrophoretically transfer the proteins onto nitrocellulose filters at 20 V for 12-15 hours. Allow filters to dry at room temperature for 30 minutes and rinse 2x for 15 minutes each in TBS. Although the avidin-biotin technique works well for probing Western blots, enhanced chemiluminescence (ECL) proved to be a more sensitive approach. The ECL method is outlined in detail in an Amersham International brochure (Amersham International, Buckinghamshire, England). In brief, block the filters in 5.0% nonfat milk for 1 hour and then rinse in TBS containing Tween 20 (two quick rinses, then 1 x 15 minutes and 2x 5 minutes). Incubate the filters for 1 hour with the primary antibody prepared in TBS plus 0.1% Tween 20. Rinse the filters as described above and incubate for 1 hour with the appropriate secondary antibody linked to HRP. Rinse the filters as described above and incubate for 1 minute in a 1 : 1 ratio of ECL chemicals (Amersham International). The blots are exposed to film for variable lengths of time depending on the intensity of the signal. Western analysis (see Fig. 4) demonstrated that the GFAP antibody recognizes a 51-kDa protein at the lesion as well as areas 2 mm rostral and caudal to the injection site. In agreement with previous reports, the neonatal rat demonstrated a moderate increase in GFAP content in response to injections of 2 /zl of PBS containing 0.5 /xg of BSA. GFAP increased dramatically, however, in response to injections of 2/zl of PBS containing 0.5/xg of CNTF. When compared to control animals, CNTF-induced GFAP was 2.5 times higher at the injection site, albeit significant increases were also observed distally. The proinflammatory cytokine TNF-a also increased GFAP near and distal to the site of injection; maximally, a 1.8-fold increase in GFAP was observed relative to the control animals. A significant increase in ED1 content was also observed in CNTF-treated and TNF-a-treated animals (Kahn et al., 1995). The use of a polyclonal antibody is often preferred for Western analysis because the sample is boiled and sonicated in the presence of detergents.

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[15] CYTOKINE-INDUCED ALTERATIONS OF CNS CELLS

A

Control kD

I

R

L

CNTF C

II

R

L

TNF a C

II

R

L

C

I

GFAP 5 1 -

B ----~

t-

75 1

GFAP

2.5

.'l=t r" -I

~"

U~ 0 3 , -.,~

m Control mNFo. [-i CNTF i i

l

50

I

,.-.._

2.1

Ec

0"~

|

e'l

Rostral

L Lesion

Caudal

FIG. 4 Western analysis of GFAP expression in PN7 rat pups receiving 2-/A intracerebral injections at PN5 of PBS containing 0.5/~g BSA (control), 0.5 ~g CNTF, or 0.5 t~g TNF-a (A). The GFAP antibody recognized a 51-kDa protein 2 mm rostral (R) and 2 mm caudal (C) to the lesion site (L). In the control, a slight increase in GFAP was observed at the injection site relative to areas distal to the lesion. Cytokinetreated animals, however, showed a dramatic increase in GFAP content; maximal levels were observed at the injection site. Quantitation was obtained through densitometry scans of the Western data (B). The largest increase in GFAP levels occurred at the site of cytokine entry, albeit significant increases were also observed in areas rostral and caudal to the lesion. Maximally, a 2.5- and 1.8-fold increase was observed for CNTF and TNF-a, respectively.

This results in a denatured protein that has a greater chance of reacting with a polyclonal anntibody that recognizes multiple epitopes. A polyclonal antibody, h o w e v e r , is likely to cross-react with other proteins on the blot. This will result in nonspecific bands that are often faint and easily r e m o v e d with stringent washing conditions. In the case of G F A P , these faint bands are m o s t likely vimentin or cytokeratins, which are k n o w n for their cross-

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PARADIGMS OF N E U R A L INJURY

reactivity. The faint band on the Western blot, therefore, is not included in the densitometry readings.

Concluding Remarks As demonstrated in this review, intracerebral injections of cytokines can provide useful information regarding the role of these factors on cells of the CNS. Antibodies to CNS-derived antigens provide a specific and sensitive tool to examine changes in cell morphology and molecular expression. These antibodies can be utilized in a variety of different applications, thus providing the researcher with a degree of versatility that is useful in attempts to understand the complex interactions of the CNS. Using the techniques given in this review, we can conclude that CNTF appears to be a likely candidate as an in vivo molecular signal capable of inducing astrogliosis. Previous studies have reported that the embryonic and neonatal rat exhibit limited astrogliosis following injury to the CNS (Barrett et al., 1984; Bignami and Dahl, 1974, 1976). The reason these cells are unable to respond following CNS trauma remains unknown; however, it is thought that an immature immune system and/or immature astrocytes with limited plasticity may be responsible (Barrett et al., 1984; DePaoli et al., 1988; Hannet et al., 1992). Because BrdU analysis did not reveal an increase in the proliferative index (Kahn and de Vellis, 1995), our results suggest that cell populations already present in the neonatal rat are plastic and are capable of responding with an increase in GFAP if given the appropriate signal. Moreover, CNTF appears to be specific for the intermediate filament GFAP; CNTF-treated animals did not experience alteration in neurofilament or vimentin when compared to the levels observed in control animals (unpublished observations, 1995). In addition to the regulation of the GFAP gene, exogenous administration of CNTF significantly enhanced the number of ameboid microglia clustering in regions of severe astrogliosis. The pleiotropic actions observed for CNTF, therefore, make it difficult to determine its exact role within the CNS. CNTF may indirectly induce astrogliosis by activating microglia. These cells could respond by releasing soluble factors that act on adjacent cell populations to evoke astrogliosis. Another possibility is that CNTF binds to receptors present on immature astrocytes to regulate GFAP directly. Previous reports have determined that the CNTF-c~ receptor mRNA is expressed solely on neuronal populations in the CNS (Ip et al., 1993). These in situ hybridization studies, however, were performed on tissue sections derived from animals in a normal state. We propose that a pathological state may be necessary to create an up-regulation in the CNTF-c~ receptor on cell populations that normally

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express the receptor at levels below the threshold of sensitivity of the methods currently used. Further studies need to be conducted to identify phenotypically those cells capable of expressing the CNTF-c~ receptor. Although in vitro studies clearly show CNTF to enhance the survival of neurons and oligodendrocytes (Ernsberger et al., 1989; Arakawa et al., 1990; Barres et al., 1993; Louis et al., 1993; Kahn and de Vellis, 1994), our current results suggest CNTF may have other roles in the in vivo environment. The pleiotropic action of this cytokine in the CNS will obviously compound any thoughts of its use in the therapeutic intervention of demyelinating diseases or in neurological disorders.

Acknowledgments The results obtained using these methodologies were provided through the talented and collaborative efforts of Julie Ellison and Graham Speight. We would also like to thank Julie Ellison for helpful comments on this review.

References Amaducci, L., Forno, K. L., and Eng, L. F., Neurosci. Lett. 21, 27-32 (1981). Arakawa, Y., Sendtner, M., and Thoenen, H., J. Neurosci. 10, 3507-3515 (1990). Balasingham, V., Tejada-Berges, T., Wright, E., Bouckova, R., and Yong, V. W., J. Neurosci. Res. 14(2), 846-856 (1994). Barres, B. A., Schmid, R., Sendnter, M., and Raft, M. C., Development 118, 283295 (1993). Barrett, C. P., Donati, E. J., and Guth, L., Exp. Neurol. 84, 374-385 (1984). Bignami, A., and Dahl, D., J. Comp. Neurol. 153, 27-38 (1974). Bignami, A., and Dahl, D., Neuropathol. Appl. Neurobiol. 2, 99-110 (1976). Bradford, M. M., Anal. Biochem. 72, 248-254 (1976). Chiu, F. C., and Goldman, E., J. Neuroimmunol. 8, 283-292 (1985). David, S., Bouchard, C., Tsatas, O., and Giftochristos, N., Neuron 5,463-469 (1990). DePaoli, P., Battistin, S., and Santini, G. F., Clin. Immunol. Immunopathol. 48, 290-296 (1988). D'Ercole, A. J., Dai, Z., Xing, Y., Boney, C., Wilkie, M. B., Lauder, J. M., Han, V. K., and Clemmons, D. R., Brain Res. 82(1-2), 213-222 (1994). Eng, L. F., Vanderhaeghen, J. J., Bignami, A., and Gerstl, B., Brain Res. 28, 351-354 (1971). Ernsberger, U., Sendtner, M., and Rohrer, H., Neuron 2, 1275-1284 (1989). Fontana, A., and Fierz, W., Immunopathology 5, 57-70 (1985). Gage, F. H., Fisher, L. J., Jinnah, H. A., Rosenberg, M. B., Tuszynski, M. H., and Friedman, T., Prog. Brain Res. 82, 1-10 (1990).

274

PARADIGMS OF NEURAL INJURY Giulian, D., Woodward, J., Young, D. G., Krebs, J. F., and Lachman, L. B., J. Neurosci. 8, 2485-2490 (1988). Giulian, D., Chen, J., Ingeman, J. E., George, J., and Noponen, M., J. Neurosci. 9, 4416-4429 (1989). Hannet, I., Erkeller-Yuksel, F., Lydyard, P., Deneys, V., and DeBruyere, M., Immunol. Today 13, 215-218 (1992). Hatten, M. E., Liem, R. K. H., Shelanski, M. L., and Mason, C. A., Gila 4, 233-243 (1991). Ip, N. Y., McClain, J., Barrezueta, N. X., Aldrich, T. H., Pan, L., Li, Y., Wiegand, S. J., Friedman, B., Davis, S., and Yancopoulos, G. D., Neuron 10, 89-102 (1993). Kahn, M. A., and de Vellis, J., Glia 12, 87-98 (1994). Kahn, M. A., Ellison, J. A., Speight, G. J., and de Vellis, J., Dev. Brain Res. 685, 55-67 (1995). Louis, J. C., Magal, E., Takayama, S., and Varon, S., Science 259, 689-692 (1993). MacMillan, V., Judge, D., Wiseman, A., Settles, D., Swain, J., and Davis, J. Stroke 24(11), 1735-1739 (1993). Nikkhah, G., Olsson, M., Eberhard, J., Bentlage, C., Cunningham, M. G., and Bjorklund, A., Neuroscience 63(1), 57-72 (1994). Perry, V. H., Brown, M. C., and Gordon, S., J. Exp. Med. 165, 1218-1223 (1987). Poindron, P., Coupin, G., Illinger, D., and Fauconnier, B., in "Methods in Enzymology" (Sidney Pestka, ed.) Vol. 78A, pp. 165-178. Academic Press, New York, 1981. Shimohama, S., Fisher, L. J., and Gage, F. H., Adv. Neurol. 60, 744-748 (1993). Smith, M. E., Somera, F. P., and Eng, L. F., Brain Res. 264, 241-253 (1983). Tedeschi, B., Barret, J. N., and Kean, R. W., J. Cell Biol. 102, 2244-2253 (1986). Timmusk, T., Lendahl, U., Funakoshi, H., Arenas, E., Persson, H., and Metsis, M., J. Cell Biol. 128(1-2), 185-199 (1995). Watts, R. G., Wright, J. L., Atkinson, L. L., and Merchant, R. E., Neurosurgery 25, 202-208 (1989).

[16]

Animal Models to Produce Cortical Cholinergic Dysfunction Reinhard Schliebs* and Volker Bigl

Introductory Remarks To study cortical cholinergic dysfunction we need to use animal models. In this chapter we will examine what such an animal model should look like and what might it tell us. The basal forebrain cholinergic system is known to play an important role in cortical arousal and normal cognitive function. Cortical cholinergic dysfunction has been implicated in cognitive deficits that occur in Alzheimer's disease, and the cholinergic projection from the nucleus basalis of Meynert to areas of the cerebral cortex is the pathway that is the earliest and most severely affected in brains from Alzheimer's patients (for review, see Ref. 4). In the rat this nucleus is not yet developed into a delineated nuclear structure but corresponds to a more heterogeneous region of cholinergic neurons that often is referred to as nucleus basalis magnocellularis (Nbm), providing the main source of cholinergic terminals to the cerebral cortex (54). Changes in markers of the neocortical cholinergic system found in brains of Alzheimer's patients are complemented by alterations in other cortical transmitter systems, including glutamate, y-aminobutyric acid (GABA), noradrenaline, or serotonin receptors (for reviews, see Refs. 8, 28, and 41), suggesting additionally (1) an important influence of the cholinergic basal forebrain system on cortical neurotransmission, and (2) a cholinergic role in cortical reorganization and in adaptive processes following injury. This is consistent with the hypothesis that the basal forebrain cholinergic system is not directly involved in the formation of learning and memory but acts as a modulatory system to control cortical information processing. Characterization of the mechanisms underlying this adaptive response may be of particular importance to elucidate the cascade of events initiated by decreased cortical cholinergic activity and to derive further rationales to intervene pharmacologically in this process, for example, with respect to finding a therapeutic strategy for treating Alzheimer's disease or to characterizing the role of the cholinergic system in cortical information processing,

* To whom correspondence should be addressed. Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PARADIGMS OF NEURAL INJURY

learning, and memory, and in cognitive behavior in greater detail. Such investigations on the functions of the central cholinergic system require adequate animal models to produce specific cholinergic deficits in vivo. This will allow a detailed evaluation of the neurochemical, neuropathological, and behavioral sequelae as well as functional implications of plastic repair mechanisms following cholinergic hypofunction, and will provide information that cannot be obtained or can only be partially obtained in humans. For example, presently there is no adequate animal model available that mimics all of the biochemical, behavioral, and histopathological abnormalities observed in patients with Alzheimer's disease. However, partial success can be achieved with so-called isomorphic models (24) representing partial parallelism between a model and some human condition. The value of such models is to define any causality of the disease and to delineate mechanisms underlying the pathological processes as well as to test for possible therapeutic strategies. A number of different paradigms have been introduced to produce cortical cholinergic dysfunction. Besides the classical techniques of lesioning cholinergic cells in the basal forebrain, a number of other approaches have been used to mimic cholinergic hypofunction or dysfunction" (1) use of aged animals, (2) pharmacological treatment with anticholinergic drugs, (3) electrolytic lesions within the basal forebrain complex, (4) devascularizing lesions of the cortex to cause retrograde degeneration of cells in the basal forebrain, (5) excitotoxin-induced lesion of the Nbm complex, (6) cholinotoxin-induced lesions of the basal forebrain complex, and (7) cholinergic immunotoxin-induced lesion of the NbM. Here, however, we will focus our attention on some of the most often used paradigms to produce cortical cholinergic deficits, including electrolytic and excitotoxic lesions, application of so-called cholinotoxins, and the recently developed method of using specific immunological targeting of unspecific toxins.

Noninvasive Procedures to Mimic Cortical Cholinergic Hypofunction Noninvasive procedures to model cortical cholinergic dysfunction involve both the use of aged animals and pharmacological intervention in cortical cholinergic transmission by systemic administration of anticholinergic drugs. The cholinergic neurons of the basal forebrain are prone to age-related degeneration (3). The resulting cholinergic hypofunction has been related to memory deficits in aged humans, rodents, and primates (see, e.g., Ref. 53). In particular, aged monkeys develop neuropathological changes comparable to those occurring during aging in humans. These age-related changes are associated with performance deficits in a number of cognitive tasks that

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resemble the impairments observed in aged humans and in demented patients, thus suggesting the value of aged primates as an experimental animal model (48). However, neurochemical changes associated with normal aging do not in each case mimic the same mechanisms underlying pathological cholinergic cell loss. Moreover, aged animals are usually associated with poor health and do not seem an appropriate model to test new therapeutic strategies. The high cost of animal care also represents a considerable limitation to using animals for research purposes. In a number of studies it has been demonstrated that systemically administered anticholinergic drugs [e.g., antimuscarinic drugs such as scopolamine (at doses of 0.1-1 mg/kg) or atropine (10-50 mg/kg)] produce deleterious effects on cognitive performance of a broad spectrum of learned behavior in rats (22) as well as humans (9, 38), suggesting an important role of adequate cholinergic input for realizing cognitive processes (18). Taken together, however, these behavioral-pharmacological studies have resulted in a broad spectrum of widely diverse and sometimes contrary data (22). This might be due to the fact that muscarinic receptors are present in virtually every region in the brain. Blocking the entire cholinergic input by antimuscarinic drugs might affect behavioral performance that could be caused by the influence of other systems and might not have a direct cholinergic basis. Further, blockade of mainly postsynaptic cholinergic input produces many effects that are not related to Alzheimer's disease, because Alzheimer's disease is mainly due to a loss of presynaptic input (for details, see Ref. 22). To study the effects of a specific cholinergic contribution to learning and memory, procedures that produce lesions in basal forebrain cholinergic regions seem to be more appropriate.

P a r a d i g m s to P r o d u c e C h o l i n e r g i c L e s i o n s in B a s a l F o r e b r a i n Lesions of the Nbm result in a cortical cholinergic denervation and have been used in a number of studies to produce an animal model that mimics some aspects of Alzheimer's disease (12, 13, 19, 50). Due to the unique topographical organization of the basal forebrain cholinergic system we are faced with a number of difficulties in order to destroy selectively and specifically the cholinergic neurons giving rise to the cholinergic basalocortical pathway: (1) Cholinergic cells in the basal forebrain do not comprise a distinct nucleus with sharp boundaries like, e.g., the locus ceruleus for noradrenergic cells, but they are distributed within a number of distinct forebrain nuclei forming an irregularly shaped band of neurons with both a rostral-caudal and ventral-dorsal extension. (2) The basal forebrain nuclei that comprise the cholinergic cell population also contain a variety of noncholinergic cells,

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for example, GABA-, serotonin-, neuropeptide- or glutamate-containing neurons, which are intermingled with cholinergic neurons. Additionally, there are also noncholinergic fiber bundles passing through the basal forebrain in the neighborhood of the cholinergic nuclei. To address the difficulties of destroying cholinergic cell populations yet leaving noncholinergic neurons or fibers intact, various cytotoxins (excitotoxins, cholinotoxins, and immunotoxins) have been used. Following a general description of the procedures to produce lesions in the Nbm, the use of these toxins, and their imitations, will be described in detail.

General Procedures to Produce Lesions in Basal Forebrain The general procedures for placing an electrolytical lesion or infusing toxins into the Nbm are described for adult male Wistar rats with body weights ranging between 150 and 200 g. The choice of the coordinates for a stereotaxic administration of the toxins is crucial. Localization of the basal forebrain cholinergic nuclei within each individual brain can vary slightly depending on strain and age of the rat. Thus, the exact coordinates should be proved in each case in preliminary experiments. The coordinates given below to place lesions into the Nbm represent values that are commonly used but must be considered as estimates (data in parentheses represent variations in coordinates based on data from various authors): 0.8 mm (0.3-0.9 mm) posterior to the bregma, 2.6 mm (2.0-3.3 mm) lateral to the sutured midline, and 7.2 mm (6.8-8.0 mm) below the dura. Following anesthesia (e.g., with 100 mg ketamine (Bayer, Lever Kusen) and 15 mg xylidinothiazine/kg body weight (Parke Davis, Berlin)), the head of the rat is placed in a stereotaxic frame (Kopf apparatus). The skull is carefully opened by drilling a small hole near the bregma (using the coordinates indicated above) with a dental drill (diameter 0.5-1 mm), without damaging the underlying dura. For electrolytic lesions, a stainless-steel electrode (diameter, 0.4 mm, which is isolated except for the last 0.5 mm of the tip) is placed into the Nbm. Lesions are made by allowing a direct current of 60/zA to flow through the electrode for 60 seconds, the other pole of the electrode being fixed on the tail (see Refs. 17, 31, 43, and 49). Sham-operated animals, in which the needle is lowered into the brain at the same place without performing the electrolytic lesion, should be used as controls. For infusion of neurotoxins into the Nbm, a 2-/xl syringe with a 31-gauge needle (Hamilton) is placed into the Nbm on one side. Stereotaxic injection of the corresponding amount of toxin in 1/zl of phosphate-buffered saline is performed at a rate of 0.1/xl/minute. After injection, the syringe is kept for

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an additional 5 minutes at the injection site to allow complete diffusion. Animals that received the vehicle alone are considered controls. For intraventricular administration of toxins, the skull is opened at bregma 1.2 mm lateral to the longitudinal suture by placing a small hole as outlined previously. Stereotaxic injection of the toxin in 3-5/zl of phosphate-buffered saline is made into one side of the lateral ventricle (coordinates from bregma: posterior, 0.8 mm; lateral, 1.2 mm) using a 10-/zl syringe with a 31-gauge needle (Hamilton). The needle is inserted to a depth of 3.4 mm below the cortical surface and the injection is performed at the rate of 1 /zl/minute. After injection the syringe is kept for an additional 5 minutes at the injection site to allow for a complete diffusion. Postsurgical care is given as described by Nilsson et al. (40). The animals to be used as controls should receive an injection of an equal volume of phosphate-buffered saline. When administering the cholinergic immunotoxin 192IgG-saporin conjugate, the control animals should be given an injection of the uncoupled immunotoxin obtained by reduction of 192IgGsaporin with 20 mM dithiothreitol overnight at 4~ (52).

Mechanical Damage of Nbm General Considerations Mechanical lesion of the Nbm (e.g., by radio frequency or electrolysis) results in damage to all neural tissue at the lesion site, including cell populations residing in the basal forebrain as well as passing (e.g., noradrenergic and dopaminergic) fiber bundles. Besides a loss of cortical cholinergic input there is a considerable reduction of dopaminergic and noradrenergic innervation of the cortex as well as degeneration of noncholinergic neurons, which are present in varying proportions depending on the lesion site. Furthermore, if the size of the lesion is not kept small enough, it encroaches on the neighboring nuclei and might damage the globus pallidus, the nucleus caudate-putamen, or hypothalamic nuclei, and even the capsula interna. This might not only lead to high mortality rates but also might considerably affect the results obtained.

Limitations The lesion is relatively nonspecific for the cholinergic system, destroying all neural tissue at the lesion site, including all passing fibers. In long-term experiments necrosis and the ensuing gliosis at the lesion site must be taken into account.

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Behavioral impairments associated with these lesions may be due to the loss of cholinergic input to the cortex, but the effects of other influences of noncholinergic nature cannot be excluded. Depending on the size of the lesion site the destruction of cholinergic cells is relatively massive, as revealed by a considerable decrease in cortical choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE).

Lesion of Basal Forebrain Nuclei by Excitotoxins General Considerations Excitotoxins are conformationally restricted analogs of the excitatory amino acid neurotransmitter glutamate [e.g., ibotenic acid, quisqualic acid, kainic acid, N-methyl-o-aspartic acid (NMDA), and c~-aminohydroxy-5-methyl-4isoxazolepropionic acid (AMPA)]. These compounds act as glutamate receptor agonists and exert their toxic action by prolonged activation of the receptors, resulting in increased influx of chloride and calcium ions, excess water entry, and osmotic lysis of the cell. The excitotoxins cited have differential affinities to distinct glutamate receptor subtypes (e.g., quisqualate is an agonist of the AMPA-type receptor, whereas ibotenic acid preferentially binds to NMDA receptors), thus the cytotoxicity of each glutamate analog is dependent on the presence of a particular glutamate receptor subtype on the neuron. This might partly explain the differential cytotoxic effects of the known excitotoxins in different regions of the brain. Quinolinic, ibotenic, and quisqualic acid destroy cholinergic cells in the ventral pallidum and substantia innominata complex, whereas quinolinic and quisqualic acid do not degenerate cholinergic neurons in the medial septum (see, e.g., Ref. 56). Ibotenate and quisqualate induce loss of neuronal cells throughout the Nbm complex, but they produce different behavioral impairments in a variety of tasks, indicating the presence of heterogeneous cell populations with differential sensitivities to a certain excitotoxin. This might be partly due to the fact that the various excitotoxins differentially affect cholinergic neurons in the basal forebrain. Quisqualate has been seen to produce a greater destruction of Nbm-cholinergic neurons compared to ibotenic acid. Cortical ChAT depletion by ibotenic acid infusion into the Nbm ranges between 27 and 46%, whereas quisqualate lesions of the Nbm result in cortical ChAT depletions by 41-74% (see Ref. 19). AMPA is even more effective in destroying cholinergic neurons of the Nbm (greater than 70%), whereas also sparing dorsal pallidum and other noncholinergic neurons in the basal forebrain (42). Ibotenic acid destroys maximally about half of the cholinergic neurons of the Nbm and a considerable number of neurons in the dorsal and ventral

[16] BASAL F O R E B R A I N C H O L I N E R G I C L E S I O N PARADIGMS TABLE I

281

Dosages of Excitotoxins to Produce Maximum Depletion in Cortical ChAT Activity a

Excitotoxin

Dose range applied (nmol/injection)

Dose to produce maximal depletion of cortical ChAT b (nmol/injection)

AMPA Ibotenic acid NMDA Quinolinic acid Quisqualic acid

0.5-25 5-50 15-120 10-180 15-180

10 (80%) 50 (65%) 120 (60%) 120 (60%) 60 (75%)

a After infusion into the rat Nbm. Data taken from Boegman et al. (5). b Percentages in parenthesis indicate reduction in ChAT activity.

pallidum and in areas adjacent to the substantia innominata (19), and induces deficits in a broad range of cognitive tests. AMPA (and to a lesser extent quisqualate) destroys a greater amount of these magnocellular cholinergic neurons, suggesting that these neurons are preferentially endowed with AMPA receptors; AMPA and quisqualate lesions of Nbm produce behavioral impairments only in selected tasks (e.g., passive avoidance retention), with AMPA inducing greater impairments than quisqualate (19). Differential placement of lesions within the Nbm and the amount ofneurotoxin applied produce varying depletions of cortical CHAT. The more posterior the lesion, the greater the depletion of cortical ChAT (55).

Applications of Excitotoxins Table I summarizes the dosages of a number of excitoxins, including the optimal dose to produce maximum depletion in cortical ChAT activity. Excitotoxins are usually directly infused into the Nbm. Depending on the experimental design, both unilateral and bilateral infusions into the Nbm can be accomplished. Some researchers have performed infusion of excitoxins at two different sites on the same side within the basal forebrain, thus destroying more rostral or caudal portions of the Nbm, using the following coordinates (1, 20, 42): (1) 0.2 mm posterior to bregma, 3.2 mm lateral to midline, 7.0 mm ventral from dura and (2) 1.0 mm posterior to bregma, 2.6 mm lateral to midline, 7.5 mm ventral from dura. The optimal time interval needed to produce a maximum reduction in cortical ChAT activity was normally 7 days after lesion, regardless of which excitotoxin was used.

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Advantages and Limitations Excitotoxins are known to destroy cell bodies in the vicinity of the injection site, but these excitotoxins spare terminals and axon passages (14). The lesion procedure used in this paradigm affects mainly cholinergic neurons because, in the rat, approximately 80-90% of the cells in the Nbm are cholinergic, and these account for 70-80% of the cholinergic input to the cortex, with a predominately ipsilateral projection (16). However, the excitotoxins are far from being selective for cholinergic neurons, which might explain why the behavioral effects depend on the toxin used to produce lesions (19, 56). It cannot be easily established whether the noncholinergic neurons within the lesion site are affected by the excitotoxins or whether these noncholinergic cells influence or contribute to the deficits in learning and memory following lesion (23, 26, 35). Quisqualic acid produces larger decreases in cortical ChAT activity as compared to ibotenic acid, but does not produce as severe deficits in spatial navigation or passive avoidance tasks as observed with ibotenic acid. Therefore, the deficits in learned behavior after excitotoxic lesions of the Nbm cannot be solely attributed to the loss of cortically projecting cholinergic neurons. It must be taken into account that the excitotoxin-induced damage to noncholinergic cells in the Nbm also contributes to these behavioral impairments. Moreover, despite a careful stereotaxic administration of the excitoxins, it is possible that the toxin also spreads out to damage adjacent structures, such as the globus pallidus, amygdala, and some thalamic nuclei; thus, degenerative effects of the infused excitotoxin might also occur far from the injection site. When interpreting the effects of an excitotoxic lesion on both neurochemical and neurophysiological sequelae or cognitive tasks, these limitations must be kept in mind.

Lesion o f Basal Forebrain by Ethylcholine Aziridinium General Considerations Ethylcholine aziridinium ion (AF64A) is a neurotoxic analog of choline; it exerts its toxic action by disrupting the high-affinity choline transport system that regulates the rate and extent of acetylcholine synthesis. At higher concentrations AF64A also inhibits AChE activity in vitro. Several authors have suggested that AF64A completely lacks selectivity for cholinergic markers (37). However, in further studies it was demonstrated that the specificity of AF64A depends on both the dosage applied and the site of injection. Local administration of AF64A at concentrations higher

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than 0.02 nmol into various brain regions was shown to produce considerable nonspecific tissue destruction at the site of injection (37). However, intraventricular injection of AF64A at low concentrations (less than 5 nmol) produces a relatively specific loss of cholinergic neurons restricted to the medial septal nucleus and the vertical limb of the diagonal band, but sparing cholinergic neurons in the Nbm, and without inducing histological damage to overlying cortex, fimbria fornix, or adjacent structures (11, 12, 27, 32, 33, 36, 44). These AF64A-induced degenerations are accompanied by decreased choline uptake and decreased ChAT and acetylcholine synthesis in the hippocampus (36).

Applications of AF64A The cholinotoxin AF64A can be applied in two ways. The optimal survival time for maximum depletion in cortical ChAT activity is about 7 to 10 days. AF64A has to be prepared immediately prior to use from 10 mM acetylcholine mustard hydrochloride, as detailed elsewhere (10). Infusion Directly into Nbm Only very low doses (0.02 nmol AF64A) seem to result in specific cholinergic cell loss as measured by AChE staining (34). When using concentrations of 0.2 nmol of AF64A and higher, this resulted in nonspecific tissue damage at the injection site (37) and in lesioning of both cholinergic and noncholinergic cells (39). Infusion of AF64A into Lateral Ventricle To obtain specific damage to cholinergic cells, AF64A must be applied at concentrations of less than 5 nmol. This leads to a decrease in hippocampal cholinergic parameters without affecting cholinergic activity in the frontal, parietal, or cingulate cortex, amygdala, or striatum (10). This seems to be due to a selective loss of cholinergic cells restricted to the medial septum, as detected by immunohistochemistry for ChAT (36).

Advantages and Limitations Intraventricular injection of low doses of AF64A (less than 5 nmol) allows for a selective lesion of medial septal cholinergic neurons, which might be useful when separately studying the role of the septohippocampal cholinergic system, particularly related to the hippocampus. The AF64A-induced behavioral effects are similar to those seen in animals with fimbria fornix lesion. Comparison of the biochemical and behavioral data obtained from fimbria fornix lesion and intraventricular AF64A injection might be helpful to eluci-

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date to what extent cholinergic hippocampal hypofunction contributes to the cognitive deficits observed in both models. Direct infusion of AF64A into the Nbm results in specific cholinergic cell loss only at a very low concentration, about 0.02 nmol. When using higher concentrations of AF64A, up to 1 nmol, both cholinergic and noncholinergic cells in the Nbm are destroyed. In conclusion, the specificity of AF64A depends on both the site of injection and the dosage applied (21, 29).

Lesion of Basal Forebrain Cholinergic System by Cholinergic Immunotoxins General Considerations Cholinergic neurons of the basal forebrain possess nerve growth factor (NGF) receptors, whereas other neurons in this region, including the cholinergic cells in the nearby striatum, do not express detectable levels of NGF receptors (25, 61). It was demonstrated that a well-characterized monoclonal antibody to the low-affinity NGF receptor, 192IgG, accumulates bilaterally exclusively in cholinergic neurons of the basal forebrain following intraventricular administration (51). Employing the properties of 192IgG, a cholinergic immuntoxin was developed by chemically linking 192IgG via a disulfide bond to the ribosome-inactivating protein saporin (192IgG-saporin; see Refs. 57 and 59; for details of preparation, see Ref. 58). Intraventricular administration of the 192IgG-saporin conjugate results in substantial reductions in ChAT activity in widespread areas of the cortex and hippocampus and in a nearly complete disappearance of ChAT-positive, NGF receptor-immunoreactive neurons in the medial septum, in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca and in the Nbm, whereas cholinergic interneurons in the striatum are not affected (6, 30, 40, 47). Seven days following injection of the immunotoxin there was a dramatic loss of AChE staining in frontal, parietal, piriform, temporal, and occipital cortices, in hippocampus and olfactory bulb, but not in the striatum and cerebellum (30, 45, 46). Noncholinergic septal neurons containing parvalbumin and noncholinergic substantia innominata neurons containing calbindin D28K or NADPH diaphorase (NADPH dehydrogenase) were not affected by 192IgGsaporin (30). The number of parvalbumin-containing GABAergic projection neurons in the septum-diagonal band of Broca complex and Nbm was not reduced following intraventricular 192IgG-saporin application (47). A dramatic increase in microglia was shown in an area corresponding to the topographic location of cholinergic neurons in the basal forebrain (47), suggesting that the immunotoxin is lethal to cholinergic cells in the nucleus basalis

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magnocellularis rather than suppressing the expression of cholinergic markers (e.g., CHAT) in these cells (7).

Applications of Cholinergic Immunotoxin The immunotoxin can be applied both systemically and by intraventricular as well as parenchymal injection. The most promising results, however, have been obtained by a single intraventricular application. Systemic injections (i.v. or salivary gland) employing 20-80/xg of 192IgGsaporin produce massive destruction of neurons in the superior cervical ganglia bilaterally; a dosage of 4-8/xg results in incomplete lesions in this region. Intraventricular injections employing 8-80 /xg of 192IgG-saporin produces severe neurological toxicity (tremor, ataxia weakness, weight losses), whereas 4-5/zg initially produces mild to moderate changes in motility and weight loss but no change in alertness. By 14 days animals are moving freely and are neurologically improved (59). A single intraventricular injection of 4/xg of 192IgG-saporin (at concentrations of 0.3-0.4 mg/ml; see also Ref. 58) will typically destroy most of the ChAT/p75NGF-positive cells in the basal forebrain; destruction is complete 7 days after injection. This cholinergic cell loss is accompanied by decreased ChAT activity in the cortex and hippocampus to about 20% of control value, with no further reduction until day 14 (6, 52). Advantages and Limitations Application of the toxin by intraventricular injection is relatively easy, with a high rate of reproducibility and safety, thus avoiding any mechanical damage of basal forebrain tissue. Small variations in the choice of the stereotaxic coordinates have no important consequences provided that the toxin is still injected into the ventricle. Parenchymal 192IgG-saporin injections induced a more pronounced gliosis than intraventricular injections (30). Injections into the substantia innominata led to damage of striatal cholinergic neurons but not when administered directly into the striatum, suggesting that specificity of 192IgG-saporin to lesion cholinergic Nbm neurons may be less after parenchymal than after intraventricular administration (30). It was found that 192IgG-saporin affects two neuronal groups outside of the basal forebrain, which express p75NGF receptors: NGF-reactive cerebellar Purkinje cells after intraventricular injection and cholinergic striatal interneurons after injections into the substantia innominata (30). There are ChAT-positive, but NGF-negative, neurons in the rat Nbm substantia innominata complex innervating the amygdala and parts of the rhinal paralimbic areas (2, 60) that are spared or only partially affected by the immunotoxin (30).

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Similarly, cholinergic neurons in the ventral pallidum and sublenticular substantia innominata not expressing p75NGF receptors are not affected by the immunotoxin. GABAergic projection neurons in the Nbm complex are not affected by the immunotoxin, although GABAergic basal forebrain neurons might also express the p75NGF receptor (15). Due to the nearly complete, selective, and specific lesions of cholinergic cells in the basal forebrain after intraventricular application, it appears that at present the 192IgG-saporin immunotoxin represents the most appropriate tool to produce cortical cholinergic hypofunction.

Summary and Conclusions Cholinergic lesion paradigms have been used to study the role of the cholinergic system in cortical arousal and cognitive function, and its implication in cognitive deficits that occur in Alzheimer's disease. In the past few years an increasing number of studies have applied neurotoxins, including excitotoxins or cholinotoxins (e.g., AF64A), by stereotaxic injection into the Nbm to produce reductions in cortical cholinergic activity. One of the most serious limitations of these lesion paradigms is the fact that basal forebrain cholinergic neurons are always intermingled with populations of noncholinergic cells and that the cytotoxins used are far from being selective to cholinergic cells. Excitoxins, when infused directly into the NbM, destroy cell bodies nonspecifically, but spare axons passing the injection site. Several excitoxins are now available (e.g., ibotenic acid, quisqualic acid, quinolinic acid, NMDA, and AMPA), all of which have differential effects on cortical ChAT activity and result in different cognitive impairments. However, the degree of cortical cholinergic deficit does not appear to correlate directly with the extent of cognitive impairment observed, suggesting that the excitotoxins differentially affect also noncholinergic projections to the neocortex. These side effects must be kept in mind when interpreting data obtained from lesion studies with excitotoxins. The selection of the excitotoxins to perform the lesion study must be based on the problem to be addressed. The specificity of AF64A in destroying cholinergic neurons depends on both the dosage applied and the site of injection. Local administration of AF64A into the Nbm at concentrations higher than 0.02 nmol produces considerable nonspecific tissue destruction at the site of injection, whereas intraventricular injection of AF64A at concentrations below 5 nmol results in cholinergic cell loss restricted to the medial septal nucleus and the vertical limb of the diagonal band, but spares cholinergic neurons in the Nbm.

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A monoclonal antibody to the low-affinity nerve growth factor receptor, 192IgG, coupled to a cytotoxin, saporin, has been described as an efficient and selective immunotoxin for the NGF receptor-bearing cholinergic neurons in rat basal forebrain (59). Intraventricular administration of the 192IgGsaporin conjugate appears to induce a nearly complete and specific lesion of neocortical and hippocampal cholinergic afferents. Other neuronal systems in the basal forebrain are spared by the immunotoxin. Neurons that express the p75NGF receptor are susceptible to the immunotoxin; these include cerebellar Purkinje cells after intraventricular administration and striatal cholinergic interneurons, but only after basal forebrain injection. The nearly complete, selective, and specific lesions of cholinergic cells in the basal forebrain after a single intraventricular application indicate that at present 192IgG-saporin represents the most appropriate tool for producing cortical cholinergic hypoactivity.

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[17]

In Vitro Studies of Liposome-Mediated

Gene Transfection K. Yang, J. Regino Perez-Polo, F. Faustinella, G. Taglialatela, and R. L. Hayes*

Introduction Liposome-mediated gene transfection in the central nervous system (CNS) is a relatively recent addition to the repertoire of DNA transfection methodologies. Carriers currently used to introduce genes into localized regions of the nervous system through stereotaxic injection include retroviral vectors (3, 28, 37), herpes simplex virus vectors (2, 11, 15, 21), adenoviral vectors (19, 10), and grafted cells (6, 17, 30). Each of these approaches to gene transfection has its advantages. However, each is also compromised by limitations. DNA transfection using lipid vesicles is an attractive method for introducing genetic information into CNS cells because of the simplicity and safety of this approach. Previously, cationic liposomes have been used to deliver various agents, including plasmid DNA (13, 25, 26, 33, 40). Furthermore, studies report liposome-mediated/3-galactosidase (/3-gal) gene transfection and expression in adult murine brain (29). The transient expression produced by liposome-mediated gene transfection may limit its application in diseases caused by genetic defects. However, liposomal transfection of trophic proteins may prove useful for treatment of CNS injury by blunting transient pathological processes and/or facilitating recovery. In addition, liposomes are free of the DNA length constraints that are typical of viralbased delivery systems. Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity, or gonadal localization after systemic delivery (26, 40).

Optimizing of Liposome-Mediated Gene Transfer in Rat Septohippocampal Cell Cultures As previous studies have suggested (for review, see Ref. 5), two important aspects of liposome-mediated gene transfection must be considered. First,

* T o w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

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the ratios of DNA to liposomes play an important role in the efficiency of gene transfection. Second, high levels of liposomes can potentially produce cell lysis. Because different cell lines respond differently to liposome-mediated gene transfer (5), investigators must optimize transfection efficiency in each cell line. Importantly, little is known about optimal concentrations of DNA and liposomes for transfection of CNS cells. Kaech et al. indicated that the efficiency of liposome-mediated DNA transfection in CNS cells is very low (16). Thus, we have initiated studies using a reporter gene,/3-gal, to optimize ratios of DNA to liposomes in CNS cell cultures and examine possible cell lysis (38). We believe these studies are a useful prerequisite to subsequent studies of liposome-mediated gene transfection of DNA into CNS cells both in vitro (38) and in vivo (see below). Previous studies indicate that the cytomegalovirus (CMV) is a prudent initial choice of viral promoter (4, 7, 20, 34). We used a pUC19-based plasmid containing a CMV promoter as an expression vector for Escherichia coli ~gal gene transfection (23). The E. coli/~-gal gene was subcloned into a unique NotI site under the control of the CMV promoter. We used the commercially available DOTMA and DOPE liposome (GIBCO-BRL, Gaithersburg, MD). The liposome formulation is a 1 : ! (w/w) mixture of the cationic lipid N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in membrane-filtered water. Primary septohippocampal cell cultures were used for in vitro studies of liposome-mediated gene transfection. Cultures were incubated for 1 week prior to transfection. By that time, astrocytes had reached confluence and were no longer actively multipiying, whereas neurons were well differentiated and stable. Our initial experiments used 10 different ratios of/~-gal DNA to liposomes for the transfection of primary septohippocampal cell cultures. Plasmid DNA [1 ~g in 100/zl Dulbecco's modified Eagle's medium (DMEM)] was mixed with cationic liposomes in ratios of 1 : 1, 1:2, 1 : 3, 1 : 4, 1:5, 1:6, 1 : 7, 1 : 8, 1:9, and 1:10 (DNA :/zg/liposomes :/zl) and overlaid on each 16-mm well of rat septohippocampal cells overnight. X-Galactosidase (X-Gal) staining was performed 2 days after transfection to calculate transfection efficiency. Cells expressing/3-gal were stained blue. We obtained the highest transfection efficiency employing a transfection concentration of 1 ~g DNA/3 /zl liposomes/well. X-Galactosidase staining was detected in cells having both neuronal and astrocytic morphology. Less efficiency transfection was observed with a transfection concentration of 1/zg DNA/1/~1 liposomes/weU. Although liposomes have not been associated with toxicity in vivo (32), treating cells with high levels of liposomes can result in cell lysis (5). Our experiments showed that cultured rat septohippocampal cells began to exhibit cell lysis when exposed to 5/zl/well or more ofliposomes. Without increasing liposome

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FIG. 1 /3-Galactosidase transfection efficiency was calculated from X-Gal staining in septohippocampal cultures at four time points after transfection with pCMV//3gal using 1/xg DNA/3/xl liposomes/well. Cell counts were conducted independently by two investigators. The values represent the mean _+ SEM of the average number of X-Gal-stained cells in each well at 1, 2, 7, and 14 days following transfection (n = 4)./3-Galactosidase expression was maximal 2 days after transfection and persisted for 2 weeks.

concentrations, we did not see any increase in transfection efficiency by increasing DNA concentrations from 1 to 2/zg/well. To further investigate the temporal profile of liposome-mediated gene transfection,/3-gal transfection efficiency was calculated from X-Gal staining in septohippocampal cultures at 1, 2, 7, and 14 days after incubation with a transfection concentration of 1 /zg DNA/3 /zl liposomes/well. Maximal X-Gal staining (>1000 cells/well) was detected 2 days after transfection (Fig. 1). We also found that/3-gal-transfected cells could continue expressing /3-gal for at least 2 weeks, although at markedly lower levels (Fig. 1). Decreased levels of/3-gal expression were partially due to cell loss caused by prolonged incubation of primary septohippocampal cell cultures. Because of the limited life span of primary septohippocampal cell cultures, we were unable to study gene expression for longer periods.

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Our studies employing the fl-gal reporter gene in CNS cell cultures further confirmed observations of other researchers that the concentrations of liposomes can influence the efficiency of transfection (5). The differential transfection efficiencies in CNS cell cultures associated with varying concentrations of liposomes are consistent with the view that the higher the net positive charge of DNA-liposome complexes is, the better the interaction with the negatively charged cell membrane will be. However, overly high levels of liposomes can cause cell lysis. Without increasing liposome concentrations, increased amounts of DNA did not improve transfection efficiency. The efficiency of the DOTMA- and DOPE-mediated pCMV/fl-gal transfection observed by us in septohippocampal cell cultures (> 1000 transfected cells per 16 mm well) exceeds previously reported transfection efficiency for fl-ga! in hippocampa! cultures employing the transfection reagents. Transfectam and DOTAP (40-200 per 35-mm well) (16). The sustained expression of fl-gal for at least 2 weeks longer suggests the potential therapeutic utility of liposomal-mediated gene transfection in CNS injury and degeneration.

Sustained Expression of Nerve Growth Factor by Liposome-Mediated Gene Transfer Exogenous supplementation of nerve growth factor (NGF) has been reported to spare neurons from death and degeneration following injury (9, 18, 24, 36) and to increase choline acetyl transferase (CHAT) activity (31, 35). Furthermore, long-term NGF administration also increases the activity of protective antioxidant enzymes in rat brain (27). The rodent hippocampus is preferentially vulnerable to a variety of central nervous system insults, including traumatic brain injury and ischemia (8). Thus, enhancing the availability of NGF following CNS injury may have significant therapeutic potential. Many different approaches, such as continuous infusion, have been developed to deliver exogenous NGF to the nervous system of mammals (9, 18, 24, 36). Although these methods have generated important information and have therapeutic potential, significant limitations imposed by protein degradation and by the blood-brain barrier restrict the clinical utility of these approaches (1). Gene transfer is another way to introduce NGF into CNS cells and tissues. As pointed out above, a number of viral vector systems have been used to introduce genes into localized regions of the nervous system. Each of these has its advantages. However, each is compromised by limitations, including concerns regarding safety and toxicity. Cationic liposomes have also been used to carry various agents into CNS cells, including plasmid DNA for gene transfer (13, 33, 40). Roessler and Davidson report liposome-mediated fl-gal gene transfection and expression in adult mouse

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brain (29). Optimal concentrations of liposomes for transfection of the/3-gal gene in primary septohippocampal cultures have been determined by our laboratory (39). The transient expression produced by liposome-mediated gene transfection may limit its application in diseases caused by genetic defects. However, liposomal transfection of trophic factors may prove useful for treatment of central nervous system injury by blunting transient pathological processes and/or facilitating recovery. Because of the simplicity, reproducibility, safety, and efficiency of cationic liposome-mediated gene transfection (5, 12, 26), we have examined liposome-mediated NGF gene transfection in vitro (38). We used a pUC 19-based plasmid containing a CMV promoter as an expression vector for NGF transfection (23, 39). The rat NGF DNA was subcloned into a unique NotI site under the conrol of the CMV promoter. We used the commercially available DOTMA and DOPE (GIBCO-BRL) liposome formulated from a 1 : 1 (w/w) mixture of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidylethanolamine in membrane-filtered water. Because of our interest in injury mechanisms in the hippocampus and the preferential vulnerability of the hippocampus to traumatic or ischemic brain injury (14, 22), we used mixed primary septohippocampal cell cultures for in vitro studies of liposome-mediated gene transfection. Cultures were incubated for 1 week prior to transfection. By that time, astrocytes reached confluence and were no longer actively multiplying, whereas neurons were well differentiated and stable. Based on our previous studies of liposome-mediated gene transfection in septohippocampal cultures (39), we employed a concentration of 1 /xg DNA/3/zl liposomes/well (16-mm well) to transfer the NGF gene to primary septohippocampal cell cultures. The purpose of these experiments was to examine systematically if liposome-mediated NGF gene transfection could produce increased expression of NGF mRNA and protein. We also sought to confirm the biological activity of the NGF protein produced following transfection. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of NGF mRNA were conducted 1 day after liposome-mediated NGF transfection of septohippocampal cultures. Increased NGF mRNA was observed in pCMV/NGF-transfected cells as compared to sham transfections. To check for possible DNA contamination during RNA preparation, we included RNA samples without performing reverse transcription. These control studies confirmed the absence of DNA contamination. NGF protein levels were examined 2 days after liposome-mediated NGF transfection using an antibody enzyme-linked immunosorbent assay sandwich (ELISA). NGF concentrations were quantified against a standard con-

295

[17] LIPOSOME-MEDIATED GENE TRANSFECTION 10000 9000 8000

[~]

Control NGF

-

*** 7 0 0 0

P < 0.001

-

6000 Q.

5000 -

u.. Q~

4000 -

v

Z 3000 -

2000 1000

3 days

7 days

14 d a y s

Time after Transfection Fro. 2 ELISA analysis of NGF protein in culture medium: Three days after NGF gene transfection, NGF protein was increased 10-fold in the medium from NGF DNA-transfected cultures. Increased secreted NGF could be detected in the medium 2 weeks after NGF DNA transfection (values represent means +_ SEM; n = 4). The medium was exchanged three times a week after gene transfection.

centration curve of pure isolated murine NGF. E L I S A studies detected dramatic increases in N G F protein in cell pallets from transfected septohippocampal cultures. Three days after N G F gene transfection, robust increases of N G F protein were detected by E L I S A in the cell culture medium. The secreted form of N G F in the medium could still be detected 2 weeks after p C M V / N G F transfection (Fig. 2). Because we routinely exchange the medium three times a week after gene transfection, the consistent detection of the secreted form of N G F in the medium suggests that septohippocampal cells express and secrete N G F for at least 2 weeks after liposome-mediated gene transfection. Rat pheochromocytoma (PC12) cells were used to confirm the specific biological activity of N G F in medium conditioned by cell cultures transfected with N G F DNA. PC12 cell medium was removed 3 hours after plating, a sufficient amount of time for cells to attach to wells, and replaced with 0.5 ml of conditioned medium collected from cultures 3 days following liposomemediated N G F DNA transfection. N G F (20 ng/ml) was added to sister wells

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PARADIGMS OF NEURAL INJURY to assay the response of the cells to exogenous NGF. Cells were observed after 33 hours for the presence of neurite outgrowth. The secreted form of N G F in the N G F DNA-transfected cell medium produced biological effects similar to those of N G F isolated from mouse submaxillary gland. However, medium from control cells incubated only with liposomes did not produce neurotrophic effects. These results represent the first reported use of liposome-mediated N G F transfection in postmitotic central nervous system cell cultures. The levels of N G F protein expressed in our transfection system are particularly high, persist for at least 14 days, and elicit prominent neurotrophic effects such as neurite growth and growth cone formation. The persistent secretion of large amounts of N G F in media after p C M V / N G F transfection suggests the potential utility of neurotrophin gene transfection for treatment of neuronal injury or degenerative disorders.

References 1. M. Barinaga, Science 264, 773 (1994). 2. X. O. Breakefield and N. A. DeLuca, New Biol. 3, 203-218 (1991). 3. K. W. Culver, Z. Ram, S. Wallbridge, H. Ishii, E. H. Oldfield, and R. M. Blaese, Science 256, 1550-1552 (1992). 4. X. J. Fang, A. Keating, J. deVilliers, and M. Sherman, Hepatology 10, 78i787 (1989). 5. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1987). 6. F. H. Gage, J. A. Wolff, M. B. Rosenberg, L. Xu, J. L. Yee, C. Shults, and T. Friedmann, Neuroscience 23, 795-807 (1987). 7. T. Giordano, T. H. Howard, J. Coleman, K. Sakamoto, and B. H. Howard, Exp. Cell Res. 992, 993-997 (1991). 8. R. L. Hayes, L. W. Jenkins, and B. G. Lyeth, J. Neurotrauma, 9, S173-S178 (1991). F. Hefti, J. Neurosci. 8, 2155-2162 (1986). lO. M. S. Horwitz, in "Fields Neurology" (B. N. Fields and D. N. Knipe, eds.), 2nd Ed., pp. 1679-1721. Raven Press, New York, 1990. ll. Q. Huang, J. P. Vonsattel, P. A. Schaffer, R. L. Martuza, X. O. Breakefield, and M. DiFiglia, Exp. Neurol. 115, 303-316 (1992). 12. P. Hug and R. G. Sleight, Biochim. Biophys. Acta 1097, 1-17 (1991). 13. S. Imaizumi, V. Woolworth, R. A. Fishman, and P. K. Chan, Stroke 21, 13121317 (1990). 14. L. W. Jenkins, K. Moszynski, B. G. Lyeth, W. Lewelt, D. S. DeWitt, A. Allen, C. E. Dixon, J. T. Povlishock, T. J. Majewski, G. L. Clifton, H. F. Young, D. P. Becker, and R. L. Hayes, Brain Res. 477, 211-224 (1989). ,

[17] LIPOSOME-MEDIATEDGENE TRANSFECTION

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15. P. A. Johnson, K. Yoshida, F. H. Gage, and T. Friedmann, Mol. Brain Res. 12, 95-102 (1992). 16. S. Kaech, J. A. Drazba, and E. Ralston, Soc. Neurosci. Abstr. 19, 1746 (1993). 17. M. D. Kawaja, M. B. Rosenberg, K. Yohida, and F. H. Gage, J. Neurosci. 12, 2849-2864 (1992). 18. L. F. Kromer, Science 235, 214-216 (1987). 19. G. leGalLaSalle, J. J. Robert, S. Berrard, V. Ridoux, L. D. Stratford-Perricaudet, M. Perricaudet, and J. Mallet, Science 259, 988-990 (1993). 20. A. P. Li, C. A. Myer, and D. L. Kaminski, Cell Dev. Biol. 28A, 373-375 (1992). 21. E. Lycke, B. Hamark, M. Johansson, A. Krotochwil, J. Lycke, and B. Svennerholm, Arch. Virol. 101, 87-104 (1988). 22. B. G. Lyeth, L. W. Jenkins, R. J. Hamm, C. E. Dixon, L. L. Phillips, G. L. Clifton, H. F. Young, and R. L. Hayes, Brain Res. 526, 249-258 (1990). 23. G. R. MacGregor and C. T. Caskey, Nucl. Acids Res. 17, 2365 (1989). 24. C. N. Montero and F. Hefti, J. Neurosci. 8, 2986-2999 (1988). 25. N. Mori and T. Fukatsu, Epilepsia 33, 994-1000 (1992). 26. E. G. Nabel, D. Gordon, Z. Y. Yang, L. Xu, H. San, G. E. Plautz, B. Y. Wu, X. Gao, L. Huang, and G. J. Nabel, Human Gene Therap. 3, 649-656 (1992). 27. G. Nistico, M. R. Ciriolo, K. Fiskin, M. Iannone, A. Demantino, and G. Rotilio, Free Rad. Biol. Med. 12, 171-181 (1992). 28. Z. Ram, K. W. Culver, S. Walbridge, R. M. Blaese, and E. H. Oldfield, Cancer Res. 53, 83-88 (1993). 29. B. J. Roessler and B. L. Davidson, Neurosci. Lett. 167, 5-10 (1994). 30. M. B. Rosenberg, T. Friedmann, R. C. Robertson, M. Tuszynski, J. A. Wolff, X. O. Breakefield, and F. H. Gage, Science 242, 1575-1578 (1988). 31. R. J. Rylett, S. Goddard, B. M. Schmidt, and L. R. Williams, J. Neurosci. 13, 3956-3963 (1993). 32. H. San, Z. Y. Yang, V. J. Prompili, M. L. Jaffe, G. E. Plautz, L. Xu, J. H. Felgner, C. J. Wheeler, P. L. Felgner, X. Gao, L. Huang, D. Gordon, G. J. Nabel, and E. G. Nabel, Human Gene Therap. 4, 781-788 (1993). 33. M. J. Stewart, G. E. Plautz, L. Del-Buono, Z. Y. Yang, L. Xu, X. Gao, L. Huang, E. G. Nabel, and G. J. Nabel, Human Gene Therap. 3,267-275 (1992). 34. T. A. Thompson, M. N. Gould, J. K. Burkholder, and N. S. Yang, Cell Dev. Biol. 29A, 165-170 (1993). 35. L. R. Williams and R. J. Rylett J. Neurochem. 55, 1042-1049 (1990). 36. L. R. Williams, S. Varon, G. M. Peterson, K. Wictorin, W. Fischer, A. Bjorklund, and F. H. Gage, Proc. Natl. Acad. Sci. U.S.A. 83, 9231-9235 (1986). 37. D. Wolf, C. Richter-Landsberg, M. P. Short, C. Cepko, and X. O. Breakefield, Mol. Biol. Med. 5, 43-49 (1988). 38. K. Yang, F. Faustinella, J. J. Xue, J. Whitson, A. Kampfl, X. S. Mu, X. Zhao, G. Taglialatela, J. R. Perez-Polo, G. Clifton, and R. L. Hayes, Neurosci. Lett. 182, 291-294 (1994). 39. K. Yang, F. Faustinella, J. J. Xue, J. Whitson, A. Kampfl, X. S. Mu, X. Zhao, G. Taglialatela, J. R. Perez-Polo, G. L. Clifton, and R. L. Hayes, Neurosci. Lett. 182, 287-290 (1994). 40. N. Zhu, D. Liggitt, Y. Liu, and R. Debs, Science 261, 209-211 (1993).

[18]

Construction and Analysis of Transgenic Mice Expressing Amyloidogenic Fragments of Alzheimer Amyloid Protein Precursor Rachael L. Neve and Frederick M. Boyce

Introduction Two pathological aspects of Alzheimer's disease (AD) that may be related, but which are not fully understood, are the accumulation of amyloid in the brain and the destruction of brain cells. Even though the relationship between these two phenomena remains to be defined with precision, it is reasonable to suspect that any information we can obtain about/3-amyloid (A4) and its derivation from the/3-amyloid protein precursor (/3-APP) will yield insights into the mechanisms by which nerve cells degenerate in the disease. One of the earliest pieces of evidence linking AD neurodegeneration and/3-APP and/or its/3/A4-containing derivatives was the finding that the/3-APP gene is on chromosome 21: virtually all individuals trisomic for this chromosome will show AD-like neurodegeneration by the age of 40. More recently, the discovery that specific point mutations in the/3-APP gene are associated tightly with some forms of familial Alzheimer's disease has contributed to the increased interest in the role of/3-APP in the disease. Our laboratory has focused on a specific aspect of/3-APP and its connection with the neuronal destruction of AD. This work evolved from our observation several years ago that the carboxy-terminal 100 amino acids of the amyloid precursor protein (/3-APP-C100, or simply C100; previously termed AB1 or /3-APP-C104) were neurotoxic (1). Other laboratories subsequently revealed that C100 was amyloidogenic (2, 3). The neurotoxicity of C100 has been confirmed by other laboratories (4, 5). We hypothesized on the basis of these data that C100 or a similar/3-A4containing fragment of/3-APP may be centrally involved in the amyloidogenesis and neurodegeneration of Alzheimer' s disease. To test this latter hypothesis, we designed and generated an in vivo model for the action of C 100, in the form of transgenic mice expressing C100 in the brain (6). These animals display neuropathology that resembles some features of Alzheimer' s disease neuropathology, lending strength to our hypothesis that C100, or a/3-APP fragment very much like it, may be the perpetrator of neurodegeneration in Alzheimer's disease. In the following pages, using our C 100 transgenic mice

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Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

[181

299

T R A N S G E N I C M I C E E X P R E S S fl-APP

RNA Splice & cDNA Polyadenylation

Promoter

r FIG. 1

A m

Diagram of a prototypic transgenic construct.

as a prototype, we describe the methodology involved in generating transgenic mice for the study of the pathological effects of amyloidogenic fragments of the Alzheimer amyloid protein precursor.

Practical Considerations The construction of transgenic mice for the study of amyloidogenesis and neurodegeneration in Alzheimer's disease entails the following pragmatic considerations: (1) How will the construct be designed and prepared for microinjection? (2) How will the resultant transgenic mice be analyzed for the presence of the transgene? (3) How will the mice be bred and maintained? (4) How will the RNA and protein products of the transgene be detected? (5) What types of histological analyses of the mouse brains should be carried out, and what are some of the technical and procedural issues involved? In the following sections, we describe methodologies that address these practical considerations for creating transgenic mice to study the neuropathological effects of amyloidogenic portions of the amyloid protein precursor.

Design of Transgenic D N A C o n s t r u c t The DNA construction for a transgenic animal requires three elements: (1) a promoter to drive expression of the transgene, (2) the transgene, and (3) RNA splicing and polyadenylation signals (Fig. 1). The choice of promoter is crucial, particularly when expression of the transgene in the brain is desired. Selection of the appropriate promoter can drive expression of the transgene in cells throughout the brain, or confine its expression to neurons or glia, or target expression to cells of a particular transmitter phenotype,

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PARADIGMS OF NEURAL INJURY TABLE I

Neural Promoters Tested in Transgenic Mice

Promoter

Specificity

Ref.

Neuron-specific enolase SCG10 L7 Rhodopsin Dopamine fl-hydroxylase Preproenkephalin Tyrosine hydroxylase S 100 gene Olfactory marker protein Dopamine-/3-hydroxylase Acetylcholine receptor a2

Panneuronal Neural Cerebellar Purkinje and retinal bipolar neurons Photoreceptor cells Sympathetic and other neurons Brain and some peripheral tissues TH-immunopositive cells Astrocytes, some neurons Olfactory neurons Noradrenergic and adrenergic cells Cholinergic subregions of CNS Neurons and astrocytes Neurons

30 31 32 33 34 35 7 36 37 38 39 40 41

c-fos Calmodulin gene II

for example. It is most economical to choose a promoter whose specificity in directing expression of a reporter gene in transgenic mice has previously been verified, given the uncertainties of predicting in vivo specificity of expression of a promoter from in vitro studies (see Ref. 7). A partial listing of neural promoters that have been tested in transgenic mice is given in Table I. For the construction of our C100 transgenic mice, we used the human dystrophin brain promoter (8), which had been shown to confer expression preferentially in the brain in transgenic mice (unpublished data of F. M. Boyce and M. Rosenberg). We elected to place the transgene under the control of the dystrophin brain promoter because dystrophin is widely expressed in the brain at relatively low levels (9) and because its expression in the cortex peaks postnatally (unpublished data of F. M. Boyce and R. L. Neve). These quantitative and qualitative features of transcripts controlled by the dystrophin brain promoter may have been important in allowing survival of the C100 transgenic mice beyond the embryonic stage. In contrast, Quon et al. (10), who overexpressed the full-length fl-APP751 cDNA in transgenic mice, used the neuron-specific enolase promoter. In their case, a promoter conferring robust expression in the brain was desirable. At least three other groups have created transgenic mice overexpressing human fl-APP under the control of its own promoter by introducing the entire fl-APP gene into the mice (11-13). Such use of an entire gene in transgenic mice often gives optimal expression of the transgene; however, most transgenic mice studies employ cDNAs or partial cDNAs. The advantages of cDNA transgenes are that they are usually easier to clone and to manipulate than are entire genes, and that cDNAs

[18]

301

TRANSGENIC MICE EXPRESS/3-APP

Dystrophin Neuronal Promoter

Mlul

Hindll,

~

C100 SV40 Splice & eDNA Polyadenylation

Hindlll ~

r

~

/N

BamHI

AAAAAA

FIG. 2 Diagram of the C100 transgenic construct.

can, with more facility, be mutated or truncated for comparison of parallel transgenics possessing the wild-type cDNA transgene with those possessing genetically altered versions of the cDNA. In our case, we wished to express only the carboxy-terminal 100 amino acids of/3-APP (which fortuitously possessed its own translation start methionine), and we had already established that it could be exogenously expressed from an amino-terminal-truncated /3-APP cDNA in cell lines in vitro. It is important, when making constructs for transgenic mice, to include not only a methionine that will be a translational start signal, but also a stop codon (if a carboxy-terminal truncation of the cDNA is planned). The cDNA transgene must be fused to an RNA cleavage and polyadenylation signal sequence at the 3' end. Many of these 3' cassettes [most of which have been derived from simian virus 40 (SV40)] include an intron as well. Inclusion of introns has been shown to enhance expression of transgenes (14). When we created our C100 transgenic construct, we added the SV40 mRNA processing signals that comprise the 800-bp BglII-BamHI fragment of pRSV-/3-globin (15). The complete C100 construct, then, in which the Rous sarcoma virus (RSV) promoter was replaced with the 3.0-kb dystrophin brain promoter, and/3-globin was replaced with C100 (0.85 kb), followed by the SV40 mRNA processing signals, was approximately 4.65 kb (Fig. 2). An additional consideration in planning the transgenic construct is to design unique restriction enzyme sites flanking the transgene. This allows the transgene to be excised from the bacterial plasmid vector sequences, which could interfere with expression. To prepare the C100 DNA for microinjection, 20txg CsCl-pure plasmid was cleaved with MluI and BamHI to release the 4.65-kb C100 transgenic construct. The digested DNA was preparatively electrophoresed on a 14-cm 0.6% Seakem GTG (FMC, Rockland, ME) agarose gel in standard Trisacetate-EDTA (TAE) buffer until the 4.0-kb vector and the 4.65-kb transgene-containing fragment were well separated. The latter band was cut out

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of the gel (after being revealed by ethidium bromide UV fluorescence) and placed into dialysis tubing with approximately 500/~1 of TAE, after which the DNA was electroeluted at 80 V for 45 minutes. A brief (1 minute) reversal of the voltage gradient ensured removal of the DNA from the walls of the dialysis tubing. The TAE buffer (containing the now-eluted DNA fragment) was removed from the dialysis tubing and the DNA fragment precipitated by the addition of 1/10 volume of 3 M sodium acetate (pH 5.0) and 2.0 volumes of 95% (v/v) ethanol. The DNA was pelleted by spinning it at 14,000 rpm for 30 minutes in a microcentrifuge, and the resultant pellet was resuspended in 50/~1 of TE (10 mM Tris, pH 7.5, 0.25 mM EDTA).

Analysis of Mice for Presence of Transgene The two mostly commonly used methods for detecting mice that possess the transgene are Southern blot analysis and polymerase chain reaction (PCR) analysis. At the time of weaning, when the approximately 3-week-old mice are tagged and males are separated from females, approximately 1 cm is clipped from the end of the tail of each mouse in a given litter (if PCR will be used to detect the transgene, care must be taken not to transfer any tissue from one tail to another, e.g., by using a pair of scissors consecutively on the mice). The tail fragments are kept on ice (if DNA preparation is to occur immediately) or frozen. DNA is then prepared from this tissue according to the following simplified protocol obtained from Dr. M. Rosenberg (personal communication, 1991). Add the tail to 600/~1 of tail buffer (500 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0; 100 mM NaC1; 1% SDS) in a 1.5-ml centrifuge tube. Add 35 p.l of 10 mg/ml proteinase K, and invert the tube or vortex it briefly to mix the contents. Incubate at 55~ for 5 hours to overnight. Then add 202/~1 of 5 M NaC1 (to bring the final concentration of salt to 1.5 M) while the tube is still warm. Add 600 /~1 of chloroform : isoamyl alcohol (24:1, v/v) and vortex each tube for 20-30 seconds. Separate the aqueous and organic layer by spinning at 14,000 rpm in a microcentrifuge for 10 minutes. A very large layer of protein and SDS will form between the aqueous and organic phases. Carefully remove the upper, aqueous layer to a new tube containing 1.5 ml of 95% (v/v) ethanol. Invert the tube two or three times, and remove the precipitated DNA with a capillary micropipette, one end of which has been heated over a flame to create a small hook, which can then be used to retrieve the DNA. Allow the ethanol to drain for 1 minute, then place the micropipette into 150/~1 of TE, pH 8.0. Allow the DNA to resuspend overnight at 4~ before subjecting it to further manipulations. The yield will be 50-100 p.g of DNA. If Southern blots are used to identify transgene-containing DNAs, the

[18] TRANSGENIC MICE EXPRESS/3-APP

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DNA should be cleaved with an enzyme(s) that will release the transgene or some portion of the construct intact. We digested our mouse tail DNAs with HindIII, which we knew should release the dystrophin brain promoter as an intact 3-kb band (see Fig. 2). The HindIII ends were put onto the brain promoter fragment artificially during the original cloning of the promoter, so that while the transgene promoter would be revealed as a 3-kb fragment on Southern blots, the endogenous promoter would appear on a DNA fragment(s) of a different size. Thus, the endogenous and transgenic dystrophin brain promoters could be distinguished by size on Southern blots. Tail DNA (30/zl, or approximately 15/xg) was digested to completion with HindIII and electrophoresed at 50 V for 18 hours. The DNA in the gel was transferred to 1.2/zm Biotrans (ICN, Costa Mesa, CA) in the presence of 20 x SSC (1 x SSC: 0.15 M sodium chloride, 0.015 M sodium citrate), and the blot probed with a dystrophin brain promoter DNA fragment radiolabeled by the random hexanucleotide priming method. Blots were washed to a maximum stringency of 0.2x SSC at 65~ with 0.1% SDS. An overnight exposure of the washed blot was usually sufficient to reveal which DNAs contained the transgene. Approximate copy number of the transgene in each line can be determined by densitometric analysis of the blots, in which the intensity of the transgene band is compared to the intensity of the endogenous dystrophin promoter band. If the polymerase chain reaction is utilized to detect the presence of the transgene, the primers must not both be internal to the promoter or to the cDNA unless they span introns, for otherwise the PCR product from the endogenous gene cannot be distinguished from the PCR product from the transgene. PCR tends to be most consistent with primers that define an approximately 100- to 300-bp fragment. The primers should be tested beforehand on control mouse DNA, and on dilutions (down to one-tenth genome copy) in control mouse DNA of the plasmid carrying the transgene. The 5' primer that we used was P6 in the dystrophin promoter (8); the 3' primer (which was within the C 100-encoding region) represented the reverse complement of base pairs 2040-2062 in/3-APP695 (see Fig. 2). PCR was carried out in 50-/xl reactions containing 50 ng of each primer and 0.5/zl (approximately 200 ng) of each tail DNA, for 32 cycles (94~ 1 minute; 50~ 1 minute; 72~ 3 minutes), followed by a 10-minute extension at 72~

Maintenance of Transgenic Mouse Colony Because of the effort and expense involved in the creation of transgenic mice, it is crucial that each transgenic line is properly maintained. Sufficient numbers of animals must be generated both for propagation of the transgenic

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line and for analysis of any neuropathology in each line. In addition, transgenic animals that are models for the effects of amyloidogenic fragments of fl-APP and for other aspects of Alzheimer's disease will almost certainly need to be aged. For these reasons, it is important that the animals are kept in a barrier or viral antibody-flee facility to avoid loss of animals to infectious agents. Our animals are housed in positive individually vented caging systems to minimize the transmission of airborne diseases, and manipulations of the mice are carried out in a laminar flow hood. We have observed neuropathology in C100 transgenic mice as early as 4.5 months postnatally, but the pathology may advance with age. Interestingly, normal aged mice do not show plaques or tangles, which are characteristic of aged human brains. The founder transgenic mice, derived from F 2 C57BL/6 • SJL/J hybrid mice, are mated with C57BL/6J mice to give rise to F~ progeny. All subsequent generations (F2, F3, etc.) are also backcrossed to C57BL/6J. Weaning is done at approximately 3 weeks of age and consists of separating each litter into separate cages of males and females. Ear tagging and typing of each offspring for the presence of the transgene are also performed at the time of weaning. Transgene-negative siblings are used as age-matched controls. Because the founder mice are derived from both C57BL/6J and SJL/J strains, it is also important to include animals from each of these strains as controls for strain variations. We determine which founder mice are carrying the transgene, and then use F1 litters to determine whether each line is expressing the transgene at the RNA and protein levels (see following). Failure to detect expression of the transgene in several F1 mice carrying the transgene suggests that the line will not be useful for further analyses. However, because expression may depend on integration site, other lines carrying the same transgene may yield expression. Thus it is important to screen founder animals for the presence of the transgene as well as F 1 animals for expression in order to minimize the number of transgenic animals that must be maintained. It is customary to maintain several expressing lines for each transgene. The presence of the transgene is monitored in each generation by PCR or Southern blotting. For long-term propagation of each transgenic line, siblings can be bred to yield offspring that are homozygous for the transgene. Because both heterozygotes and homozygotes are positive for the transgene, homozygotes are usually detected by test breeding with C57BL/6J mice. Homozygotes will yield litters with all progeny carrying the transgene, whereas heterozygotes will yield mixed litters. When a homozygous male and female have been identified, they may be bred to yield homozygous offspring. Creation of a homozygous line reduces the need for further transgene typing. However, it is wise to check for the presence and expression of the transgene periodically, because errors in breeding may occur, and because expression of the transgene may be extinguished with time.

[18] TRANSGENIC MICE EXPRESS/3-APP

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Detection of RNA and Protein Products of Transgene To assess the tissue specificity of the expression of C100 under the control of the dystrophin brain promoter, we first examined the transgenic mice for the presence of RNA transcribed from the transgene. Total RNA was prepared using 100-500 mg of tissue by the guanidinium thiocyanate procedure (with adaptations) (16). The final step involved precipitation of the RNA with 1/2 volume of ethanol, which preferentially precipitates RNA but not DNA. Because the dystrophin brain promoter drives the expression of relatively low levels of dystrophin in the brain, we anticipated that similarly low expression of the transgene might make detection of the transgene RNA by Northern blots difficult. We chose to use reverse transcription coupled with PCR (RT-PCR), using the same primers that were used to analyze the tail DNAs. It is important to confirm that the 5' primer, if part of the promoter region, is also represented in the expected RNA transcript. RNA (1 ~g) from each tissue was treated with DNase I (0.3 U//A in a volume of 13.1 ~1) at 37~ for 20 minutes to remove possible contaminating DNA, and then at 65~ to inactivate the DNase. CH3HgOH (3 ~1, 0.1 M) was added to remove RNA secondary structure; 1.55/~1 of 0.7 M 2-mercaptoethanol was then added, after which cDNA was synthesized from each RNA sample in a 25-~1 reverse transcription reaction that consisted of 50 mM Tris (pH 8.2 at 42~ 50 mM KC1, 6 mM MgCI 2, 10 mM dithiothreitol, 1000 U/ ml Promega (Madison, WI) Biotec RNasin, 1 /~g of 3' primer (identical ~o the 3' primer used in the tail DNA PCR reactions), 400 ~M dNTPs, and 350 U/ml Life Sciences avian myeloblastosis virus reverse transcriptase. After incubation of these reactions at 41.5~ for 2 hours, the reactions were placed at 65~ for 10 minutes to inactivate the reverse transcriptase; 1 /~1 of each cDNA reaction was used as template in a 50-/.d PCR reaction mix containing 200 ng of each of the primers (identical to the primers used for PCR analysis of tail DNAs) and 0.25 ~1 Taq polymerase (Perkin-Elmer, Norwalk, CT). The reactions were subjected to 40 cycles of PCR (94~ 1 minute; 50~ 2 minutes; 72~ 3 minutes) and 40 ~1 of each reaction was electrophoresed on a 1% agarose gel in TAE buffer and transferred to 1.2-~m Biotrans (ICN) in the presence of 20• SSC. The filter was then hybridized with an internal oligonucleotide probe that was 32p-labeled using T4 polynucleotide kinase (U.S. Biochemicals, Cleveland, OH). We used RT-PCR to assess expression of the C100 transgene in brain, muscle, heart, and liver of animals from nine different founder lines. The expected 320-bp RT-PCR product was seen at highest levels in the brain, in all lines evaluated. Although the transgene was transcribed at low levels in other tissues in some of the lines, in all transgenic animals its expression was - 10 times higher in brain than in any other tissue examined. As expected, we were unable to detect the transgene transcript on RNA blots.

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PARADIGMS OF NEURAL INJURY

46K

----

3 0 K "-'-p,-

14.3K

----

FIG. 3 Detection of C100 protein in the brains of transgenic mice. Immunoblot of the antibody 10D5 against total brain homogenates of three nontransgenic (-) and two transgenic (+) 9-month-old mice in lines 2 and 3 is shown.

Attempts to detect the C100 transgene protein product in the brains of the transgenic mice highlighted one of the difficulties often encountered during the characterization of a transgene product, which is the inability to distinguish between endogenous and transgene protein products by immunocytochemical or immunoblot analysis. In this instance, our efforts to detect the C100 were hampered by the fact that available antibodies to human C100 (expressed by the transgene) also reacted with endogenous mouse flAPP. Among the endogenous processing products of mouse fl-APP, as of human fl-APP, are amyloidogenic carboxyterminal fragments of fl-APP that have a similar size to the product of the C100 transgene and are detected by most antibodies to human C100. However, we were able to obtain a monoclonal antibody to made human ~/A4 1-15 (10D5, gift ofD. Schenk, Athena Neurosciences, Inc.) that does not react with mouse fl-APP. Brains of 9-month-old mice were rapidly homogenized and 20/~g of each homogenate was electrophoresed and transferred to PVDF membranes. The membranes were immunostained with the monoclonal antibody 10D5, and enhanced chemiluminescence was used to detect positive immunoreactivity at a band of approximately 28 kDa only in lanes containing homogenates of positive transgenic mouse brains (Fig. 3). We believe that this band represents a C 100 dimer, largely because the predominant portion of the C 100 that we generate in bacterial or baculovirus systems migrates at 28 kDa (about 10% migrates at 15 kDda; unpublished results of R.L.N.).

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[18] TRANSGENIC MICE EXPRESS fl-APP TABLE II

Epitope Tags Tag

Sequence

Ref.

FLAG Streptavidin-affinity tag Influenza virus hemagglutinin a-Tubulin epitope HSV-Tag

AspTyrLysAspAspAspAspLys SerAlaTrpArgHisProGlnPheGlyGly TyrProTyrAspValProAspTyrAlaSerLeu GluGluPhe GlnProGluLeuAlaProGluAspProGluAsp GluGlnLysLeulleSerGluGluAspLeu

17 42 43 44 45 46

c-myc

To improve our ability to detect the C100 transgene protein product in mice, we have constructed a second transgenic mouse, expressing a cDNA that we term FLAG/3-APP-C100. We have fused the coding sequence for a hydrophilic 10-amino acid sequence termed FLAG (17) onto the aminoterminus of C100. We have successfully expressed this fusion fragment in cells, and have shown that it retains its neurotoxicity in culture, and that it binds to the specific C 100 receptor (18). We will be able to distinguish the transgene product from the endogenous/3-APP gene product in the transgenic mice expressing FLAG/3-APP-C 100 by using monoclonal antibodies to FLAG (M2 and M5, available commercially from VWR, Greenbelt, MD) or polyclonal antibodies to FLAG that we have made to detect FLAG/3-APP-C 100 and its metabolic derivatives in vivo. We have tested the FLAG antibodies immunocytochemically, and they demonstrate specificity, with the polyclonal antibodies showing particularly robust immunoreactivity to the FLAG epitope, both immunocytochemically and on immunoblots. We will use these antibodies to determine the location and state of aggregation of FLAG/3-APP-C100 in the transgenic mice. We have illustrated the use of epitope tags by describing the rationale for, and the design of, our FLAG/3-APP-C 100 transgenic mice. Additional epitope tags have been described, however, which are useful for the same purpose. A partial listing of such tags is given in Table II. Selection of the appropriate tag will depend on whether the investigator wishes to use it at the amino terminus or the carboxy terminus of the transgene protein (or even internal to the protein), and on availability and characteristics of the antibody.

Histological Analyses of Transgenic Mouse Brains Expressing Amyloidogenic Fragments of/3-APP in Brain Classic histological stains for Alzheimer's disease neuropathology have historically been those that detect amyloid plaques and neurofibrillary tangles, the two major features of Alzheimer's disease neuropathology that form the

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basis for postmortem diagnoses. During the past decade, hov,ever, we have developed antibodies and cloned genes that enable increasingly sophisticated analyses of features of Alzheimer's disease that precede the end-stage plaques and tangles. As a result, a broader definition of the neuropathology characteristic of this disease has emerged, as exemplified by the analyses that we carried out on our C 100 mice (6). We used an affinity-purified antibody, E1-42 (19), raised against a peptide representing the 42-amino acid/3/A4 fragment, to detect amyloid deposition in the transgenic mouse brains. This antibody does not recognize normal human/3-APP, but distinctively immunoreacts with pathologic structures specific to Alzheimer's disease and (to a lesser extent) normal aged brain. These include the amyloid cores of neuritic plaques, as well as the so-called diffuse amyloid deposits that are not revealed by conventional histological stains for amyloid. Immunostaining of control mouse brains with E1-42 showed that in mouse brain, in contrast to human brain, this antibody recognizes normal structures to some degree in that it displayed light homogeneous staining of cell bodies. Both quantitative and qualitative differences in the pattern of E1-42 (/3/A4) immunoreactivity were exhibited in transgenic mouse brains. Intensely E1-42 immunoreactive material was seen in neuronal cells throughout the brains of all transgenic mice tested. In most cases the/3/A4 immunoreactivity occurred as punctate deposits within neurons that have a rounded, compact appearance. Such staining was clearly visible in Ammon's horn of the hippocampus and in the stratum oriens. The intracellular accumulation of/3/A4 was particularly prominent within the hilus, where the immunoreactivity was seen not only in the cell bodies but also in abnormal processes. The emergence of/3/A4 immunoreactivity in the neuropil may represent a stage of amyloid deposition later than that observed in the cell soma, because it was seen only in the three lines expressing highest levels of the transgene. Punctate accumulations of/3/A4 immunoreactivity in dystrophicappearing fibers were dramatically apparent in the stratum radiatum of the CA2/3 region of the hippocampus in these lines. When we analyzed these same mice with additional antibodies to/3/A4 and its subdomains, we discovered that they exhibited patterns of immunoreactivity somewhat different from those displayed by the antibody E1-42. For some antibodies, the difference was merely quantitative, whereas with others we detected no differences between transgenic and control animal brain. When these antibodies were used to probe immunoblots of E1-42 peptide, we discovered that only those antibodies recognizing aggregated forms of E1-42 revealed/3/A4 deposits in the transgenic mice. The antibodies that immunodetected only the E 1-42 monomer on immunoblots did not show abnormal immunoreactive structures in the brains of the mice. Interestingly,

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most antibodies that recognized only the E1-42 monomer were made to subdomains of the j3-amyloid fragment (e.g., 1-28) or to the coupled 1-42 peptide. Those detecting aggregates of/~/A4 comprised primarily antibodies generated with uncoupled 1-42 peptide. We had previously shown that staining of Alzheimer's disease brains with F5, an antibody to the carboxy-terminal nine amino acids of/~-APP, exposed intracellular aggregates of this epitope in secondary lysosomes in pathologically afflicted regions of Alzheimer's disease brain, such as in the CA1 neurons of Sommer's sector (20). Hence, we might expect to detect a similar phenomenon in the transgenic mice. And indeed, staining of the brain sections with the antibody F5 showed a striking change in the subcellular localization of the F5 epitope that was particularly evident in the CA2/3 region of the hippocampus in transgenic mice. Whereas control mice showed homogeneous light F5 immunoreactivity predominantly of the neuronal somata in this region, the F5 immunoreactivity in the transgenic mice presented as dark punctate accumulations in subcellular compartments that extended markedly into the neuronal processes. Adjacent Nissl stained sections did not reveal detectable gross abnormalities in the area of altered F5 staining, suggesting that the disorganization evident in the immunostained sections mainly involves the neuropil in transgenic mice of this age. In transgenic mice from the three lines expressing highest levels of the transgene, the cells in the CA2/3 region showed particularly dense reaction product in the neuropil, and the reactivity in the soma took the form of larger accumulations, as if the punctate vesicular immunoreactive material had fused or aggregated. The appearance of the F5 immunoreactivity in these cells was very similar to what we observed in Alzheimer's disease brains (20). F8, an independent antibody made to the carboxy terminus of/3-APP (gift of D. Schenk, Athena Neurosciences), exhibited the same abnormal pattern of immunoreactivity revealed by the F5 antibody. The immunocytochemistry is carried out as follows. F1 backcross progeny from six different founder lines (ages 3 weeks to 4 months) and founders only from three additional lines (6 months of age) are analyzed histologically. We also analyzed eight age-matched C57BL/6 and SJL controls. Mouse brains are either immersion fixed (in cases in which we use half of the brain for RNA analysis) or are perfused with freshly prepared 4% paraformaldehyde (w/v) buffered with 0.1 M sodium phosphate, pH 7.2, and postfixed overnight at 4~ before cryoprotection in 30% sucrose in 0.1 M sodium phosphate, pH 7.2. The brains are frozen in a dry ice-acetone bath maintained at -40~ then processed immediately or stored at -70~ The tissue is cut at 50-~m intervals on a sliding microtome into ice-cold Tris-buffered saline (TBS: 50 mM Tris, pH 7.5,300 mM sodium chloride). Human brain tissue is obtained in formalin and is cryoprotected and cut as described for

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the mouse tissue except that the TBS used for the human tissue contains only 145 mM sodium chloride. E 1-42 immunocytochemistry on mouse and human sections, and F5 immunocytochemistry on human sections, are carried out as follows. Sections are pretreated with 1% H202 in TBS (100 mM Tris, pH 7.5, 145 mM sodium chloride) for 20 minutes, rinsed for 5 minutes in TBS and 15 minutes in TBSA (TBS plus 0.1% Triton X-100), blocked for 30 minutes in TBS-B (TBS-A plus 2% BSA), and incubated overnight at room temperature in primary antibody diluted 1" 750 in TBS-B. Sections are then rinsed for 15 minutes in TBS-A and 15 minutes in TBS-B, reacted with the secondary antibody (biotinylated goat antirabbit IgG, 1:250 in TBS-B according to manufacturer's specifications for the Elite kit (Vector Labs, Burlingame, CA), rinsed for 15 minutes in TBS-A and 15 minutes in TBS-B, reacted with horseradish peroxidase (HRP)-conjugated avidin-biotin complex (as specified for the Elite kit; Vector Labs), rinsed 3x 5 minutes in TBS, and visualized using Vector Labs HRP substrate (diaminobenzidine) kit. Nickel chloride is included with the chromogen for F5. F5 and F8 immunocytochemistry on mouse brains is carried out as follows. The brain sections are pretreated with 3% H202 in TBS (50 mM Tris, pH 7.5, 300 mM sodium chloride) for 20 minutes, blocked for 1 hour in 0.3% Triton X-100, 20% goat serum in TBS, washed 2• 15 minutes in TBS, and incubated for 40 hours at 4~ in primary antibody diluted 1 : 1000 in 0.1% Triton X-100, 20% goat serum (TBST). Sections are then rinsed 2x 15 minutes in TBS, reacted with the secondary antibody (biotinylated goat antirabbit IgG, 1"250 in TBST according to manufacturer's specifications; Vector Labs), rinsed 2x 15 minutes in TBS, reacted with HRP-conjugated avidin-biotin complex (as specified; Vector Labs), rinsed 2x 15 minutes in TBS, and the reaction is visualized using 0.05% diaminobenzidine (Sigma, St. Louis, MO) plus 0.8% nickel chloride as a chromogen. For all antibodies used, sections incubated in parallel without primary antibody fail to develop any staining. Sections are mounted onto chrom-alum subbed slides, covered, and photographed with Kodak (Rochester, NY) T-MAX film. We do not know whether the /3/A4 immunoreactive aggregates or the abnormal subcellular buildup of the carboxy-terminal portion of/3-APP as revealed by staining with the F5 antibody derive from the endogenous/3APP or from the product of the transgene. This question can begin to be answered if the product of the transgene is tagged, as described previously. With regard to our new FLAG/3-APP-C100 transgenic mice, double staining with the FLAG antibody and either the E 1-42 or the F5 antibody may reveal whether the abnormal immunoreactivity is generated from the product of the transgene.

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We showed previously that the enlarged intracellular organelles into which the F5 immunoreactivity segregates in the disease are probably fused lysosomes (20). The dense F5 immunostaining in these enlarged organelles was particularly prominent in regions of the hippocampus that were heavily invested with pathology, such as Sommer's sector (CA1), in which the heavy staining of the pyramidal cells was accompanied by atrophy of many of these cells (20). Although Nissl and Bielschowsky stains did not reveal gross neuronal death in any of the transgenic brains at 4-6 months of age, it is possible that aggregation of the F5 epitope into enlarged lysosomes may presage neuronal degeneration. To determine whether the lysosomal system is indeed involved in the pathology in our transgenic mice, it is necessary to carry out immunoelectron microscopy to confirm that the F5 aggregates in the transgenic mice, as in AD brain, are fused lysosomes. Enzymatically active lysosomal proteases have been detected in association with senile plaques in Alzheimer's disease, and immunoreactivity for certain of these enzymes is abnormally increased in degenerating neurons in the disease (21). It will be of interest to look, in transgenic mouse models for Alzheimer's disease, for abnormalities of the lysosomal system that resemble those seen in the disease. Thioflavin S histochemistry was employed for identification of amyloid deposits in the mouse and human brains. Sections were incubated for 20 minutes in 1"1 100% ethanol'chloroform, and then rinsed 3x 1 minute in 95% (v/v) ethanol, 3 minutes in 70% (v/v) ethanol, 3 minutes in 50% (v/v) ethanol, 3 minutes in H20. Sections were then incubated for 4 minutes in 1% thioflavin S (w/v) (Sigma) in H20 and differentiated by rinsing in 80% ethanol. The thioflavin S histochemistry showed no abnormal fluorescence of structures in the control mice, or in six of the transgenic mouse lines. However, the mice from the three lines with highest brain expression of the transgene displayed prominent thioflavin S fluorescence around blood vessels, which we also observed in Alzheimer's disease brains stained with thioflavin S. This fluorescence suggests that amyloid has accumulated in the cerebral blood vessels of these transgenic animals. Cytoskeletal alterations, particularly with regard to the phosphorylation state of the microtubule-associated protein tau (r), should be evaluated in any animal model for the effects of amyloidogenic fragments of/3-APP. We have used the monoclonal antibody Alz50 to reveal cytoskeletal pathology in the brains of mice that had received transplants of C100-producing PC12 cells (22). The Alz50 antibody was originally identified on the basis of its selective immunoreactivity with a neuronal antigen in the brains of Alzheimer disease patients (23). A modified form of the microtubule-associated r protein is present in Alzheimer' s disease brain, (24), and Alz50 has been hypothesized to recognize a modification of r that occurs early in the sequence of events

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PARADIGMS OF NEURAL INJURY leading to neurofibrillary degeneration (25, 26). Caution should be exercised, however, when using Alz50 to assess animal models of Alzheimer's disease (27). This antibody stains normal structures in both human and animal brains, so that careful controls must be carried out to show that a particular pattern of Alz50 staining that is suspected to be abnormal is indeed not observed in control brains. Moreover, because it is a mouse monoclonal antibody, an antirodent secondary antibody is normally used in conjunction with it. This antibody will, of course, nonspecifically react with endogenous rodent immunoglobulins, necessitating careful controls for such an artifact. During the past several years, numerous additional antibodies that define pathological phosphorylated epitopes of r and that reciprocally mark the normal lack of phosphorylation at these epitopes have been defined, and are reviewed in two articles (28, 29).

Acknowledgment This work was supported by NIH Grant NS28965 (R.L.N.).

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12. B. E. Pearson and T. K. Choi, Proc. Natl. Acad. Sci. U.S.A. 90, 10578 (1993). 13. J. D. Buxbaum, J. L. Christensen, A. A. Ruefli, P. Greengard, and J. F. Loring, Biochem. Biophys. Res. Commun. 1997, 639 (1993). 14. R. D. Palmiter, E. P. Sandgren, M. R. Avarbock, and D. D. Allen, Proc. Natl. Acad. Sci. U.S.A. 88, 478 (1991). 15. C. Gorman, R. Padmanabhan, and B. H. Howard, Science 221, 551 (1983). 16. R. L. Neve, P. Harris, K. S. Kosik, D. M. Kurnit, and T. D. Donlon, Mol. Brain Res. 1, 271 (1986). 17. K. S. Prickett, D. C. Amberg, and T. P. Hopp, Biotechniques 7, 580 (1989). 18. M. R. Kozlowski, S. F. Spanoyannis, S. P. Manly, S. A. Fidel, and R. L. Neve, J. Neurosci. 12, 1679 (1992). 19. B. J. Cummings, J. H. Su, J. W. Geddes, W. E. Van Nostrand, S. L. Wagner, D. D. Cunningham, and C. W. Cotman, Neuroscience 48, 763 (1992). 20. L. I. Benowitz, W. Rodriguez, P. Paskevich, E. J. Mufson, D. Schenk, and R. L. Neve, Exp. Neurol. 106, 237 (1989). 21. A. M. Cataldo and R. A. Nixon, Proc. Natl. Acad. Sci. U.S.A. 87, 3861 (1990). 22. R. L. Neve, A. Kammesheidt, and C. F. Hohmann, Proc. Natl. Acad. Sci. U.S.A. 89, 3448 1.1992). 23. B. L. Wolozin, A. Pruchnicki, D. W. Dickson, and P. Davies, Science 232, 648 (1986). 24. A. Nieto, I. Correas, E. Montejo de Garcini, and J. Avila, Biochem. Biophys. Res. Commun. 154, 660 (1988). 25. K. Ueda, E. Masliah, T. Saitoh, S. L. Bakalis, H. Scoble, and K. S. Kosik, J. Neurosci. 10, 3295 (1990). 26. V. M.-Y. Lee, B. J. Valin, L. Otvos, and J. Q. Trojanowski, Science 251, 675 (1991). 27. P. Davies, Neurobiol Aging 13, 613 (1992). 28. M. Goedert, Trends Neurosci. 16, 460 (1993). 29. E.-M. Mandelkow and E. Mandelkow, Trends Biochem. Sci. 18, 480 (1993). 30. C. W. Wuenschell, N. Mori, and D. J. Anderson, Neuron 4, 595 (1990). 31. S. Forss-Petter, P. E. Danielson, S. Catsicas, E. Battenberg, J. Price, M. Nerenberg, and J. G. Sutcliffe, Neuron 5, 187 (1990). 32. J. Oberdick, R. J. Smeyne, J. R. Mann, S. Zackson, and J. I. Morgan, Science 248, 223 (1990). 33. D. J. Zack, J. Bennett, Y. Wang, C. Davenport, B. Klaunberg, J. Gearhart, and J. Nathans, Neuron 6, 187 (1991). 34. E. H. Mercer, G. W. Hoyle, R. P. Kaput, R. L. Brinster, and R. D. Palmiter, Neuron 7, 703 (1991). 35. D. M. Donovan, M. Takemura, B. F. O'Hara, and M. T. Brannock, Proc. Natl. Acad. Sci. U.S.A. 89, 2345 (1992). 36. W. C. Friend, S. Clapoff, C. Landry, L. E. Becket, D. O. Hanlon, R. J. Allore, I. R. Brown, A. Marks, J. Roder, and R. J. Dunn, J. Neurosci. 12, 4337 (1992). 37. B. L. Largent, R. G. Sosnowski, and R. R. Reed, J. Neurosci. 13, 300 (1993). 38. S. Morita, K. Kobayashi, T. Mizuguchi, K. Yamada, I. Nagatsu, K. Titani, K. Fujita, H. Hidaka, and T. Nagatsu, Mol. Brain Res. 17, 239 (1993).

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PARADIGMS OF NEURAL INJURY 39. P. Daubas, A.-M. Salmon, M. Zoli, B. Geoffroy, A. Devillers-Thi6ry, A. Bessis, F. M6deville, and J. P. Changeux, Proc. Natl. Acad. Sci. U.S.A. 90, 2237 (1993). 40. B. Ont6niente, P. Horellou, I. Neveu, I. Makeh, F. Suziki, C. Bourdet, G. Grimber, P. Colin, P. Brachet, J. Mallet, P. Briand, and M. Peschanski, Mol. Brain Res. 21, 225 (1994). 41. K. Matsuo, H. Ikeshima, K. Shimoda, A. Umezawa, J. Hata, K. Maejima, H. Nojima, and T. Takano, Mol. Brain Res. 20, 9 (1993). 42. T. G. M. Schmidt and A. Skerra, Prot. Engineering 6, 109 (1993). 43. Y.-T. Chen, C. Holcomb, and H.-P. H. Moore, Proc. Natl. Acad. Sci. U.S.A. 90, 6508 (1993). 44. D. K. Stammers, M. Tisdale, S. Court, V. Parmar, C. Bradley, and C. K. Ross, FEBS Lett. 283, 298 (1991). 45. K. Kuchler, H. G. Dohlman, and J. Thorner, J. Cell Biol. 120, 1203 (1993). 46. V. J. Isola, R. J. Eisenberg, G. R. Siebert, C. J. Heilman, W. C. Wilcox, and G. H. Cohen, J. Virol. 63, 2325 (1989).

[19]

Golgi Technique Used to Study Stress and Glucocorticoid Effects on Hippocampal Neuronal Morphology Ana Marfa Magarifios, Eberhard Fuchs, Gabriele Fltigge, and Bruce S. McEwen

Introduction More than a century ago, Camilo Golgi introduced the "reazione nera," the black reaction, or silver impregnation technique (12), which has become an invaluable tool in helping to understand the morphology of the nervous system. This technique is used to study isolated neurons in order to visualize their dendritic arbor structures, branching patterns, and density of spines. Given the high quality of morphological detail that can be obtained with this method, it also lends itself to being combined with electron microscopic procedures. In this way, it is possible to study the synaptic organization of neurons with great detail (2, 3, 29, 30). For a long time, Golgi's approach faced serious criticism; its unpredictability was the main concern. Today, modified versions of the original technique allow us to obtain much better results. Not only can the Golgi technique visualize normal neuronal structures, but it also permits the identification of atrophic neurons or neurons undergoing plastic change in response to natural stimuli or hormonal manipulations or as a result of certain diseases. Basically, the Golgi technique involves treatment of fixed neural tissue with potassium dichromate, followed by exposure to silver nitrate. The resulting impregnation consists of a reduced silver salt. In contrast, damaged and degenerating neurons are visualized by another technique that is based on the fact that tissue elements in traumatized neurons will catalyze the reduction of silver salts. This method, the silver degeneration technique, takes advantage of this property and labels only damaged cells. This brief review summarizes the uses of the Golgi technique for studies of stress-induced alterations in hippocampal neuronal morphology, but only after first considering the distinction between the modern version of the Golgi method and the methods for studying degenerating neurons and glial cells. Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Degeneration Staining Methods for Neural Tisuse Because the classical Golgi method and the silver degeneration stain provide different information and yet are similar in some respects, it is important to consider how to discriminate between neurons damaged by primary injury in vivo and those damaged by secondary tissue changes induced by alterations in blood flow, oxygen supply, or uneven fixation. Even removing the brain immediately after perfusion can lead to artifacts. Thus, close attention should be paid in order to avoid distortion of the brain as well as to rule out the fact that the injury-induced reactions that produce the affinity for silver may take place in insufficiently fixed brain tissue (4). A recently developed method for silver degeneration staining has demonstrated how to minimize the "dark" neuron reaction as an artifact of brain manipulations at the time of perfusion (11, 31). Using appropriate controls and carefully regulated experimental conditions, it was shown that in normal, untreated brain tissue no cell body axons or dendrites of any neuron type are stained. Furthermore, the silver impregnation method can be potentially useful in detecting and assessing different stages of progressive neuronal damage. Another advantage of this method is the visualization of dendritic arborizations, which provides information about the different neuron types undergoing degeneration. It should be noted that other common histological techniques, such as cresyl violet staining, have been used to demonstrate traumatized neurons. Indeed, these cells are usually characterized by their darkly stained condensed chromatin and light or absent cytoplasm. The fact that the various processes are unable to be observed makes it difficult to recognize different neuronal types. Both cresyl violet and silver degeneration techniques have been used successfully in studying adrenalectomy-induced cell death in the dentate gyrus of the hippocampus in rats (13, 15, 26).

Applications of Single-Section Golgi Method to Investigate Effects of Stress and Glucocorticoids In the remainder of this chapter, we describe our studies on hippocampal morphology using a modification of the single-section Golgi impregnation technique (10). In particular, we examine the effect of chronic stress and glucocorticoid hormones on the structure of neurons in the rat hippocampus. Furthermore, using a model of psychosocial stress, we applied the same histological studies to examine hippocampal neuronal morphology in the tree shrew (Tupaia belangeri). Tree shrews are placental mammals whose brains

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combine primitive as well as primate-like features (18, 20). Because part of the central nervous system of tree shrews resemble primates, this species was formerly considered to be phylogenetically related to primates (14, 17). However, according to more recent evidence, tree shrews are classified as a separate order (Scandetia or Tupaiidae) (27). Confrontations between dominant and subordinate animals proved to induce effectively atrophy of dendrites of hippocampal neurons that are similar to those produced by glucocorticoid administration.

Materials and Methods

Experimental Animals For the chronic-resistant stress studies, male Sprague-Dawley rats (CD strain, Charles River, Kingston, NY), weighing 290-300 g at the beginning of the experiments, are housed in groups of three per hanging metallic cage with ad libitum access to food and tap water. Animals are maintained in a temperature- and light-controlled environment (12/12 hours, light/dark cycle; lights on from 0700 to 1900 hours). After handling the animals daily during 1 week, they are randomly assigned to experimental groups. For the psychosocial stress studies, male tree shrews (German Primate Center, G6ttingen, Germany) are housed individually on a regular day-night cycle (lights on from 0800 to 2000) with water and food ad libitum. Experimental Treatment Groups Restraint Stress in Rats Experiments are performed during the light period of the light-dark cycle. Rats are assigned to the following experimental groups: 1. Unstressed control groupmthe rats remain in their cages except for body weight recordings. 2. Chronic-restraint stress control groupnconsists of 6 hours/day (from 10 a.m. to 4 p.m.) restraint of the rats for 3 weeks in wire mesh restrainers, secured at the head and tail ends with clips. During the restraint sessions, the rats are placed again in their home cages. 3. Drug-treated chronic-restraint stress group--the rats receive daily restraint stress for 21 days as described above. Three different sets of rats receive daily injections of one of the following treatments: (1) 10 mg of corticosterone (CORT) in 250 ~1 of sesame oil, subcutaneously, (2) phenytoin (40 mg/kg), dissolved in propylene glycol, or (3) tianeptine (15 mg/kg), dis-

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solved in propylene glycol. Treatments (2) and (3), are applied prior to the restraint sessions and the injections are given intraperitoneally. Psychosocial Stress in Tree Shrews Removal of the opaque partition between the cages of two males unknown to one another results in an active competition for control of the enlarged territory. After establishment of a stable dominant/subordinate relationship, the two males are separated by a transparent wire mesh. The wire mesh is removed every day for 1-2 hours. Morning urine samples are collected and the animals are weighed daily. This period of psychosocial stress lasts for 28 days. Control animals are housed in separate quarters in the animal facility. [For details see Fuchs et al. (9).]

Single-Section Golgi Staining Procedure Fixation of Brains At the end of the treatment period, the rats are deeply anesthetized with Metofane (Pitman-Moore, Mundelein, IL) and transcardially perfused with 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer with 1.5% (v/v) picric acid. The brains are then postfixed in the perfusate overnight at 4~

Potassium Dichromate Treatment Using a Vibratome, 100-/zm-thick sections are cut into a bath of 3% w/v potassium dichromate dissolved in distilled water. Sections are then processed according to a modified version of the single-section Golgi impregnation procedure (10). Brain sections are then incubated in an aqueous solution of 3% w/v potassium dichromate overnight, at room temperature. The following day, the tissues are rinsed twice--but only for a few s e c o n d s ~ i n distilled water and mounted onto plain slides.

Construction of Assemblies and Silver Nitrate Treatment After mounting tissue sections onto slides, the excess of potassium dichromate is carefully wiped off the tissue edges. A coverslip is glued over the section at the four corners and these assemblies are then incubated in a 1.5% w/v silver nitrate solution dissolved in distilled water overnight and in the dark. The construction of the assemblies is a critical step. If the sections are too wet, the undesired formation of crystals on top of the tissue sections is likely. If, on the other hand, the tissue is dried excessively, not enough potassium dichromate will be available to react with the silver nitrate solution and no impregnation will be evident. Thus, a critical degree of humidity is

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essential in order for the reaction to occur. Another crucial concern is to avoid trapping air bubbles between the tissue and the glass of the assemblies. If this happens, the silver nitrate solution will not diffuse evenly and certain areas will not be impregnated.

Initial Analysis of Impregnation It is possible to follow the progress of the labeling method with a light microscope. A quick examination will give information about the number and density of Golgi-impregnated cells in areas of interest. If the impregnation is not satisfactory, it is still feasible to recycle the sections: the crystals should be removed from the tissue by treating them with 1% sodium thiosulfate three separate times for 15 minutes each with constant agitation, followed by three separate washes in distilled water for 5 minutes each. From our experience, this recycling procedure produces adequate results but the quality of the tissue impregnation is not comparable with that obtained initially. In other words, in the case of quantitative evaluation of dendritic spines we do not recommend the recycling procedure because many structural details are lost and the results can be seriously underestimated. After 2 days of incubation in silver nitrate, the assemblies are carefully dismantled. Then the tissue sections are rinsed in distilled water and dehydrated through an ascending series of ethanol concentrations (95 and 100%). The sections are then defatted in Histoclear (Americlear), mounted onto gelatinized slides, and coverslipped under Permount (Fisher Scientific).

Data Analysis Slides containing brain sections are coded prior to quantitative analysis. This renders the experiment completely blind and the code is not broken until the analysis is completed. In order to be selected for analysis, Golgi-impregnated neurons must possess the following characteristics: (1) a location in the appropriate subregion of the rostral hippocampusunamely, within the region of the area CA3 extending from a point just below the apex of the lateral bend in the pyramidal cell layer to a point directly ventral to the most lateral extension of the upper limb of the dentate granule cell layer; (2) a dark and consistent impregnation throughout the entire length of the dendrites; and (3) relative isolation from neighboring impregnated cells, which could interfere with the analysis. Moreover, in order to avoid a partial picture, only cells located in the middle third of the tissue section are analyzed. For each brain, six to eight pyramidal cells from CA3c are selected. Each selected neuron is traced at 400 x magification using a light microscope with a camera lucida drawing tube attachment. From each pyramidal cell drawing, the number of dendritic branch points of each dendritic tree is determined.

320

PARADIGMS OF N E U R A L INJURY

In addition, the length of the dendrites is determined for both apical and basal dendritic trees using a Zeiss interactive digitizing analysis system. Means are determined for each variable and for each brain. The resulting values are analyzed using a one-way ANOVA with Tukey HSD posthoc comparisons.

Results

Effects of Chronic-Restraint Stress and Glucocorticoids We have used Golgi impregnated tissue to assess the effects of chronicrestraint stress on the structure of the hippocampus. Close attention was paid to the selection of an equal number of the various cell types within each treatment group in order to validate the subsequent comparisons (7). All cells being analyzed exhibited optimal impregnation quality, regardless of the subtype they belonged to, or the degree of atrophy they displayed. CORT treatment during 21 days resulted in significantly fewer apical dendritic branch points in CA3 pyramidal neurons as contrasted with their control counterparts (36). In contrast, no differences in the number of CA3 pyramidal cell basal dendritic branch points were detected with CORT treatment. Quantitative analysis of CA1, CA2, and dentate gyrus neurons revealed no significant differences between treatment groups in their branching patterns. CORT treatment also resulted in a significantly reduced total apical dendritic length in CA3 pyramidal neurons (Fig. 1). Daily restraint stress for 3 weeks induced an identical atrophic effect of hippocampal CA3 pyramidal neurons (33). Once again, this morphologic change was not observed in neurons belonging to other areas of the hippocampus, namely, CA1, CA2, or dentate gyrus. Furthermore, the atrophy was restricted to the apical dendritic tree of CA3 neurons (Fig. 2).

Effects of Pharmacological Treatmets Watanabe et al. (34) showed that daily administration of phenytoin, an antiepileptic drug that interferes with excitatory amino acid release and excitatory synaptic transmission (25), prevented the atrophic branching pattern and the shrinkage in apical dendrites of CA3 area induced by both chronic-restraint stress and CORT treatment (Figs. 1 and 2). Another drug, tianeptine, led to the same result (35); this atypical antidepressant, known for facilitating serotonin uptake (21), was able to interfere with the atrophic effect. Recent results showed that a third drug, the steroid synthesis inhibitor,

[19] GOLGI METHOD: STRESS AND GLUCOCORTICOIDS

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FIG. 1 Effects of corticosterone or corticosterone plus phenytoin on (A) dendritic branch points in CA3c pyramidal neurons of the rat hippocampus and (B) thymus/ body weight ratios (mean +_ SEM); ,, significant difference from control; p < 0.05. Reprinted with permission from Ref. 34. Y. Watanabe, E. Gould, H. A. Cameron, D. C. Daniels, and B. S. McEwen. Phenytoin prevents stress- and corticosteroneinduced atrophy of CA3 pyramidal neurons. Hippocampus 2(4), 431 (1992). Copyright 1992, Churchill Livingstone, New York.

322

PARADIGMS OF NEURAL INJURY

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FIG. 2 Effect of a 21-day daily restraint stress and phenytoin plus stress on dendritic length (A) and dendritic branch points (B) in CA3c pyramidal neurons of the rat hippocampus (mean +_ SEM); ,, significant difference from control; p < 0.05. Reprinted with permission from Ref. 34. Y. Watanabe, E. Gould, H. A. Cameron, D. C. Daniels, and B. S. McEwen. Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons. Hippocampus 2(4), 431 (1992). Copyright 1992, Churchill Livingstone, New York.

[19] GOLGI METHOD: STRESS AND GLUCOCORTICOIDS

323

cyanoketone, also blocked completely the chronic restraint-induced CA3 atrophy, and the apical branching pattern did not differ from control animals (19). Under these conditions basal levels of CORT were not affected but the stress-induced CORT response was blocked.

Effects of Psychosocial Stress After 28 days of daily confrontations, the Golgi impregnation studies showed that the apical trees of CA3 neurons in subordinate tree shrews were atrophic compared to control ones, as judged by the lower number of branch points and the decreased total dendritic length observed (8). In this study we also quantified the spine density in both basal and apical CA3 dendrites but no significant differences were detected between groups.

Discussion The atrophy observed in the hippocampus of either chronically stressed or CORT-treated rats might be considered as a very early pathophysiological transformation of these neurons, because adjacent sections stained with cresyl violet showed no significant number of pyknotic neurons or other signals of neuronal degradation. It could be argued that, because the Golgi technique does not label all neurons contained in a given area and the criteria followed to choose representative cells might rule out neurons that are poorly impregnated and perhaps atrophic, the results might reflect characteristics of a subgroup of nonrepresentative cells. This seems n o t to be the case in view of the Nissl stain results mentioned previously. Moreover, we have evidence employing the same impregnation technique that demonstrates the reversibility of the atrophy following the termination of stress. It is interesting to note that beyond the intrinsic differences in the nature of the stresses applied, two phylogenetically distant species showed the same specific atrophic pattern in response to two qualitatively different types of chronic stressors, namely, restraint and psychosocial confrontation. Yet the same altered morphological change was observed in spite of the fact that the restraint stress led to habituation of CORT secretion whereas the social confrontation seems not to induce tolerance in the cortisol response. In order to understand the physiological significance of this phenomena, we should note that CA3 apical dendrites are the main postsynaptic target for the mossy fibers (MF), which originate in the granule cells of the dentate gyrus (DG). The DG, in turn, receives excitatory amino acid (EAA) input from the entorhinal cortex (EC) (1, 5). Thus, CA3 neurons play a crucial role in mediating excitatory transmission between the EC, the DG, and the

324

PARADIGMS OF NEURAL INJURY CA1 pyramidal cells of the hippocampus. Moreover, numerous studies show that stress and CORT induce excitatory amino acid release and other EAAmediated metabolic actions (16, 22-24, 28). These pathways are known to be involved in aspects of learning and memory (6). In this respect, the morphological changes were correlated with altered performance of rats in solving maze tasks (32). However, not only do CA3 neurons receive EAA transmission through mossy fibers, but hippocampal interneurons are known to be heavily innervated too. Thus, an excess of EAA stimulation inducing atrophy of the postsynaptic elements in CA3 neurons is a p o s s i b l e ~ b u t not sufficient--explanation. In summary, it has yet to be established whether these morphological effects are a consequence of a potentiating role of glucocorticoids on detrimental effects of EAA neurotransmission, a result of the lack of integrity of inhibitory counterbalances, an effect of cortisol/corticosterone on other neurotransmitter systems (GABA, serotonin, etc.), or a protective response, given that a smaller dendritic surface would prevent CA3 pyramidal cells from being excessively stimulated by EAA. Nevertheless, our strategy to investigate and understand how the adult brain deals with stress is increasingly focusing on dendritic remodeling effects and their correlation with behavioral responses.

References 1. S. A. Bayer, in "The Rat Nervous System" (G. Paxinos, ed.), Vol. 1, p. 335. Academic Press, New York, 1985. 2. T. W. Blackstad, Z. Zellforsch 67, 819 (1965). 3. T. W. Blackstad, "The Interneuron" (M. Brazier, ed.), p. 391. UCLA Forum in Medical Sciences, Los Angeles, 1969. 4. J. Cammemeyer, Histochemistry 56, 97 (1978). 5. B. J. Clairbone, D. G. Amaral, and W. H. Cowan, J. Comp. Neurol. 246, 435 (1986). 6. H. Eichenbaum and T. Otto, Behav. Neural Biol. 57, 2 (1992). 7. J. M. Fitch, J. M. Juraska, and L. W. Washington, Brain Res. 479, 105 (1989). 8. E. Fuchs, G. Fl~igge, H. Uno, B. S. McEwen, and A. M. Magarifios, N. Y. Acad. Sci. Abstr. (1994). .

10. 11. 12. 13. 14. 15.

E. Fichs, O. J6hren, and G. FliJgge, Psychoneuroendocrinology 18 (1993). P. L. Gabbott and J. Somogy, J. Neurosci. Methods 11, 221,557 (1984). F. Gallyas, M. Tsu, and G. Buszaki, J. Neurosci. Methods 10, 159 (1994). C. Golgi, Arch. Ital. Biol. 3, 285. (Reprinted in ibid. 97) (1883). E. Gould, C. S. Woolley, and B. S. McEwen, Neuroscience 37, 367 (1990). D. E. Haines and D. R. Swinder, J. Hum. Evol. 1, 407 (1972). D. Jaarsma, F. Postema, and J. Korf, Hippocampus 2(2), 143 (1992).

[19] GOLGI METHOD: STRESS AND GLUCOCORTICOIDS

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16. M. T. Lowy, L. Gault, and B. K. Yamamoto, J. Neurochem. 61, 1957 (1993). 17. W. P. Lucket, "Comparative Biology and Evolutionary Relationship of Tree Shrews." Plenum, New York, 1980. 18. J. S. Lund, D. Fitzpatrick, and A. L. Humphrey, in "Cerebral Cortex" (A. Peters and E. G. Jones, eds.), Vol. 3, p. 157. Plenum, New York, 1985. 19. A. M. Magarifios and B. S. McEwen, Neuroscience 69, 89 (1995). 20. R. B. Martin, in "Primate Origins and Evolution" (R. B. Martin, ed.), p. 191. Chapman and Hall, London, 1990. 21. T. Mennini, E. Mocaer, and S. Garratini, Naunyn-Schmiebergs, Arch. Pharmachol. 336, 478 (1987). 22. D. R. Packan and R. M. Sapolsky Neuroendocrinology 51, 613 (1990). 23. R. M. Sapolsky, "Stress, the Aging Brain and the Mechanisms of Neuron Death." MIT Press, Cambridge, Massachusetts, 1992. 24. R. E. M. C. Schasfoort, L. A. De Bruin, and J. Korf, Brain Res. 475, 58, 1988. 25. J. Skerrit and G. Johnston, Clin. Exp. Pharmacol. Physiol. 10, 527 (1983). 26. R. S. Sloviter, G. Valiquette, G. M. Abrams, C. Ronk, A. I. Sollas, L. A. Paul, and S. A. Neubort, Science 243, 535 (1989). 27. D. Starck, "Vergleichende Anatomie der Wirbeltiere auf Evolutionsbiologischer." (Grundlage, ed.), Vol. 1. Springer-Verlag, Berlin and New York, 1978. 28. B. A. Stein-Behrens, E. M. Elliot, C. A. Miller, J. W. Schilling, R. Newcombe, and R. M. Sapolsky, J. Neurochem. 58, 1730 (1992). 29. W. K. Stell, Anat. Rec. 153, 389 (1965). 30. W. K. Stell, Am. J. Anat. 121, 401 (1967). 31. A. Van den Pol and F. Gallyas, J. Comp. Neurol. 296, 654 (1990). 32. M. Villegas, C. Martinez, V. N. Luine, and B. S. McEwen, Brain Res. 639, 167 (1994). 33. Y. Watanabe, E. Gould, and B. S. McEwen, Brain Res. 588, 341 (1992). 34. Y. Watanabe, E. Gould, H. A. Cameron, D. C. Daniels, and B. S. McEwen, Hippocampus 2(4), 431 (1992). 35. Y. Watanabe, E. Gould, H. A. Cameron, D. C. Daniels, and B. S. McEwen, Eur. J. Pharmacol. 222, 157 (1992). 36. C. S. Woolley, E. Gould, and B. S. McEwen, Brain Res. 531, 225 (1990).

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Index

ACAS 570 interactive laser cytometer, 145-146 N-Acetylaspartate, in normal brain and cerebral ischemia, 215 Acetyl-L-carnitine, effects on Alzheimer's disease brain, in vivo 31p MRS, 205-206 Acidity, cytosolic, and reactive oxygen species generation, 245 Adenylate kinase release, cell injury assessment, 9 Age effects on cerebral glucose/energy metabolism, 132-133 on serotonin neuronal system, 116-117, 121-122 Aluminum chloride, neurotoxicity, astrocyte role, 75-76 Alzheimer's disease /3-APP expression in transgenic mouse brain, 307-312 1H MRS in vitro studies, 203 31p MRS studies acetyl-L-carnitine treatment effects, 205-206 in vitro, 203 in vivo, 204-205 3-Aminobenzamide, effects on NAD + depletion, 14-18 a-Aminohydroxy-5-methyl-4-isoxazoleproionic acid, lesioning of basal forebrain nuclei, 280-282 /3-Amyloid protein precursor, expression in transgenic mice animal maintenance, 303-304 brain amyloidogenic fragments, histological analysis, 307-312 C 100 neurotoxicity, 298 DNA construction, 299-302 polymerase chain reaction, 302-303 protein product detection, 305-307 RNA detection, 305-307 Southern blot analysis, 302-303 Anesthesia, for studies of cerebral glucose/energy metabolism, 132 Antibodies, to/3/A4 fragment, detection of amyloid deposition, 308-309, 308-312 Antioxidants, intracellular, determination, 252-254 J3-APP, see j3-Amyloid protein precursor Apparent diffusion coefficient vs. cerebral blood flow in ischemic cortex, 219-222

changes after ischemia, 223-224 characteristics, 211 comparison with blood flow autoradiography for early stroke assessment, 217-224 threshold determination, 218-219 in vasogenic edema, 224 Arginine, depletion, methods, 40 Ascorbic acid, intracellular, and oxidative status, 253 Astrocytes heavy metal accumulation in cultured astroglia atomic absorption spectroscopy, 136-139 cellular fluorescence imaging, 144-146 gap junctional intercellular communication, 159-164 glutathione content, 146-153 intracellular Ca 2+ measurements, 153-159 kinetics constants, 139-143 mitochondrial membrane potential, 146-153 lead gliotoxicity, 74-75 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine effects, 76-77 role in aluminum chloride neurotoxicity, 7576 Astrogliosis, C6 glial cell models, 69-71 Autoradiography blood flow, comparison with apparent diffusion coefficient, 217-224 for local cerebral glucose utilization, 129 Avian sarcoma virus-induced glioma model (rodent), 85-87 Avidin-biotin technique, for cultured cells, 47

Basal forebrain, cholinergic lesions cholinergic immunotoxins, 284-286 ethylcholine aziridinium, 282-284 excitotoxins, 280-282 general procedures, 278-279 mechanical damage, 279-280 Benzoic acid, hydroxylation, for reactive oxygen species determination, 249-250 Biochemical markers A2B5, 58 galactocerebroside, 47, 58-59 glial fibrillary acidic protein, 47, 58-59

327

328

INDEX

glutamine synthetase, 58, 66-67 myelin basic protein, 47 3',5'-cyclic-nucleotide phosphohydrolase, 58, 66-67 transferrin, 47 vimentin, 58 Blood flow, cerebral vs. apparent diffusion coefficient in ischemic cortex, 219-222 autoradiography, comparison with apparent diffusion coefficient, 217-224 correlation with diffusion-weighted imaging, 231-235 and diffusion-weighted imaging hyperintensity, 231-235 flow indices, calculation, 228-227 mismatch with diffusion-weighted imaging, 222 Bulbectomy, olfactory effects on p75 NGFRexpression in olfactory bulb, 102-104

procedure, 98 Buthionine sulfoximine, inhibition of glutamine synthesis, 173-174, 254

genes regulating apoptosis, 2 Calcium, intracellular, measurement interpretation, 156-159 limitations, 156-159 method, 155-156 rationale for, 153-155 0-Carotene, intracellular, and oxidative status, 253 Catalase, role in reactive oxygen species detoxification, 253 Cell cultures, primary neurons nitric-oxide synthase expression, 32-33 procedure, 30-32 tissue culture plate preparation, 28 Cell death assays, cell counting, 34-35 reactive oxygen species in, 3-7 Cell lines developed from nitrosourea-induced glioma (rodent), 88 PC12, nerve growth factor effects on oxidant injury (rat pheochromocytoma), 10-19 Cell survival, assessment methods, 8-9 Cell viability, assessment methods, 8-9 Cerebral blood flow, see Blood flow, cerebral C a e n o r h a b d i t i s elegans,

Cerebral glucose/energy metabolism, see Glucose/ energy metabolism, cerebral Cerebral perfusion pressure, control of cerebral blood flow, 124-125 Chemiluminescence method, Western analysis, 270 Cholinergic lesions, basal forebrain cholinergic immunotoxins, 284-286 ethylcholine aziridinium, 282-284 excitotoxins, 280-282 general procedures, 278-279 mechanical damage, 279-280 Cholinergic neurons, aspirative transection of timbria fornix materials, 109-110 surgical procedure, 110-112 Chronic-restraint stress, effects on hippocampal morphology, 320 Ciliary neurotrophic factor, induction of astrogliosis, 272-273 Copper, 67Cu(II) efflux from glial cells kinetic parameters, 142 time dependence, 142 uptake by glial cells kinetic parameters, 140-141 time dependence, 140 Correlation time, in magnetic resonance spectroscopy, 186 Corticosterone, effects on hippocampal morphology, 320-321 Cresyl violet staining, 316 Crosslinking, protein-phospholipid, 6 Cyanoketone, effects on hippocampal morphology, 323 Cyclic GMP, for nitric oxide formation in cultures, 36 Cyclin D1. expression in dying cells, 2 Cystathionase, assay, 170 Cysteine bound, assay, 169-170 in central nervous system, 167-168 chemical forms in vivo, 169 metabolic pathway, 168 total, assay, 169-170 Cytokines, injection into CNS animal preparation, 262 immunocytochemistry, 266-269 model system, 261-262 procedure, 262-264 tissue fixation, 265-266

INDEX tracer dyes, 262 Western analysis, 269-272 Cytomegalovirus promoter, for/3-gal gene transfection, 291 Cytometer, interactive laser, ACAS 570, 145-146 Cytotoxicity, glial cell aluminum chloride effects, 75-76 C6 cells, 59-71 ethanol effects, 72-74 lead effects, 74-75 methylmercuric chloride effects, 77-78 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine effects, 76-77 primary cultures, 56-59 Dichlorofluorescein, assay of reactive oxygen, 249 Diffusion-weighted imaging, s e e Magnetic resonance imaging, diffusion-weighted 5,7-Dihydroxytryptamine, serotonergic lesions applications, 121-122 experimental design, 117-118 functional impact of denervation, 121 intracerebral, 120-121 intraventricular, 118-120 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide reduction applications, 9 in PC12 cells, 10 DNA damage fragmentation internucleosomal, 3 during oxidant injury in PC12 cells, 13-14 poly(ADP-ribose) synthase activation, 38 strand breakage, 6-7 Dye-exclusion measurements, of cell viability, 9 Dyes, tracer, for cytokine injections, 262 Edema, vasogenic, 224 Eicosanoids, production, 245-247 Electron spin resonance detection of free radical species, 248-250 low molecular weight iron compounds, 254 Energy homeostasis, reactive oxygen species effects, 6-7, 13 Energy metabolism, cerebral, s e e Glucose/energy metabolism, cerebral Enzyme-linked immunosorbent assay, nerve growth factor after liposome-mediated transfection, 294-295 Enzymes, mitigating oxidative damage, 252-254

329 Ethanol, gliotoxicity, 72-74 Ethylcholine aziridinium, lesioning of basal forebrain nuclei, 282-284 N-Ethylnitrosourea, glioma model (rodent), 81-85 Excitotoxins interactions with reactive oxygen species, 3 lesioning of basal forebrain nuclei, 280-282 Fenton reaction, OH (hydroxyl radical) formation, 4 Fimbria fornix, aspirative transection, 108-109 materials, 109-110 surgical procedure, 110-112 Fixation, tissue cryoprotection, 265-266 perfusion technique, 265 sectioning, 265-266 Fluorescence imaging ACAS 570 interactive laser cytometer, 145-146 for cell viability, 9-10, 35 rational for, 144 Fluorometry glutathione assay, 253-254 tryptophan content of solubilized proteins, 252 Free induction decay, 181-182 Freeze-thaw methods C6 glial cells, 61-66 oligodendroglia data interpretation, 52-53 experimental procedures, 45-48 immunocytochemical analysis of cultures, 50-52 observation of samples, 48-50 Galactocerebroside markers, 47 in reanimated glial cultures, immunofluorescence, 50-53 Gap junctional intercellular communication, measurement interpretation, 162-164 method, 159-161 rationale for, 159-160 Genes, C a e n o r h a b d i t i s e l e g a n s , role in apoptosis, 2 Gene transfer, liposome-mediated cytomegalovirus promoters, 291 nerve growth factor expression characteristics, 293-296 transfection efficiency, 291-293

330

INDEX

Glial cells biochemical markers, 57-59 C6 glioma cell passage procedure, 60-61 early passage, 67-69 freezing, 61-63 glial phenotype characterization, 66-67 late passage, 67-69 thawing, 63-66 oligodendrocytes, freeze-thaw method data interpretation, 52-53 freezing medium, 45 immunocytochemical characterization, 47-48, 50-52 medium, 46 preparation, 45 preparation for freezing, 45 reanimation, 46 sample fixation, 50 sample observation, 48-50 primary cultures E3H-derived immunocytochemical characterization, 58-59, 62-63 preparation, 57 E15CC-derived immunocytochemical characterization, 58-59, 62-63 preparation, 57 mixed astrocyte-oligodendrocyte, 56-57 Glial fibrillary acidic protein expression after cytokine injection into CNS immunocytochemistry, 267-269 Western analysis, 269-272 markers, 47 in reanimated glial cultures, immunofluorescence, 50-53 Glioma models, rodent avian sarcoma virus, 85-87 cell lines developed from, 88 nitrosourea compounds, 81-85 transplantation, 87-92 Glucocorticoids, effects on hippocampal morphology, 320 Glucose/energy metabolism, cerebral age effects, 132-133 anesthetics, 132 biochemical analyses, 130-131 blood flow control mechanisms, 124-125 enzyme activities, 131-132

in experimental animals, 129-132 in humans, 125-129 in vivo tissue studies, 129-130 Kety-Schmidt technique, 126-127 nuclear magnetic resonance in experimental animals, 131 in humans, 128 physiological steady state, 125 positron emission tomographic techniques, 127-128 postmortem studies, 128-129 substrate utilization, 129 Glutamate neurotoxicity, NO-initiated, model for, 28 y-Glutamyltransferase, assay, 252 Glutathione chemical forms in vivo, 169 cytosolic, analysis, 146-150, 152-153 high-performance liquid chromatography assay, 172-173 inhibitors, 173-174 intracellular, and oxidative status, 253 metabolism, 171 role in protection, 171 sources, 170-171 spectrophotometric assay, 172 tissue levels, determination, 254 Glutathione peroxidase in oxidative injury, 5 role in reactive oxygen species detoxification, 253 Golgi method, single-section staining procedure, 318-320 Greiss reaction, for nitric oxide formation in cultures, 36 Heavy metals, effects on cultured astrogial cells atomic absorption spectroscopy, 136-139 cytosolic glutathione content, 146-150, 152-153 fluorescence imaging, 144-146 gap junctional intercellular communication, 159-164 intracellular Ca 2§ content, 153-159 mitochondrial membrane potential, 146-148, 150-152 transport kinetics, 139-143 Hemoglobin, reduction, 41 High-performance liquid chromatography biochemical analysis of cerebral glucose/energy metabolism, 130-131

INDEX cysteine, 173 glutathione, 172-173 Hippocampus, neuronal morphology chronic-restraint stress effects, 320 glucocorticoid effects, 320 pharmacological treatment effects, 320-323 psychosocial stress effects, 323 single-section Golgi staining, 318-320 Hydrogen peroxide, toxicity in PC12 cells, nerve growth factor effects, 11-18 Ibotenic acid, lesioning of basal forebrain nuclei, 280-282 Immunocytochemistry amyloidogenic fragments of fl-amyloid protein precursor expression in transgenic mouse brains, 307-312 C6 glial cells, 67 characterization of reanimated oligodendroglia, 47-52 ED1 monoclonal antibody expression after cytokine injection, 267-269 E3H- and E15CC-derived cultures, 58-59, 62-63 glial fibrillary acidic protein expression after cytokine injection, 267-269 Immunofluorescence, characterization of reanimated oligodendroglia, 47-52 Immunohistochemistry, p75 NGFRexpression in olfactory system after bulbectomy, 98-101, 103-104 Immunotoxins, 192IgG-saporin conjugate, lesioning of basal forebrain nuclei, 284-286 Indicator-dilution method, for mean transit time measurement, 226-227 In vitro injury models, oligodendroglia freeze-thaw method, 45-47 immunocytochemical characterization, 47-53 Ion regulation, reactive oxygen species effects, 6 Iron compounds, low molecular weight assay, 254 in neurological disorders, 244 Ischemic penumbra and chemical shift imaging spectroscopy, 215-217 therapeutic window, 210-211 Kety-Schmidt technique, 126-127 Kinetics constants interpretations, 142-143 limitations, 142-143

331 measurement methods, 140-142 rationale for measurement of, 139-140 k-space substitution method, 227-228 Lactate cerebral tissue, in stroke, 183-184 proton resonances, 183 Lactate dehydrogenase, release for cell injury assessment, 9 in PC12 cells, 10 Larmor frequency expression for, 180 field dependence, 182 Lead accumulation in cultured astroglia atomic absorption spectroscopy, 136-139 cellular fluorescence imaging, 144-146 gap junctional intercellular communication, 159-164 glutathione content, 146-153 mitochondrial membrane potential, 146-153 gliotoxicity, 74-75 Lipid peroxidation, cytotoxic effects, 6 Lipofusin, formation, 251 Magnetic moment, nuclear, 178-179 Magnetic resonance imaging contrast agents, 227 diffusion-weighted, 211-213, 218 correlation with cerebral blood flow, 231-235 mismatch with cerebral blood flow, 222-223 sensitivity and specificity, 218 in vivo glioma metabolism (human), 91 perfusion techniques, 226 cerebral blood flow-diffusion-weighted imaging hyperintensity, 231-235 contrast agents, 227 diffusion-weighted spin-echo image, 226 flow indices, 228-227 k-space substitution, 227-228 relative mean transit time and cerebral blood flow images, 226-228 region of interest analysis, 230-231 Magnetic resonance spectroscopy applications Alzheimer's disease brain, 203-206 freeze-clamped Fischer 344 rat brain, 201-203 chemical shifts N-acetylaspartate in cerebral ischemia, 215 applications, 183

332

INDEX

and ischemic penumbra, 215-216 lactate in stroke, 213-215 lactate resonances, 183-184 correlation time, 186 free induction decay, 181-182 information contained in spectrum, 186-187 in vitro

quantitation of spectra, 194-195 signal identification, 193-194 in vivo

quantitation of spectra, 195-197 signal identification, 194 spatial localization depth-pulse technique, 190-191 spin-echo technique, 191-193 Larmor frequency, 180 line broadening chemical exchange, 188-189 correlation time effects, 190 field inhomogeneity, 188 magnetic field effects, 179-180 nuclear magnetic moment, 178-179 nuclear spin types, 179 radio frequency pulse application, 179-180 resonance frequency of a nucleus, 185 sample preparation, 197 saturation of nuclear magnetization, 181 sensitivity, 187 signal identification, in vitro spectra, 193-194 spin coupling, 187 spin relaxation, 185-186 Magnetic resonance spectroscopy, 1H instrumental conditions, 198 in vitro, Alzheimer's disease brain, 203 perchloric acid of brain tissue, 183 Magnetic resonance spectroscopy, 31p freeze-clamped Fischer 344 rat brain, 201-203 instrumental conditions, 197-198 in vitro

Alzheimer's disease brain, 203 perchloric acid of brain tissue, 184 in vivo

acetyl-L-carnitine treatment of Alzheimer's disease brain, 205-206 Alzheimer's disease brain, 204-205 with depth-pulse localization technique, 198-199 with spin-echo localization technique, 199-200 with spin-echo pulse sequence, 191-193

Mean arterial blood pressure, control of cerebral blood flow, 124-125 Mean transit time, blood through brain, indicatordilution method, 226-227 Membrane potential, mitochondrial, analysis, 150-153 Messenger RNA, nerve growth factor, after liposome-mediated transfection, 294 Metal ions, multivalent, 245 N-Methyl-D-aspartate, neurotoxicity, attenuation, 27-30 N-Methyl-D-aspartic acid, lesioning of basal forebrain nuclei, 280-282 Methylmercuric chloride, gliotoxicity, 77-78 N-Methylnitrosourea, glioma model (rodent), 81-85 1-Methyl-4-phenyl- 1,2,3,6-tetrahydropyridine, Parkinsonism-inducing effects, astrocyte role, 76-77 Mitochondria function, cytotoxicity assays for, 35 membrane potential, analysis, 150-153 superoxide anion production, 3-4 Monochlorobimane, reaction with glutathione, 253-254 Monoclonal antibodies 192IgG-saporin conjugate, lesioning of basal forebrain nuclei, 284-286 ED1, immunoreactivity after cytokine injection into CNS, 267-269 04, 47, 51-52 Myelin basic protein markers, 47 in reanimated glial cultures, immunofluorescence, 50-53

NAD + depletion nerve growth factor effects, 13-18 in NO-initiated glutamate neurotoxicity, 28 poly(ADP-ribose) polymerase inhibitor effects, 13-18 NADPH-diphorase stain, for nitric-oxide synthase in primary neuronal cultures, 32-33, 39 Nerve growth factor effects on oxidant injury in PC12 cells, 10-19 expression after liposome-mediated gene transfer, 293-296 liposome-mediated gene transfer in septohippocampal cultures, 290-293

INDEX Nerve growth factor receptor p75 NGFRexpression, unilateral bulbectomy effects bulbectomy procedure, 98, 102 immunohistochemistry, 98-101, 103-104 intranasal irrigation, 98, 101-102 P75 NGFRinteraction with neurotrophins, 1-2 Neurotoxicity aluminum chloride, astrocyte role, 75-76 fl-amyloid protein precursor C100, 298 glutamate, NO-initiated, 28 N-methyl-D-aspartate, attenuation, 27-30 and peroxynitrite formation, 28 Nitric oxide chemistry, 36-37 donor reagents, 41 formation, levels of regulation, 27 targets, 37-38 toxicity, in primary neuronal cultures cell death assay, 34-35 exposure, 33-34, 39-40 nitric-oxide synthase activation, 36 valence states, 36-37 Nitric-oxide synthase activation characterization, 27 criteria for, 36 in primary neuronal cultures, NADPH-diphorase stain for, 32-33, 39 Nitrosourea glioma models (rodent), 81-85 Nuclear magnetic resonance, techniques for cerebral glucose/energy metabolism in experimental animals, 131-132 ex vivo in vitro technique, 131 in humans, 128 Nuclei, properties, magnetic resonance spectroscopy-related, 180 Nucleus basalis magnocellularis, lesioning cholinergic immunotoxins, 284-286 ethylcholine aziridinium, 282-284 excitotoxins, 280-282 general procedures, 278-279 mechanical damage, 279-280 Oligodendrocytes glial cultures, freeze-thaw method data interpretation, 52-53 freezing medium, 45 immunocytochemical characterization, 47-48, 50-52

333 medium, 46 preparation, 45 preparation for freezing, 45 reanimation, 46 sample fixation, 50 sample observation, 48-50 lead gliotoxicity, 75 Oxidant injury experimental induction methods, 8 PC12 cells, nerve growth factor effects, 10-19 Oxidases, role in reactive oxygen species generation, 247 Oxidative stress, quantifiable parameters, 245-247 Partition pressure of carbon dioxide in arterial blood (paCO2), 124-125 Perchloric acid extract 1H MRS spectrum, 183 lap MRS in vitro spectrum, 184 31p MRS in vivo spectrum, 184 Peroxynitrite formation, and neurotoxicity, 28 synthesis, 42 Phagocytosis, role in reactive oxygen species generation, 247 Phenytoin, effects on hippocampal morphology, 320 pH levels, and reactive oxygen species generation, 245 Phosphorylation, oxidative, 245 Platelet-activating factor, C6 glial cell response, 69-71 Poly(ADP-ribose) polymerase activation, DNA damage-related, 38 inhibitors, effects on NAD + depletion, 13-18 Polyclonal antibodies, for Western analysis, 270-271 Polymerase chain reaction reverse transcription, s e e Reverse transcriptionpolymerase chain reaction transgene detection, 302-303 Positron emission tomography, techniques for cerebral glucose/energy metabolism, 127-128 Promoters cytomegalovirus, for fl-gal gene transfection, 291 neural, tested in transgenic mice, 300 Prooxidant status direct assays with electron spin resonance, 248-250

334

INDEX

dyes for, 244 indirect indices, 252-254 oxidation products of cellular constituents, 250-252 Propargylglycine, inhibition of glutamine synthesis, 174 Psychosocial stress, effects on hippocampal morphology, 323 Quinolinic acid, lesioning of basal forebrain nuclei, 280-282 Quisqualic acid, lesioning of basal forebrain nuclei, 280-282 Radio frequency pulse, application in magnetic resonance spectroscopy, 179-180 Radiolabeled precursors, for cell viability, 9-10 Reactive oxygen species cellular effects, 6-7 direct assay, 248-249 benzoate hydroxylation, 250 with dichlorofluorescein, 249 endogenous sources, 3-4, 245-247 cytosolic activity, 245 eicosanoid production, 245-247 metal ions with multivalence potential, 245 oxidases, 247 oxidative phosphorylation, 245 phagocytosis, 247 enzymes mitigating oxidative damage, 252-254 estimation using oxidation products of cellular constituents, 250-252 glutamine synthase assay, 252 protein tryptophan residue degradation, 252 exogenous sources, 4 interactions with excitotoxins, 3 intracellular antioxidant determination, 252-254 metabolism, 4-5 Resonance, defined, 180 Reverse transcription-polymerase chain reaction C100 transgene RNA and protein products, 305-307 nerve growth factor mRNA, 294 Ribonucleic acid, C 100 transgene, RT-PCT detection, 305-307 RT-PCR, s e e Reverse transcription-polymerase chain reaction Saporin, 192IgG-saporin conjugate, lesioning of basal forebrain nuclei, 284-286

Saturation of nuclear magnetization, 181 Septohippocampal cell cultures liposome-mediated gene transfer in, 290-293 nerve growth factor expression after transfection, 293-296 Serotonergic neurons, 5,7-dihydroxytryptamineinduced lesioning experimental design, 117-118 functional impact of denervation, 121 intracerebral, 120-121 intraventricular, 118-120 Silver degeneration staining, 316 Singlet oxygen, sources, 8 Southern blotting, transgene detection, 302-303 Spectrophotometry for biochemical analysis of cerebral glucose/energy metabolism, 130-131 glutathione assay, 172 Spin, nuclear, 179 Spin coupling, characteristics, 187 SPINECHO research pulse sequence, 192 Spin-lattice relaxation, characterization, 185 Spin-spin relaxation, characterization, 186 Stroke, ischemic early diagnosis/assessment apparent diffusion coefficient and blood flow autoradiography, 217-224 factors affecting, 209-210 early treatment, 210-211 entry time, 210-211 ischemic penumbra, 210-211 magnetic resonance imaging techniques animal model, 225-226 diffusion-weighted imaging, 211-213 magnetic resonance spectroscopy, 213-217 perfusion imaging, 224-235 therapeutic window, 210-211 Superoxide anion, production in mitochondria, 3-4 Superoxide dismutase, role in reactive oxygen species detoxification, 253 Superparamagnetic iron oxide contrast agent, 227

Tetrazolium salt reduction, for cell viability assessment, 9 Thanatins, characterization, 2 Therapeutic window, in ischemic stroke, 210-211 Thiol oxidation, protein inactivation by, 6 Tianeptine, effects on hippocampal morphology, 320

INDEX a-Tocopherol, intracellular, and oxidative status, 253 Transection, aspirative, fimbria fornix, 108-109 materials, 109-110 surgical procedure, 110-112 Transferrin markers, 47 in reanimated oligodendroglial cells, immunofluorescence, 50-53 Transgenic mice,/3-APP expression animal maintenance, 303-304 DNA construction, 299-302 polymerase chain reaction, 302-303 protein product detection, 305-307

335 RNA detection, 305-307 Southern blot analysis, 302-303 Transplantation glioma models (rodent), 87-92 Transverse relaxation, 186 Triton X-100, intranasal irrigation with, 98, 101-102 Tryptophan residues, determination, 252 Valence states, of nitric oxide, 36-37 Vasogenic edema, apparent diffusion coefficient, 224 Western blotting, glial fibrillary acidic protein expression after cytokine injection, 269-272

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FIG. 2 Double immunofluorescence of primary glial cultures. (A and B) Cultures 2 weeks after reanimation; (C and D) nonfrozen 4-week-old sister cultures. Reanimated cultures were characterized by the presence of GC + cells, the majority in clusters (A); these cells had cell processes. Single GC + cells are seen in B (again no processes observed). The presence of GFAP + cells with long processes was very frequent (45% approximately). Magnification: x200.

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FIG. 3 Triple immunofluorescence for the expression of GFAP (green), Tf (red), and 0 4 (blue) in oligodendroglial cultures 3 days after reanimation. (A-C) Cultures fed with DF-10; (D-F) sister cultures fed with GDM. Cells in DF-10 were flat and without distinct cell processes. 100% of the cells in the cultures expressed GFAP, Tf, and 0 4 at different intensities. Diffuse staining was observed for both GFAP and 0 4 whereas Tf + vesicles were frequently seen on the cell membrane or extracellularly (arrows). Cells fed with the glial development medium also shared the expression of the three antigens, GFAP, Tf, and 04. However, the cell morphology was different from DF-10 cells. (D) Weak expression of GFAP at the level of the cell body. Interestingly, 0 4 and Tf staining revealed the preservation of myelinlike sheaths. Both antigens were expressed in the cell by all the cells as well as in membranous cell processes (arrowheads). Magnification: x200. Other cells had modest cytoplasmic extensions (small arrows).

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