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ADVANCES IN SPACE BIOLOGY AND MEDICINE

Volume 7

1999

This Page Intentionally Left Blank

ADVANCES IN SPACE BIOLOGY AND MEDICINE Editor:

SJOERD L. BONTINC Goor, The Netherlands

VOLUME 7

1999

JAI PRESS INC. Stamford, Connecticut

Copyright 0 1999 /A/PRESS INC. 100 Prospect Street Stamford, Connecticut 06901

All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 0-7623-0393-X Manufactured

in

the United States of America

CONTENTS LIST OF CONTRIBUTORS

vii

INTRODUCTION TO VOLUME 7 Sjoerd L. Bonting

xi

Chapter 1 SURVEY OF STUDIES ON HOW SPACEFLIGHT AFFECTS RODENT SKELETAL MUSCLE Monika B. Fejtek and Richard J . Wassersug

1

Chapter 2 IS SKELETAL MUSCLE READY FOR LONGTERM SPACEFLIGHT AND RETURN TO GRAVITY? Danny A. Riley

31

Chapter 3 NUTRITIONAND MUSCLE LOSS IN HUMANS DURING SPACE F L IGHT T P Stein

49

Chapter 4 HORMONAL CHANGES IN HUMANS DURING SPACE F L ICHT Felice Strollo

99

Chapter 5 GROWING CROPS FOR SPACE EXPLORERS ON THE MOON, MARS, OR IN SPACE Frank B. Salisbury

131

Chapter 6 ELECTROPHORESIS IN SPACE )ohann Bauer, Wesley C. Hymer, Dennis R. Morrison, Hidesaburo Kobayashi, Ceoffry b!E Seaman, and Cerhard Weber

163

Chapter 7 TEACHING OF SPACE LIFE SCIENCES Didier A. Schmitt, Pierre Frangon, and Peter H.U. Lee

21 3

V

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS lohann Bauer

Max Planck lnstitut fur Biochemie Martinsried, Germany

Monika B. Fejtek

Department of Anatomy and Neurobiology Dalhousie University Halifax, Nova Scotia, Canada

Pierre FranGon

International Space University France

Wesley C. Hymer

Center for Cell Research Penn State University University Park, Pennsylvania

Hidesaburo Kobayashi

Department of Chemistry Josai University Sakado, Japan

Peter H. U. Lee

Brown University School of Medicine Providence, Rhode Island

Dennis R. Morrison

NASA, Johnson Space Center Houston, Texas

Danny A.Riley

Department of Cellular Biology and Anatomy Medical College of Wisconsin Milwaukee, Wisconsin

Frank B. Salisbury

Department of Plants, Soils, and Biometeorology Utah State University Logan, Utah

Didier A.Schmitt

European Space Agency, ESTEC Noorwij k, the Netherlands

vi i

...

LIST OF CONTRIBUTORS

Vlll

Ceoffry VE Seaman

Emerald Diagnostic Eugene, Oregon

IF? Stein

Department of Surgery University of Medicine and Dentistry of New Jersey Stratford, New Jersey

Felice Strollo

Postgraduate School of Aerospace Medicine University "La Sapienza" and Endocrine and Metabolic Department Italian National Research Centers on Aging Rome, Italy

Richard 1. Wassersug

Department of Anatomy and Neurobiology Dalhousie University Halifax, Nova Scotia, Canada

Cerhard Weber

GmbH, Klausnerring 1 7 Kirchheim, Germany

INTRODUCTION TO VOLUME 7

This is being written just after receiving the joyful news that the first part of the International Space Station has been successfully launched. When, after many more launches, the station will be complete and fully operational in the year 2004, a new era will begin for space life sciences research with greatly expanded opportunities for high quality experiments. During the past several years there has been a shortage of flight opportunities for biological and medical projects. And those that were available usually had severe restrictions on instrumentation, number of subjects, duration, time allotted for performing the experiments, and possibility for repetition of experiments. It is our hope and expectation that this will change once the International Space Station is in full operation. The advantages of a permanent space station, already demonstrated by the Russian Mir station, are continuous availability of expert crew and a wide range of equipment, possibility of long-term experiments where this is warranted, increased numbers of subjects through larger laboratory space, proper controls in the large 1-G centrifuge, easier repeatability of experiments when needed. The limited number of flight opportunities during recent years probably explains why it has taken so long to acquire a sufficient number of high quality contributions for this seventh volume of Advances in Space Biology and Medicine. While initially the series was aimed at annually appearing volumes, we are now down to a biannual appearance. Hopefully, it will be possible to return to ix

X

INTRODUCTION

annual volumes in the future when results from space station experimentation are beginning to pour in. Meanwhi I e, the purpose of the series remains unchanged. As explained in the Introduction to the first volume, this purpose is (1) to bring authoritative reviews of the findings and accomplishments in this field to a wider group of scientists than the relatively small group of biologists and physiologists currently involved in space experimentation; (2) to cover the entire field of biology, human (incl. medical aspects), animal, plant, cell and molecular; (3) to appeal to the wider life science community by discussing not only theproblemsinvestigatedandtheresultsobtained, but also some of the technical aspects and limitations peculiar to space research. The present volume would seem to satisfy this threefold purpose. The first three chapters deal with muscle. Fejtek and Wassersug provide a survey of all studies on muscle of rodents flown in space, and include an interesting demography of this aspect of space research. Riley reviews our current knowledge of the effects of longterm spaceflight and reentry on skeletal muscle, and considers the questions still to be answered before we can be satisfied that long-term space missions, such as on the space station, can be safely undertaken. Stein reviews our understanding of the nutritional and hormonal aspects of muscle loss in spaceflight, and concludes that the protein loss in space could be deleterious to health during flight and after return. Strollo summarizes our understanding of the major endocrine systems on the ground, then considers what we know about their functioning in space, concluding that there is much to be learned about the changes taking place during spaceflight. The many problems of providing life support (oxygen regeneration and food supply) during extended stay on the Moon, on Mars, or in space by means of plant cultivation are discussed by Salisbury. The challenges of utilizing electrophoresis in microgravity for the separation of cells and proteins are illustrated and explained by Bauer and colleagues. Finally, the chapter on teaching of space life sciences by Schmitt shows that this field of science has come of age, but also that its multidisciplinary character poses interesting challenges to teaching it. I gratefully acknowledge the invaluable support of Dr. Augusto Cogoli, Zurich, Switzerland, in bringing out this volume. He has handled the acquisition of the contributions and the correspondence with the authors, gently pushing them to submit their chapters within a reasonable length of time. It was our intention that after this volume he would take over the editorship. Unfortunately, the publisher has decided to discontinue the series because of low sales figures. In ending the editorship of the series, I wish to thank all contributors to the seven volumes brought out. I have carried out this task in the conviction that it is important to have a regular series of indepth review articles on space biology and medicine. Hopefully, with this research getting in full swing on the International Space Station it may be possible to resurrect the series. Sjoerd L. Bonting Editor

Chapter 1

SURVEY OF STUDIES ON HOW SPACE FLIGHT AFFECTS RODENT SKELETAL MUSCLE

Monika B. Fejtek and Richard J. Wassersug ....................

2

........................ . . . . . . . . . . . . . . 11

A. Biological Considerations . . . . . . . . . . . . . . . . . . B. Demographic Considerations . . V. Conclusions . . . . . . ........................

. . . . . . . . . . . . . . 23 References . . . . . . . . .

Advances in Space Biology and Medicine, Volume 7, pages 1-30. Copyright 0 1999 by JAl Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

1

MONIKA B. FEJTEK and RICHARD J. WASSERSUG

2

Progress, j u r ,from consisting of change, depends on retentiveness.. .those who cunnot renzember the past are condemned to,fulfil it.

--George Santayana ( I 863- 1952)

1.

INTRODUCTION

We have undertaken a comprehensive survey of the published literature on rodents exposed to microgravity in spaceflight experiments. The purpose of this survey was twofold. Firstly, we were interested in learning what was known about the effects of exposure to microgravity on the histology of rodent skeletal muscle. This was meant to serve as background to our own investigation of body wall muscle from rats that flew on NASA's Space Shuttle in July of 1995.' The second purpose of this study was to examine a variety of issues on the demography of science that pertains to biological research in space. All research in space can be distinguished from ground-based studies by the high cost of the endeavor. By current estimates, it costs in excess of $lOK to place one kilogram of any material, biological or otherwise, into orbit. This enormous cost continually draws attention to space exploration and legitimately leads to speculation, if not cynicism, about whether the rewards are commensurate with the effort. Our survey provides a database for beginning to examine in an objective way what we have learned from space exploration within one well-defined area of biology, that is, rodent myology-the scientific study of rodent muscles. We have reviewed virtually all the primary scientific literature on space research concerning rodent myology, from its inception as a subdiscipline of space biology approximately a quarter of a century ago. Ironically, the single item which makes space biology expensive-the high cost of access to space-also makes topics within the discipline sufficiently circumscribed that the literature can be reviewed in toto. Thus, our first goal was to determine, as objectively as possible, what we have learned from spaceflight experiments on rodent muscle. It is immediately clear from our survey that only a small percentage of rodent skeletal muscles have actually been examined after spaceflight. These few muscles, however, have been studied in detail, both functionally and morphologically. Several reviews have summarized the results of these studies showing that microgravity does indeed affect rodent skeletal muscle, most notably causing atrophy of muscle t i ~ s u e . ~ . ~ However, our survey shows that there is also much variation in experimental parameters such as flight duration, type and size of animal enclosure, and time to postflight data collection. We demonstrate here that the variation in these parameters can affect the biological results. Furthermore, we show that this large variance in experimental design prohibits scientists from pooling the results of different spaceflight missions in a manner that might promote deeper insight into the time course of muscle deconditioning during flight and muscle recovery postflight.

Table 7. Sllldj &

(Refi

Flighr & Yew

Invesngutor.\

1 Cosmo\ 60.5 Ilyind-Kakuevaet (6.7) 1973 u1. (Russia)

2

Cosmo\605

(8)

1973

Literature Summary of Spaceflight Effects on Rodent Skeletal Muscle

MI \ston Lengrh (Day,)

Aninid Encloure

Enclosure Size

Control.!

21.5

BIOS-l

S Individuali

Chamber

10x20 cm cylinder

Simulation (X). Vivarium (8)

Savik & Rokhlenko (Rusiia)

22.5?

Neiterov & Tigranyan (Russia) Oganesyan & Eloyan (Russia)

227

BIOS-1 Chainher

(#)

5 Individual\ Synchronous (1 I?), 10x20 cm Vivarium (I17)

Nuniber & sex of

Srmrn, Age & Flight Kurr Body Wetght IS

W1star

Mule

7 7

ll

Wurur

Mule

1

Synchronous (?), Vivarium ( 7 )

12 Male

Wistar

Synchronous (?), Vivarium ('?), Centrifuge (?I

1

Mule

Pmtjlrghf Collernon (Hour$)

48 (X) 648 (7) _

I

Musclec Examined

Fiber CSA

SDN

GPD

Other

Soleus

U red,

U.

fi.

U mass. U MDH.

mtermed X X NIA

X. X NIA

X. X. NIA

U mas\

EDL MG, QF. BB, Dia Soleu\ 48 i'?) 648 (?)

cylinder

3 (9)

Cosmos605 1973

4 (10)

Cosmoi605 1973

5 (11)

Co\mo\605 1973

Portugalov & Petrova (Runia)

21,s

6 (12)

Cosmos605 1973

Kazaryan el ul. (Rusk) Baranski & Marcrniak (Poland)

21.5

22'?

BIOS-I Chamher BIOS-I Chamber

5 Individual\ 10x20 cm cylinder 5 Individuals 10x20 cm cylinder

'?

200-250 g Wistar ? >

48 (7) 648 ( 5 ) 48 (?) 648 (7)

BlOS-1 Chamher

5 Individuals 10x20 cm cylinder

Simulation (8, 7 ) . Vivarium ( S O )

14 Male

Wistur

?

48 ( 8 ) 648 (6)

BIOS-I Chamber

5 Individuals 10x20 cm cylinder 5 Individuals 10x20 cm cylinder

Simulation (lo), Vivarium (20)

10 Male

Wistar ?

48 ( 5 ) 648 ( 5 )

Gastrocnemiur DIaphragm Plantarn Gastrocnemius Triceps, QF. "Poiterior thigh," Semi-mh, FDE Biceps hrachii Soleus Plantarii

X.

n permeability, edema of capillaries: atrophy; necrori\. X. no changes Na', K ' . ~ g ' + ,~ a ' +in any of the muscles. I? cathepsin activity (48 F, C).

NIA

NIA

NIA

NIA

NIA

NIA

NIA

NIA

NIA

U LDH,,?, I? LDH4,5. LDH, (48 F), U LDH3,4, fi LDH,

NIA

NIA

NIA

U m a s , U stroina,

X

(648 F).

7 (13)

Cosmos 690'! 1974

21"

BIOS-l Chamber

I

Vivarium (15)

1s

Wistar

Male

7

"lmmediately"?

Soleus

EDL Soleus

fi T prot. fi stroma. NIA

NIA

NIA

U volume sarcomerc. mitochondria, SER, \yn ves; I? glycogen.

-200 g Quudriceps

U volume SER, syn ves

Diaphragm "Posterior thigh'

U volume syn ves. NIA

NIA

NIA

U pho\pholipids (24 F);

Quadricepi

NIA

NIA

NIA

U T prot, n ATPase (24,

(red, white fib).

8 (14) 9 (15, 16)

Co\iuo\690 1974 Co\mo\690

1974

Belitskaya (Russia) Gayenkaya e f a/. (Runla)

20.5

20.5

BIOS 1 Chamber BIOS-1 Chamher

5 Individuals 10x20 cm cylinder 5 Individuals 10x20 cm cylinder

Synchronou\ (?), Vivarium ('?)

? Rad

Wisfur

MUlP

7

24 (?) 624 ( 7 )

Simulation ('?Rad), Vivarium (?)

? Rad

Wivur

Male

?

fi pho\pholipids (624 S).

1

24 ('1) 624 ('?)

7

Soleus Gastrocnemiui

624 Siin), fi LDH4,5 sup (Sim). U \ap, T prot, atm: fi LDH sap, AST activ?. no change glvcoeen

Table 7. StUdJ

(RefJ

Fltgkt & Year

10 (17)

Costno\ 690 1974

&

11 (18)

Co\mo\ 690 1974

12 Cosmos782 (cited 1975 in 19) 13 Co\mo\7X2 (20) 1975

14 (8)

Cosmos 782 1975

Investrgntor\

Nesterov & Tigranyan (Rus\ia) Ilyina-Kakueva & Portugalov (Russia)

Baranski et a/. (Poland) Marciniak (Poland)

Mission Length (Daysj

20.5

20.5

19.5

21?

(Continued)

Number Animal Enclo\ure

Enclosure Size

Conrrols

& Sex of

Strain, Age &

(#i

Flight Rat,!

Body Weight

BIOS-I Chamber

5 Individuals 10x20 cm cylinder

Svnchronous ( 8 Radl, Vivarium (22)

10 Rad Male

Wistar

BIOS-I

5 Individuals

Chamber

10x20 cm cylinder

BlOS-l Chamber BIOS-I Chamber

Savik & Rokhlenko (Russia) Ushakov ef a/. (Russia)

19.5

19.5

BIOS-l Chamber

BlOS-1 Chamher

BIOS-I Chamber

15 (21)

Cosmos782 1975

16 (20)

Cosmos936 1977

Marciniak (Poland)

21’!

17

Co\rnor916 1977

BIOS-l Chamber

18 (22)

Co\mos 936 1977

Baranski ec u l (Poland) Vlasova ef ul. (Russia)

18.5

(19)

18.5

BIOS-I Chamber

5 Individuals 10x20 cm cylinder 5 Individuals 10x20 cm cylinder

Simulation (6 Rad), Vivarium (12)

6 Rad Male

Wisfur 7

?

Male

Other

NIA

NIA

NIA

u K+INa+ 124 F).

24 (’?) 624 (?)

Diaphragm Soleus

U red, intermed

u

n

Gastrocnemius

X.

X

X.

X.

X

X.

?.

7

7.

?

Quadriceps

NIA

NIA

NIA

4-6 (’?) 600 (?)

11

Wistar ? ?

Diaphragm Soleus

NIA

NIA

NIA

Gastrocnemiu: Quedriceps

NIA

NIA

NIA

I1 Male

Wistar 63 days -212 8

8-10(6) 600 ( 5 )

7

Wi\tar 62 days -200 g Wistar 62 days 200 g Wistar 62 duys 215 g

?

Soleu\

NIA

NIA

NIA

”Immediately”?

Soleus

NIA

NIA

NIA

6 600

Quadriceps

NIA

NIA

NIA

Malc

Vivarium (?)

’! Malc

Synchronous (9). Vivarium (’!), Onboard Centrifuge (?)

7

Mule

t K+Nd+, U H20m l k g (24 F). X. U HBD, U NADHD, U fib diam, endomysium, fi connective tissue. U fiber diam, Ti connective tissue. X. “slight changes in m u d e fibers & axon endings of NMJ.” U re1 VOI, #, area/vol of syn ves in axon ends. U re1 YOI. # syn ves & # mito in axon ends. U # functional capillaries: atrophy. X. iso. leu, val, tyr, phe, thn, gly (8-10 F); fi phe, asp, glu (8-10 S ) ; U iso, leu, thn, tyr, glu. rer (600 S). re1 VOI. #, area/vol syn ves; re1 v o ~area/ , vol mito i n axon ends. U #, vol mitochondria, glycogen, myofih vol. altered NMJ. U iso, leu, val, thn, scr, met, tyr, phe, asp. glu, gly (6 F); ser. met, asp, thn, glu (600 F); U iso, leu, met, tyr, phe. glu, pro (6 C).

n

EDL, BB Quadriceps Diaphragm

Mule

(7)

GPD

Soleus Plantaris

8-10

10x20 cm Vivarium ( I I ? ) cylinder 5 Individuals Synchronous (1 I), 10x20 cm Vivarium (9) cylinder

Vivarium

SDH

24 110) 624 (5)

Wistar 63 days -212 g Wistar 63 days -200 g

5 Individual\ Synchronous (1 I?)

5 Individuals 1 Ox20 cm cylinder 5 Individuals 10x20 cm cylinder 5 Individuals 10x20 cni cylinder

Fiber CSA

?

Mule Vivarium (?)

Muscles Examined

200-250 g

7

?

1

Postflighr Collection (Hours)

n

u

U

n

u

n

u

19 (23)

Cosmos 936

20

Cosmos936 1977

(24, 25j 21

(26)

22 (27)

1977

Cosmo\ 936 1977

Cosmm 936 1977

23 Cosmos 1 I29 (28) 1979

24 Co\mos 1 129 (29) 1979

Oganov er u / (Russia)

18.5

Chui& Cast I ema n (USA) Ne\terov et al. (Russia)

18.5

Nosova er ril. (Russia)

Oganov er a/. (Russia)

Szilagyi er rrl (Hungfly)

18.5

18.5

18.5

18.5

BIOS-I Chamher

5 Individuals Synchronous (5.6) 10x20 cm Vivarium (5.10) cylinder

BIOS-1 Chamber

5 Individuals

BIOS-I Chamber

BIOS-I Chamber

BIOS-2 Chamber

B10S-2 Chamber

10x20 cm cylinder 5 Individuals 10x20 cm cylinder

5 Individuals 10x20 cm cylinder

Synchronoo\ ( 5 ) , Vivarium ( 5 ) Synchronous (?), Vivarium (?), Onboard Centrifuge (?) Synchronous ( S ) , Vivarium ( 5 ) , Onboard Centrifuge ( 5 )

group of 10 Synchronous (7). 66x22~16cm Vivarium (?)

group of 10 66~22x16cm

Synchronous (?), Vivarium (?)

4-5

Male

5

Male ?

Male

5 Male

Wisrur 62 d a j s 215 g

5-9 600

wicrar 62 days 215 g Wistar 62 day\ 215 g

'\everal('

Wistur 62 dajs 215 g

6 600

6 600

Soleus Brachiali\ Triceps EDL EDL

NIA

NIA

NIA

V PU, V force. fi PO. fi force? (F, S ) U PU, ti force'!.

U fat

NIA

N/A

Gastrocnemius

NIA

N/A

NIA

Tibialis anterior Quadriceps Soleus

fiber diameter, no significant #slow fibers seen. fi RP metah, fl G6PD. fl 6PGD. fl GAPD (6 F, S, C), fl TK (6 S , C) fi 6PGD (6 F, S , C).

NIA

N/A

NIA

fl LDH, ALT (6 F, C);

X.

X.

ll atm (600 F, c), cyto AST (600 F). fi cyto AST, U LDH (6 C); iimito AST (600 F)

Quadriceps

1

Male

'?

Male

Wistar 85 d a y -250 g

6 696

Wistar 85 days 300-360 g

6 144 696

X.

Gastrocnemius Soleus

NIA

NIA

NIA

Brachialis EPL Triceps Soleus

N/A

NIA

NIA

U ma% (F, S),fi Po, U force, fl MLC (fa\t). V mass (F, s). U mass (F, s). b inass (F, S), U force. U mass, U con (6, 144 F; 6 S),

Brachiah\

mass,

fi MLC (fast). V con (6, 144

F: 6 S), U MLC (fast).

u mass, u con (6 F, S),

EDL Triceps

25

Cosmos I129

(30)

1979

Mailyan ef a / (Rusia)

18.5

BIOS-2 Chamber

group of 10 Synchronous (6-8) 66122x16 crn Vivarium (6-8)

6-8 Mule

Wistar 85 dajs 300-360 g

10 144

"Posterior thigh

696 Ouadriceos

N/A

N/A

N/A

fl MLC (fast). Umass(to696),Ucon(6, 144 F: 6 S ) , ti MLC. coef, rate OP; resp control, fi time OP (10 F); U rate OP, fl time OP (144 F) (mito sus). X. X.

U

(conllnrred)

(Continued)

Table 1.

26 Comas 1129 (31) 1979

Takac\ el ul. (Hungary)

18.5

BIOS-2

group of 10

Synchronou\ (‘’1,

7

Wl.51 U I

6

Soleu\ Brachiah\

NIA

NIA

NIA

n MLC (fast/ilow F, S) MLC n h1l-C

EDL Tricep\

27 Co\mo\ I129 (32) 1979

Rapcrak ef a/. (Hungaiyj

18.5

BIOS-2 Chambcr

group of 10 66~22x16cm

Synchronous (3, Vivarium (”1

7

Male

Wi\tar K5 duly -300 g

6 144 696

Soleu.

(fdct),

NIA

NIA

NIP.

Chui & Castlcman (USA1

18.5

29 Comas 1514 (34) 1983

Rapc\ak er a / . (Hungary)

5

30 Co\mos 1514 (35) 19x3 31

(36381

Cosmos 1514 1983

Mailyin era/. (Rus\ia)

5

Holy& Mouiuer (France)

5

BIOS-2 Chamber

BIOS-2 Chambei-

group of 13 Synchronou\ (25). 6 6 x 2 2 ~ 1 6cm Vivarium ( 2 5 )

group of 10 66x22~16cm

Synchronour (71, Vivarium (7)

25 Mule

7 Femalc

Wistar 85 dms

6 (7). 144 (h), 144 +

Gastrocnemiu5

iminob. (7). 696 ( 5 )

W i . 3 mr

’?

Soleus. Bra Tricep\ M G , EDL “Posterior thigh

prepnant G I 4 350 g

BIOS-2 Chanibcr

group of 10 66~22x16cm

Synchronou\ (51, Vivarium ( 5 )

5 Fcinale

W,rr‘rr pregnant G I 4

I0

BIOS-2 Chamher

group of lo? 66x22~16cm

Synchronous (5). Vivarium ( S ? )

ti

W15tar pre,qnanr G14 288 g’?

6

Female

fi slow, fast

NIA

NIA

NIA

NIA

(6 F)

250.300 g

NIA

Bahakoba el ill.

(Ru%ia)

7

BIOS-2 Chamber

group of 10” 66x22~16cm

Synchronow I?), ? Vivarium (’!) Mali,

Diaphragm Soleu\ Ga\lrocnemiu\ Dbaphragm

u

u

u n m \ . V,. b V,, u mT (F, S). X.

NIA

NIA

NIA

U mitochondrial protein

N/A

NIA

NIA

U

(F), c o d ~r rate OP, re\piratory rate (s). Uman mass, U fiber diamcter, & mT, Po (Fl; U CaBA (S). d mass, mT. X. synaptic coiilact. depenlregen in NMJ. \ynaptic reconrtruct. synaptic recon\truct.

n

Solell\ LG

n

X.

u

u

Planlain5

32 Co\mos 1667 1985 (39)

(dow F. S). U mas\, b mT (6, 144 F, S), V, (6, 144 F). ma% mT, V, (6 F, S). U ma\\ (6 F, S). U ma\\, U mT, U V, (6, 144 F. S). \low fiber\ located in 3 regions, U \low/fa\t ratio

u

Brachiah\ EDL Tricep\

2X Co\mo\ 1129 (25, 1979 331

nMLC(fa\t P, S)

U MLC (fast). ll MLC

NIA

NIA

NIA

33

Co\mo\ 1667

(34)

1985

Rapcwh PI

a/.

BIOS-2 Chanrhei

group of 10'' 6 6 x 2 2 ~ 1 6ciii

Synchronou, ( 7 ) . V l \ a n u m (7)

7

Mi,\irir

Male

100 drrj5 320-150 g

(Hungary)

34 Co\nio\ 1667 (36 19x5 38)

Holy & Mounlei iFrancc)

BIOS-2 Chamher

proiip of 10 6 6 x 2 2 ~ 1 6cin

Synchionow (71, Vivauum ( 7 )

7 Male

Wi\rai I00 d u x 298 g"

6

Dcsplanche\

UIOS-2 Chamher

group of l o ? 6 6 x 2 2 ~ 1 6cm

Synchninou\ ("), 2r Vivarium ( ? )

7 Male

wistar 100 J u s 310.350 g

4-8

PI

ill.

(France) BIOS-2 Chamber

group of 10 6 6 x 2 2 ~ 1 6cm

Simulation (7)". Vivarium ( 7 ) ?

7 Male

Wi\tar 100 days -132 g

3-8

Spacelab 3 51-R 19x5

Rilcy t t id. (USA)

RAHF

24 individuals 10.5x11 5x28 cm

Simulaiion (7)

7 Male

12-16 (Sim 60-64)

Spacelab3 51-B 1985

Martin ef a1

24 indix'iduak 10.5xl l.Sx28 cm

Simulation ( 6 )

SpmgueDawley 85 day\'? 382 g Sprugue-

Co\mo\ 1667

1985

37 (42, 33) 38

RAHP

(USA)

6 Male

Soleu,. Bra

Triccp MG, E D 1 Solcur I .c Plantari\ Diaphragm Soleu\ (41)

Soleus Gastrocncmiu5 PlanLari5 Tricep. B5, Bra EDL. OF Soleus

EDL 11-17

Dowlej 50 days -252 g"

Soleus Adducror longu\ Plantan\ EDL

39 (45 -47)

Spacelab 3

Muyacchia

51-B 1985

era/.

40

Spacelab 3

(48, 49)

51-5 1985

RAHF

(USA)

Steffen & Musacchia (US'%)

RAHF

24 individuals 10 5x11.5x28 cm

Siinulation ( 5 ) . BodySuipended (10)

24 individuals Simulalion ( 7 ) 10.5~11.5~28 cm

5

Male

7 Male

N/A

NIA

NIA

SpragueDawley 85 day\'! 360-410g

12

SpragucDawley 85 d q s ? 360-410 g

12

NIA

NIA

NIA

NIA

NIA

NIA

NIA

NIA

NIA

X

U tih h a m , U Po U mas\. U mas\ X.

U ma\\. U V ' T ~ P CI , Ir % ~ y p eIla. U m a s , U V ~ T Y I,~ C U HAD activity.

u ma\\.

V ma\\. U ma\\. U mass. X.

U SO, FOG. uS0,FOC. FC. U light. dark ATPd\e. light. dark.

u

k light, dnrk.

X. X X

U mass.

fl Iighl,

U mass, % dark ATPa\e. & ma\\, R % dark

dark. fl light. dark. X.

tl hght

ATPax X.

4TPase X.

NIA

NIA

Soleui

uslow-

ED1

twitch. fast-twitch. U fa\ttwitch. NIA

NIA

fi mas. mitochondria, fl TAP, n CAP.

X.

IJ light, dtrk. fl dark

U light ATPasc

Ge\trocncmius

U mT (F. S). X.

NIA

MG

Soleus

u ma\\. b v,. b v,, X.

EDL ( 7 )

Garenko Cf ol. (Russia)

36 (41)

(44)

4-8

NIA

n

U mass.

umas

u

m a s , fi fiber & capillary density (fast).

U mass, riher X capillary den\ity (fast). II protein. DNA, U RNA. U aag/arp. glmiglu, gly. hi\, ly\. RNA, U plmiglu

n

u

Table 7. Miston Length

Stdy & (Ref.)

41 (501

Fligki & Yeor

Spacelab 3 51-B 1985

lnvesrrwiifors

(Day,)

An~mul Eniloure

Hennkwn

7

RAHF

Enclo~ure Size

24 indi\idual\

Conrrols

i#J Simulation ( 6 )

et iil

(Continued)

Number & S r x I$ Strain, Age & Fliwht Rafr Aodi Wrwhi

-

6 Mule

cm

(USA)

SpmgueDawle)

PosfflrRhf Collection iHour.sl

Mus~lr\ Examrned

Fiber CSA

SDH

CPU

Ofher

12

Soleus Gastrocuemiu\ Plantaris EDL T i b l a h anterior Soleus

N/A

NIA

N/A

TI tyrome, 11 glycogen 11 growth, fi glycogen

, J

42 (51)

Spacelab 3 51-B 1Y85

Ti\chler rf a/. (USA)

7

RAHF

24 individuals Simulation (6) 10.5~11.5~28 cm

6 MUlP

Sprugue Dawlej ?

12

U growth,

NIA

NIA

NIA

)

U

Plantans

43

Martin (Canada)

7

(52)

Spacelab 3 51-B 1985

Manche\ter et a/. (USA) Miu ef al. (USA)

12.5

12.5

Riley ef nl. (USA)

12.5

44

Cosmos 1887

(53)

1987

45

Co\mo\ 1887 1987

(54,

24 individuals Simulation ( 6 ) 10.5~11.5~28 cm

6 Male

Sprugue

BIOS-2 Chamhei

group of I0 66x22~16cin

Synchronou\ (2)

2 Male

48

BIOS-2 Chamber

group of 10 66x22~16cm

Synchronous ( 5 )

Wistur 84 days -300 g Wufar 6'4 days -300 g

BIOS-2 Chamber

group of 10 66x22~16cm

Basal (?), Vivarium ( q ) , Synchronous (3)?

Wistur 84 days -300 g

48

RAHF

5

Male

55)

46 Covnos 1885 (56 1987 -58)

47 Cosmo.; IR8i (59, 1987 60)

Baldwin Pf ul. (USA)

12.5

BIOS-2 Chamber

group of 10 66x22~16cm

Synchronous (3, Vivarium (5)

3? Male

5

Male

11-17

Dawlej 50 days 382 g

Wistx 84 day r -300 g

48

48

U

NIA

N/A

N/A

'?.

NIA

NIA

u

4 light, total intermed, activity dark ATPa\e. X. MG LIight, dark. Soleiis b. N/A Adductor longus U FG 7. Soleus

u

Plantaris EDL Vastus intermed Vastus lateralis

U

U

Gastrocnemius

EDL Tibialir anterior Soleus Adductor longus Plantan.; Tihialis anterior EDL MG Soleus Tibyah\ anterior

glycogen. glycogen. Tt glycogen. U ala, U glm, U glu, asp, U mal, TI glm/gIu U glm, glu, U mal, iT glmiglu. b glm, glu, U asp, iT glm/glu. U glm, U g ~ u . glu, TI gIm/glu. U mass, protein, HP. U m a s , proLein, [HP]. U mas\, protein, HP. U mass, protein U mass, protein, HP. U mass. TI HK?. iT HK, A oxidative enzymes?. 11 9% intermed ATPa\e fibers, 11 atrophy.

u growth.

U. UFOG. NIA

N/A NIA NIA

fi mean activity (dark ATPase). X.

X.

N/A

11 necrotic fibers.

11 c/o intermed, u 9% SO,

U mitochondria1 area. X. X

NIA

U myofih protein, U isomyosin, l? ATPase. l? Q intermed, 9% fast.

u

48 Co\mos 1887 (61, 1987 62) 49 Coarnos 1887 (633) 1987

Musacchia er ul. (USA) Holy er al. (France)

12.5

SO Cosmo\ 1887 (64) 1987

Desplanches et a/. (France)

12 5

51 Cosmos 1887 (65) 1987

Bell er ul. (Canada)

12.5

12.5

BIOS-2 Chambcr

group of 10 66x22~16cm

BIOS-2 Chamber

group of 10 66x22~16cm

BIOS-2 Chamber

group of 10 66x22~16em

BIOS-2 Chamber

group of 10 66x22~16cm

Basal ( 5 ) , Vibarium ( S ) , Sqnchronous ( 5 ) Synchronou\ (?)

5

W,r1nr

Mule

84 d q s -300 g Wi\tar 84 drqs -300 g Wistar 84 days -330 g

7

Male Synchronou\ (S), Vivarium ( 5 )

Synchronou\ ( 5 )

5 Male

5 Male

wistu,. 84 dujs -300 g

48

X.

Vastus medialis

NIA

NIA

fl capillary density”.

U LPL?. 48

Soleu\

NIA

NIA

NIA

48

Plantaris Soleu\

U Type 1,

NIA

NIA

X.

EDL 48

Soleus

IIx, IIb. NIA

A capillary density,

U # caplfiber

IIa, IIc.

U Type I, IIa, IIb. U Type IIa

Plantari\

u fiber diam. Po,

n fast MLC, TnC?.

u # caplfiber. u # caplfiber. D mcan

NIA

NIA

il\ynaptic contact, degedregen in NMJ. synaptic reconstruct. synaptic reconstruct. U mass (TS), PO (F). U mass (F, s). U mass (F, TS, S) U mass (TS, s). U mass (F, S) U mass (F, s),PO (F). A capillary density.

activity

so, u

activity & distrib

52

Cosmos 1887 (39) 1987

Babakova er al. (Russia)

12.5

Oganov el a/. (Russia)

12.5

54 Cosmos 1887 Ilyina-Kakueva

12.5

53 (66)

(67)

Cosmos 1887 1987

1987

55 Cosmo\ 1887 (68) 1987

56 (69, 70)

Cosmos 2044 1989

(Russia)

BIOS-2 Chamber

group of 10 66x22~16em

Synchronms (?>, Vivuriurn ( 7 )

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

Basal ( 5 ) , Vivarium (7). Synchronous ( 5 ) , Suspended (7)

BIOS-2 Chamber

group of 10 66x22~16cm

Synchronous ( 5 ) Vivarium ( 5 )

Booth et ul (USA)

12.5

BIOS-2 Chamber

Musacchia ef al. (USA)

14

BIOS-2 Chamber

Basal ( S ) , Synchronous ( 5 ) Vivarium ( 5 ) group of 10 Basal ( 5 ) , 66x22~16cm Vivarium ( S ) , Synchronous ( 5 ) Tail-Suspended ( 5 )

group of 10 66x22~16cm

? Mule

Wistar 84 duys -300 g

48

48

5

Wtslnr

Male

84 days -300 g

5 Male

Wistar 84 days -334 g

5 Mule 5

Male

Wfstar 84 day,\ -330 8 Wistar 109 days -321 g

Soleu\

NIA

FOG. NIA

NIA

NIA

NIA

NIA

NIA

48

Gastrocnemiu\ Diaphragm Soleus Triceps LG MG Biceps brachii EDL Soleus

42

U Ia, 11, intermed. X. U Type 1Ic X. NIA

NIA

Gastrocnemius Quadriceps Biceps brachii Triceps

NIA

NIA

X. X. U mass.

7-12

Vastus medialis

k Type I (F,

NIA

NIA

fl fiber density,

u

TS), Type I1 (F)

n capillary density

A triglycerides, A LDH activity (connnued)

Table 1. Mission Lenglh

StLldj &

Night

(RefJ

& Year

Muscles Exumined

Fiber CSA

SDH

Wistar 109 davs 330 ,q

8-12

Soleus

U slow fihers

u total

5 Male

Wistar 109 days -321 g

8-12

5 Male

Wistar 109 days -321 g Wistar 109 days 330 g Wistar 109 days 330 g

8-12

Tihialis anterior Soleus

8-9

Soleus

U (F, TS)

8-11

Tibialis anterior Adductor longus

U SO (F,

Wlstur I09 days 330 g

8-12

EDL Vastus intermed

tSO(F) NIA

NIA

Wistar 109 days 330 g WIStaI 3 months? -330 g

8-11

LG Triceps Adductor longus

NIA

NIA

8-12

Soleus

NIA

NIA

Wistar IOY days 330 g

8-12

LG EDL Soleus

NIA

NIA

Enclo,ure Sire

Controls

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

Synchronous ( 5 ) . HindlimbSuspended ( 5 )

5 Male

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

Synchronous ( 5 ) . HindlimbSuspended ( 5 )

14

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

14

BIOS-2 Chamber

Synchronou\ ( 5 ) . HindlimbSuspended ( 5 ) groupof 10 Synchronous (2), 66x22~16cm Tail-Suspended (2)

14

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

(DuysJ

57 Cosmos 2044 (71. 1989 72)

Ohira er al. (USA1

14

58 Cosmos 2044 (73) 1989

Jiang et al. (USA)

14

59 Co\mo\2044 (74) 1989

Talmadge et ul. (USA) Chi et al. (USA)

61 Co?mo\2044 (76 1989 -78)

Riley et al. (USA)

62 Cosmos2044 (79, 1989 80)

Thomason rt 01.

63 Cosmo\2044 (61) 1989

Postflight Collection (Hours)

Animul Enclosure

lnvectrgntors

60 Cosmos 2044 (75) 1989

(Continued)

Nu,flhel& Sex of Flight Rut,

14

BIOS-2 Chamber

(#J

Basal (3, Vivarium (51, Synchronous ( 5 ) Tail-Suspended ( 5 )

group of 10 Synchronous (5?), 66x22~16cm Tail-Suspended

2 Male

5 Mule

57

Male

(52

(USA) 14

BIOS-2 Chamber

group of I0 6 6 x 2 2 ~1 6cm

64 Cosmos 2044 (82, 1989 83)

Daunton ef ul. (USA) Stevens er ul. (France)

14

BIOS-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

65 Cosmos 2044 (84) 1989

Lowry et ul. (USA)

14

BIOS-2 Chamber

Basal (3, Vivarium ( 5 ) , Synchronous ( 5 ) Synchronous ( 5 )

groupof 10 Synchronous ( 2 ) , 6 6 x 2 2 ~1 6cm Tail-Suspended (2)

5 Male

5 Male

2 Mule

Strain, Age & Body Weight

MG

Plantaris

Tibialis anterior

GPD

total (F, HS). activity activity slow fibers fast fibers (F, HSJ. (HS) X. U intermed U fast, U total fibers (F, activity HS). (HS). X. X. X NIA NIA NIA

NIA

X

NIA

n

Other

U % slow fibers. t % intermed fibers (F)

U mass, Ti fast ATPase (FJ.

X.

n 8 total Type IIx (F,

HSJ, U % fiber Type I, IIa (F, HS). (F, TS). 8 mass, t HK (F, TS), d 3KA (FJ. X. 7. % SO (F)?. NIA

U

TS) SO (F. TSJ

X

NIA

B TPH activity. U =-actin mRNA (F, TS), U cyt-c mRNA (F), fl cyt-c mRNA (TS). U =-act mRNA (F. TS).

X. myofiber atrophy & dismay, necro\i\, altered NMJ. U fiber diameter, NIA Po, CaBA fast. U Po, U CaBA. X l? (F, TS). U fiber size (F, TS), HK (F, TS), U 3KA (F). fi Type IIa, PFK Type I, IIb (F)?, IIb (F, TSJ. thiolase (F)”

NIA

U

U

n

n

U

66 Cosmo\ 2044 (85) 1989

Oganov ef a/. (Russia)

67 Cosmos 2044 IIyina-Kakueva & (86) 1989 Burkovskaya (Russia) 68 Cosrno\ 2044 (87 1989 -89) 69

Cosmos2044 1989

Stauber er a/.

14

14

BIOS-2 Chamber

14

BIOS-2 Chamber

14

BIOS-2 Chambei

(USA)

Baldwin er u/ (USA)

B10S-2 Chamber

group of 10 6 6 x 2 2 ~1 6cm

Sy h ro n ouh ( 5 ) . Vivarium ( 5 ) , Tail-Suspended ( 5 )

group of 10 Synchronous (5 Inj). 66~22x16 cm Vivarium ( 5 Inj), Tail-Suspended ( 5 InJ) group of 10 Synchronous ( 5 Inj), 6 6 x 2 2 ~1 6cm Vivarium ( 5 Inj), Tail-Suspended (5 I j ) , Basal ( 5 ) group of I0 Synchronous (5), 6 6 x 2 2 ~1 6cm Vivarium (3, Tail-Suspended ( 5 )

5

wirur

MU/?

109 duy\ 330 g

NIA

5 Male

WlStaI 109 days

“immediately”?

Vastus intermed

NIA

N/A

X.

330 8

group of 5 24.5X43.7X51 cm

Simulation (6)

6 Male

PSE-1 STS-41 1990

Backup er ul. (USA)

4

AEM

group of 5 Vivarium‘! (1 2) 24.5~43.7~51

8 Male

SLS-1 STS-40 1991

Baldwin

SLS-I STS-40 1991

Haddad

cm

et al.

(USA)

20 Male

cm 9

AEM

group of 5 Vivarium (20) 2 4 . 5 ~ 4 3 . 7 1~ 5

cm

NIA

NlA

AEM

(USA)

EDL Brachialis Soleus

NlA

4

24 individuals Vivarium (20) 10.5x11.5~28

(F, TS),

Gastrocnemius

Simulation (51, 5 , 6 + GH group of 5 24.5~43.7~51 Simulation + GH (6) Male

20 Male

SpragueDawley 35 days 125-135 g SpragueDawley 35 days 125-135 g SpragueDawley 35 days -120 g SpragueDawley ? 80 g SpragueDawley ? 80 g

li mas), F,, fiber diam

U V, (TS)

Gastrocnemius Triceps

8-12

Tidball & Quan (USA)

RAHF

N/A

Wistar 109 d a y 330 g

PSE-I STS-41 1990

9

NIA

5 Inj Male

cin

et ul.

NIA

4-7?

hang e r a / . (USA)

AEM

Soleus

Wistar 109 dam -300 g

5 hJ Male

PSE-1 STS-41 1990

4

8-12

4.5-6

NIA

NIA

Vastus lateralis

X,

Vastus medialis

U (TS)

Soleus

U(F,Fi

X.

N/A

GH).

U mass (F)’). U reparation area & new fiber thickness (F, TS). fi macrophages, blood vessels (F)?, iT mast cells (F, s)?,iT myofiber repair (TS)?. fi ATPase (F), fi % slow myosin (TS). 8mass (TS), fi myofiber protein (F). U mass, myo prot (F), ?I slow myosin (TS). U mass (F, F + GH), fi % intermed (3/5F).

U MTJICSA,

4.5-6

Plantari\

NIA

NIA

NIA

4-6

Biceps hrachii

NIA

NIA

NIA

no change GAP mRNA. U actin mRNA?.

6 (10) 216(10)

Vastus intermed Vastus lateralis

NIA

NIA

N/A

U mass. U mass, U paimitate

N/A

NIA

fi # fibroblasts?.

oxidation red, white.

6 (10) 216 (10)

Tihiall\ anterior Vastus intermed

Vastus lateralis

Tihialis anterior

X. NIA

U mass (6,216). U MHC mRNA, d MHC protein Type Ilb. MHC mRNA Type IIa, IIX; MHC protein Type IIa, IIb. X

U

(continued)

Table 7. Studv &

(Ref.J 75

(96 -98)

Flight & Year

SLS-1 STS-40 1991

Mission Length /nvesfi#utor.\

(DaysJ

Animal Enclosurz

Riley

9

AEM

et ul

(USA) RAHF

Enclo\ure Sire

Controls

group of 5

Simulation (I51

24 5X43.7X5 1

i#J

cm 24 individual\

(Continued)

Number & Sex of FIighr Rut\

5 AEM 10 RAHF Male

Strum, Age &

Posrflighr Collection

Body Weight

(Hourc)

Muscler Examined

Fiber CSA

SDH

GPD

Other

SpragueDewley 58 days 250-310 g

2.3-6.8 216

Soleus 4dductor longus

U (2-216).

N/A

NIA

U muscleibody mass. U musclcibody mass,

b (2-216).

fi % nonmyofiber area.

U macrophages, sarc lesions, regeneration. muscleibody mass.

10.5~11.5~28

cm

76 (99)

77 (100, 101)

SLS-I STS-40 1991 PARE-I STS-48 1991

Esser B Hardeman (Australia) Tischler

9

5.4

RAHF

AEM

er a1

(USA)

24 indibiduals Simulation (10) 10.5~11.5~28 cm group o f 5 Asynchronous'? (8). 2 4 . 5 ~ 4 3 . 7 ~ 5Tail-Swpended 1 (8) cm

group of 5 Asynchronous" (XI, 24 5 ~ 4 3 . 7 ~ 5Tail-Suspended 1 (8) cm

78 (102)

PARE-I STS-48 1991

Henrikseu erul. (USA)

54

79 (93)

PSE-2 STS-52 1992

Backup et al. (USA)

10

AEM

group of 5 Simulation (6) 24.5~43.7~51 cm

80 (103)

PARE-2 STS-54 1993

Lee er al.

6

AEM

group of 5 24.5x43.7x51 cin

81 (104 -107)

PARE-2 STS-54 lY93

AEM

(USA) CaioLzo er al. (USA1

6

AEM

group of 5 2 4 . 5 ~ 4 37x51 c in

Simulation (6)

10 Mule

SpragueDawley

6

8 Female

8 Female

Mule

SpragueDawley 26 days 60-65 g SpragueDawley 42 day\ 180 g SpragueDawley

6 Male

-200 g SpragueDawley

6 Male

6

2-3 3

N/A

NIA

Soleus

2-3.3

Plantaris Gastrocnemius Tibialis anterior Soleus EDL

fl fastmRNA?,

n/Uslow rnRNA'!. fi fa\t mRNA?.

N/A

NIA

NIA

U mass (F, TS), 1prot

(F, TS), TI glucose UPtake (+ insulin F, TS) U glucose uptake (+ B - insulin F). mass. U ma\\

U

X. NIA

N/A

NIA

U mass, fi IFV (F, TS). X.

?

Biceps brachii

NIA

NIA

NIA

no change GAP or actin mRNA.

3-8

Diaphragm

NIA

NIA

NIA

fi CS?, lipid peroxidation byproducts. X.

U Type I,

NIA

NIA

U mass, U Type IIa

Intercostal5 3-9+

Soleus

Type 11.

1

250 g

X. NIA

EDL

?

Sirnulation (61

U

X.

U (2-7).

EDL

7

250.310 8 SpragueDawley 26 days -62 g

EDL Diaphragm Soleus

Soleus (In

\LtU]

NIA

U

MHC protein.

fi 5% hybrid fibers.

n V,, U twitch time,

U max power, U force

SLS-2 STS-58 1993

Allen e f ul. (USA)

14

SLS-2 STS-58 1993

Ohiraer ul. (Japan)

14

RAHF

24 Individuals

Slrnlllallon ( 5 )

10.5~11.5~28 cm RAHF

SLS-2 STS-58 1993

5 Male

Ba\al (51, 24 individuals 1 0 . 5 ~ 1 1 . 5 ~ 2Synchronous 8 (10) cm

10 Male

24 individuals IO.Sx11 5x28 cm

5 Male

Basal (h), Simulation (6)

SpragueDawley 5K days 250-310 R SpragueDawley 5K day5 285 g SpragueDawley 58 day, 250-310 g

5

Soleus

b Type 1,

NIA

NIA

Ilx, b %Type 1. myonuclear # Type I .

Plantariz

NIA

b.

N/A

Pmax of P-adrenoceptor (F v\ B )

4

Soleus

NIA

NIA

NIA

b m a s , b myofih prot, b Po, Vmax, b Type I

Plantaris

14

RAHF

24 individual\ SimulaLion (10) 10.5~11.5~28 cm

16 Male

SpragueDawley 58 days 250-310 g

Inflight day13 (6) 5.3-6.3 336

Soleus Adductor longus

n

n

MHC mRNA, Type IIx MHC mRNA b mass, b myofih prot, Type IIx MHC mRNA. b mass, b myofib prot, Type IIh MHC mRNA. fi Type IIB MHC mRNA. b musclehody mass.

n

Tihialis anterior Riley er al. (USA)

u

5 (5) 216 ( 5 )

Vastus intermed

SLS-2 STS-58 1993

u mass, n 7' i Type Ila,

Type L/Il hybrid.

b (113, 5-

u

336). (113, 5336).

NIA

N/A

u musclehody mass, n Fnonmyofiher area & sarc lesion (5-7).

EDL revintiom

X.

b musclchody mass

(In order of appearance by column) General- X=no efibcl, NlA=nat applicable, %nformation queWonable 01 not a r a h b l e , riolur=mformatmn obtained from \ourcc\ othcr than the referenced papcr(r) Flifihf & Year. PSE=Physmlogical Systems Expcnments, SLS=Spacelah Life Sciences, PARE=Physdagical Anatomical Rodent Enperimcnta Anrmal Enclosure RAHF=Rc%arch Animal Holding Facility, AEM=Animal Enclowre Module Conlrols Rad=madmed with 800 cad on flight day 10, InJ=crash injury 2 days pnor 10 flight; GH=exogmoua growth hormone Srrricn, Age & Body Weighi Glrl=gestatmn day 14 P m j l l g h r Collvctron (Hours) Sm=iimulation control Murcles Exnmined EDL=cxtensor digitorurn longus, MG=medtal p\trocncmiu\, QF=quadnceps femons, BB=bicepi brachu, Dia=diaphragm, Scmi~mb=semimemhrano~u\, FDE=ertenwra 01 the forelimb digttr; EPL=extencor polluc!i longu5,

Bra=hrachmlir, LG=lateral gabtiocncmiua Fther CSA CSA=cm\\-sectiond arcs, ~nlrrmud=inrcrmcdial~fiber type, F=flight animal, SO=dow twitch oxidative fibers, FOG=fast twitch onidatire-glycolytic fibers: FG=ta?t twitch glycolytic fcber\, Typz I=ribers expressing $low isoforms of the myosin heavy cham. Type Ila,lIc,llb,lln=fibei\ enprzaaing one of the tasl sttormi of m y o m heavy chain, TS=tail-suspendcd control, HS=hindlimb-\u\pended control SDH; SDH=succmate dehydrogenaw (oxidative enzyme) GPD GPD=glycerophosphate dehydrogenax (glycolytic enzyme) Other- MDH=malate dchydiagcnase (oxidatwe eniyme). C=centnfuge control, LDH=lactale dehydrugenase (glycolytic enzyme), T prot=tranivcrse tubule protem. SER=\mooth endoplasmic reliculum, syn \~cs=synapticvec~clec chronow ~ o i i t m l\ap=sari.oplasmic , protein, airn=actomyosin. AST=aspanate ammotran\ferilae (amino acid mclahohsrn). HBD=hydconybutyiiitr dchydrogcnare (Ilpld metabolism), NADHD=NADH, dehydrogemase (axidatwe enryme). diam=dtameter, NMJ=neuromu\cular p n c t m n , re1 \ol=re181we volume; rnito=mitochondna. ~ w = ~ \ o l e u c i i i e leu=leucinc, , val=vahne. tyr=tyrorine. phe=phenylalanine. tyn=threonine, gly=glycme, aap=aqnrtate, glu=glutamatc. aer=sennc, myotih=myofiber. cnetaboliam (carbohydrate metabolism), GbPD=eluco\e-h-pho\philtr dehydrogcnase (carbohydrate metaholiim), hPGD=h-pho\phoglucunirtr dehydrugenaie car^ met=methionme, pro=prolme. Po=iwmetric tension, RP=eneymo of nbosc~S~pho$phate bohydrate metabolism). CAPD=glyreraldehyde-~-ph"~phate dehydrogcnaie (carbohydrate metaholi\m); TK=tran\kelalaac (carbohydrate metaholi\m), ALT=alanmc aminotranrferase (dmino acid metabolism), cyto=cytoplaamic, MLC=myoain light chain, con=conlrachlity. cocf=corfficienr. OP=oxidatwe phmphorylatmn, reap=reipirlory, mito \us=mitochondnal m\penamn, mT=ATP-Ca induced maximum tenuon, CaBA=caIcium binding affinity, V,=contractmn \eloc!ty, V,=rebnatmn \ e l u u l y , dcgen=degcneration, rcgen=regeneiation, HAD=l~hydroxyacyl-CoAdehydrogcnara (oxidative enzyme), TAP=tnpeptidylammopeptldase (myofibnl breakdown), CAP~illcium-.sti\.atrd protraie (myatihn I hreakdown), a\gla\p=a\par,igine-a\partatc, glm/glu=glutamine-glutamafe, hia=hi,lidmc, lys=lys,nc: ala=alanine, mal=maliite, HP=hydroxyprolme (collagenous protein), HK=hcnukina,c (glycolyttc emyme), L P L = h p o p ~ o t elipaae ~ (tnglyccnde metabollim), TnC=troponm C (calcium-bmdq contractile protein), 3KA=3-kctoacid-CoAtianrferarr (kctune body metabolim), TPH=lymromal Inpeptidy1 peptide hydrolaw (protcm breakdown), cyrK=cytochiome c, PFK=pho~phofructokinaae(glycolylic cn?yme): F,=contractile force, MTI=myotendinous JU"C~~O", GAP=glyceraldehyde-3-phorphate dchydrogcnax (glycolytic enzyme), MHC=myosm heavy cham, sarc=wrcomcre; IFV=mIcrrtit~alflutd YoIumz. CS=cmate \yntha\e (onidatire metabolism), V,,,,=maxlmum qhonenmg velocity. pmrx=maximum binding capacity. B=bu\al control

MONIKA B. FEJTEK and RICHARD J. WASSERSUG

14

In addition to examining the status of our scientific knowledge about the effects of spaceflight on rodent myology, we have used our database to explore aspects of the demography of space biology. We specifically looked for patterns in the size and nationality of the community of scientists interested in the effects of spaceflight on muscle tissue. There are a variety of questions that we address on the demography of the scientists that help define the discipline such as the following: 1 . Where do most experimenters reside? 2. How large and diverse is the core research community? 3. How accessible are the essential resources for doing research within this discipline?

We have thus used our survey to determine who has, in the past, been privileged with access to the prized commodity of biological material exposed to spaceflight.

II.

PROCEDURE AND SOURCES

The data for this survey (Tables 1-3) were compiled from a variety of literature sources. The most valuable resources were the Spaceline and Medline databases, Souza and colleagues’ Life Into Space and NASA’s Technical Memoranda. In Table I the technical terminology used in the source publications was maintained rather than standardized. This was purposely done to avoid errors of interpretation and to reveal the diversity of terms used in the literature (e.g., for muscle names, fiber types, suspension models). The survey includes spaceflights through the year 1993.

’

111.

FINDINGS

The results are given as a summary of the major points presented in and drawn from Table I . Each paragraph below summarizes the results from a column in the table, read left to right. All abbreviations are defined in Table 1. From 106 references, the results of 85 studies on the effects of spaceflight on rat skeletal muscle have been compiled. There are at least four more studies (I 12115) plus 17 review papers (one in Russian) on the topic, all of which we could not obtain and have thus not listed in Table 1 . There were a total of 16 spaceflight missions spanning a 20-year period from 1973 to 1993 that included rats for muscle studies. Nine of these were Russian-based Biocosmos missions (56%) and seven were U.S. missions (44%). The first U.S.-based mission was not until Spacelab-3 in 1985. Of the 85 studies listed in Table I , 39 are U.S.-based (45.9%) from 25 different investigators and 26 are Russian studies (30.6%) from 15 different investigators.

Spaceflight Effects on Muscle

15

There are six French studies (7.1%) from three different investigators, five Hungarian studies (5.9%) from three different investigators, five Polish studies (5.9%) trom two different investigators, two Canadian studies (2.4%) from two different investigators, one Australian study ( I .2%), and one Japanese study (1.2%) (see Table 2). The number of investigators is based on either the first author of a paper or the Principal Investigator listed in a NASA Technical Memorandurn. When the studies are counted by number of major investigative groups, the breakdown is as follows: United States = Baldwin et al.: five flights, seven studies, 12 references; Edgerton et al.: five flights, seven studies, nine references; Riley et al.: five flights, five studies, thirteen references; Musacchia et al.: three flights, four studies, nine references; and Tischler et al.: two flights, four studies, five references. Russia = Ilyina-Kakueva et al.: six flights, six studies, eight references; and Oganov et al.: six flights, six studies, seven references. Oganov has been an author on a total of 32 references (16 USA, 10 Russia, five Hungary, one Canada) and Ilyina-Kakueva has been an author on 30 references ( 1 7 USA, nine Russia, two France, one Poland, one Canada). The United States participated in Russian Biocosmos flights for the first time during the Cosmos 936 mission in 1977 (one group) (33,107) then on Cosmos 1129 in 1979 (same one group) (33,110). The nationality of the 16 spaceflight missions was as follows: Cosmos 605, all Russia; Cosmos 690, RussiaPoland; Cosmos 782, Poland/Russia, Cosmos 936, PolandRussidUSA; Cosmos 1 129, Russia/Hungary/USA; Cosmos I5 14, HungaryIRussialFrance; Cosmos 1667, RussidHungaryIFrance; Spacelab-3, USAICanada; Cosmos 1887, USAFrance I RussiaICanada; Cosmos 2044, USAPranceIRussia; PSE-I, USA; SLS-I, USA/

Table 2.

Country

France

Hungary Poland

Canada Australia

Japan

Number o j Studirs 39 26 6

USA Russia

Note:

Flight Opportunities of the Major Space-faring Nations and Their Activities

5 5 2 1 I

Number of Flights

Number of Investigritors

Number of Groups *

11

2s

5

9 4 3 3 2

1s 3 3

S

1

1

2 2 1 1

2 1 1

Number of Srudies

27 8 6 5 5

. .

. .

._

. .

. .

. .

*This column represents the number of major investigative groups in each country.

MONIKA B. FEJTEK and RICHARD J. WASSERSUG

16

Australia; PARE-1, USA; PSE-2, USA; PARE-2, USA; SLS-2, USA/Japan. A total of 18 investigators/groups have had multiple flight opportunities. Spaceflight mission duration ranged from as little as 4 days up to 21.5 days, with an average of 12.4 days. Four different enclosure types were used to house the animals. Two of these housed rats individually: BIOS-1 cylinders (four flights, 11 studies) and RAHF boxes (three flights, 14 studies); and two housed rats in groups of five (AEM, five flights, 10 studies) or ten (BIOS-2, 5 flights, 38 studies) animals per box. Most studies used at least a ground simulation or synchronous control; seven studies (8.2%) used only a vivarium control. Suspended controls were used in 16 studies (18.8%). The number of rats flown per mission ranged from two to 25 averaging 7.6 animals. Male rats were used on 14 of the 16 missions; females were used only on Cosmos 1514 and PARE-1. In total, muscle samples were taken from approximately 160 rats flown on the 16 spaceflight missions. All US-based missions utilized Sprague-Dawley rats whereas all Russian-based Biocosmos missions used Wistar rats. The age of the animals ranged from 26 to 109 days (average 78.2 days) with many unknowns (as indicated by question marks in Table 1). Weights ranged from about 60 g to about 400 g (average -272 g), although these data were not specified in several studies. Postflight data collection times ranged from 2 to 3 hours to 48 hours (average - 18 hours), with only one instance of inflight collection (i.e., on SLS-2 in 1993).

Table 3. .4naromical Location

Hindlimb

Forelimb

Other Notes:

Rodent Skeletal Muscles Examined After Spaceflight Musc~le*

Fiber Composition

Function

Soleus EDL Gastrocnemius Plantaris Tibialis anterior Quadriceps -Vastus intermed -Vastus lateralis -Vastus medialis Adductor longus Posterior thigh -Semi-mb Triceps Brachialis Biceps EPL FDE Diaphragm Intercostals

slow fasthntermed fasthtermed faasthtermed fasthtermed mixed slow/intermed fasthntermed fasthtermed slow fast fast fasthtermed fasthntermed fasthntermed fast fast slow/fast fast

plantarflexor dorsiflexor/toe extensor plantarflexor plantarflexor dorsiflexor extensor extensor extensor extensor adductor flex knee/extend hip flex kneekxtend hip extensor flexor flexorhpinator extensor/abductor extensor respiratory respiratory

*Abbreviations: EDL=extensor digitorurn longus, Semi~mb=semimemhranosus, EPL=extensor pollucis longus, FDE=extensors of the forelimb digits

# Studies

56 30 30 18 13 13

6 4 3 7 4

1

Spaceflight Effects on Muscle

17

The longest delay, as a percentage of flight time, occurred on Cosmos 1887 where data were collected 2 days postflight of a 12.5 day mission (16%). This is followed by Cosmos 605, where data collection occurred 2 days after a 21.5 day mission (9.3%). A total of 16 different muscles have been examined in the spaceflown rat, and 14 of those are limb muscles (see Table 3). The frequencies that each muscle has been investigated are in descending order: soleus (56 studies = 65.9%), extensor digitorum longus (EDL; 30 studies = 35.3%), gastrocnemius (30 studies = 35.3%), quadriceps (26 studies = 30.6%), plantaris (18 studies = 21.2%), triceps/tibialis anterior (13 studies each = 15.3%), diaphragm (12 studies = 14.1%), brachialis (nine studies = 10.6%), biceps (eight studies = 9.4%), adductor longus (seven studies = 8.2%), "posterior thigh" (four studies = 4.7%), semimembranosus forelimb digit extensors (FDE) and extensor pollucis longus (EPL)/intercostals (one study each = 1.2%). Note that some studies examined only the lateral (LG) or medial (MG) head of the gastrocnemius and these were counted together with studies that examined the entire gastrocnemius muscle. Similarly, studies that examined only components of the quadriceps muscle, that is, vastus intermedius, vastus lateralis, and vastus medialis, were counted together with studies examining the entire quadriceps. Approximately five different terminologies were used to define muscle fiber type. A total of 22 studies (25.9%) measured fiber cross-sectional area (CSA). The most common result is a decrease in CSA after exposure to microgravity (although two studies showed an increase), particularly in slow and intermediate fiber types. EDL shows the most mixed results as far as which fiber type changed. A total of 10 studies (1 1.7%) measured activity of the oxidative enzyme succinate dehydrogenase (SDH). Most found a decrease after spaceflight, but two studies showed an increase in SDH activity. There were also 10 studies ( I 1.7%) that measured activity of the glycolytic enzyme glycerophosphate dehydrogenase (GPD). All of these found an increase in GPD activity after spaceflight. The most common finding overall was a decrease in muscle mass reported in 32 studies (37.6%). Many studies also examined other metabolic enzymes, muscle proteins and contractility.

IV. A.

DISCUSSION

Biological Considerations

With the exception of Biocosmos missions 605,690, and 1887 (where the postflight tissue collection was delayed by 24 to 48 hours; see Table l), spaceflights with rodents onboard have been of long enough duration to document changes in muscle tissue associated with reduced loading (these changes have been most recently reviewed in references 3,4, and 116-1 18.) However, a conspicuous weakness in the data is that missions have been either too short or the animals too old

18

MONIKA B. FEJTEK and RICHARD J. WASSERSUG

to reveal the effects of microgravity on developing rodents. The fact that the average mission to date has been only 12.4 days long and the longest one 21.5 days supports the belief that longer duration flights, such as may become available with the International Space Station, are warranted. Rodent experiments in space have made use of four different flight habitats. This diversity in enclosure design is one source of noise in the muscle data. Ground controls are another problem. Not all investigators appear to have maintained ground controls in enclosures identical to their flight hardware. This last point is a serious one since four of the studies listed in Table 1 (numbers 15, 26, SO, 68) reveal cage effects-that is, muscle properties are affected by enclosure type, independently of exposure to microgravity. We have found a cage effect in our own research with rats that flew on the Space Shuttle in 1995.' Cage effects appear to originate from two sources. Rats that are housed in simple, flat-bottom vivarium cages have their movement largely restricted to a single plane. In contrast, rats in enclosures such as NASA's AEM, with footholds on the walls and ceiling, can move through a more complex three-dimensional environment. Such flight chambers provide both more and different exercise opportunities for the rats.' The second source of cage effects comes from animals housed singly versus those in groups. Two of the four spaceflight habitats house rats singly, whereas the other two house them in groups. Animals in groups move about differently than those maintained in isolation. Inflight video of rats in NASA's AEM'19 confirms, for example, that rats in microgravity continuously climb over each other and in so doing rotate their torsos seven times more often than in normal gravity. For several of the older studies listed in Table 1 , it was simply not possible to tell what type (or types) of enclosures were used to house control animals. As indicated in Table I many studies failed to give basic information on the number, sex, strain, age, and weight of the rats flown. In the majority of the cases, however, this information was extractable from other publications related to the same spaceflight. Whereas the majority of the rodent spaceflight experiments were flown by the Russians via their unmanned Biocosmos satellite series, these vehicles lack the precision landing of the U S . Space Shuttle. Consequently, the postflight collection times have been far more variable for the Biocosmos rodents than for those housed on the Space Shuttle. Perhaps the most significant improvement in the execution of space biological research came with the capability to collect tissue samples inflight. That technical advance, however, was only realized in 1993 with the U.S. SLS-2 flight. There are in excess of 100 different striated muscles in the rat. The effects of extended exposure to microgravity has thus been examined in less than 16% of these muscles (see Table 3). The soleus muscle has been and continues to be the archetypal slow fiber muscle, which shows dramatic shifts in fiber type composition after extended periods of unloading. Since this is the "model" muscle for

Spaceflight Effects on Muscle

19

examining the effects of spaceflight on skeletal muscle, its functional and morphological properties have been analyzed after orbital flight no less than 56 times! The diagnostic procedures for muscle fiber typing have evolved over the past few decades from metabolic enzyme markers (e.g., SDH and GPD) to more discriminating myosin heavy chain analyses. Nevertheless, the majority of studies to date have reached the same conclusion but with different histochemical and biochemical procedures. The most notable result is that skeletal muscle does significantly atrophy during spaceflight. There i s also often an accompanying shift from slow to fast muscle properties after extended exposure to unloading, and there is a related enhancement of glycolytic capacity. Contractile properties of skeletal muscle are also altered in a similar direction, that is, contraction velocities of slow muscles become “faster”, and contraction force decreases with observed decreases in muscle size. More details of the effects of spaceflight on rodent skeletal muscle are reviewed in references 2-4, 1 16-1 I 8,120- 126, and I33 with reviews of specific missions in references 127-130. Our survey shows that not only are these conclusions ineluctable, but that they have been independently demonstrated many times.

B.

Demographic Considerations

A primal (if not “the” primal) motivation for scientific exploration is that a given topic has not been previously explored. Indeed scientific papers commonly begin with a statement to the effect that a particular topic has been “poorly studied” or “not previously investigated”. In this regard the large number of studies that have been performed on rat skeletal muscle exposed to microgravity came as a surprise to us. We did not anticipate so many studies nor for that matter had many biologists who had been involved in spaceflight research. We informally asked many of those scientists how many studies they estimated had been performed in this area and none guessed more than sixty. Our estimate of approximately 89 studies may be low for several reasons. Firstly, it only reflects studies published on spaceflight missions prior to 1994. Since then there have been several more space shuttle flights that included rats from which muscle samples were taken (e.g., the joint U.S. National Institute of Health-NASA rodent experiments). The results from most of those flights have yet to be published. Secondly, studies that either failed for some technical reason or yielded negative results are unlikely to have been published. Although great effort was made to find all the relevant literature, it is still possible that we overlooked some studies published in technical documents and/or languages other than Russian or English, the languages of the major space-faring nations. Our results confirm that research in space biology has been published too often in obscure journals (e.g., not listed in Current Contents) and technical memoranda, not commonly tracked by the major scientific indexing services. Thus, on one hand, the completion of our survey was greatly facilitated by the Spaceline

20

MONIKA B. FEJTEK and RICHARD J. WASSERSUC

database, which was a particularly helpful lead to the non-English literature. On the other hand, this database only identified 51 % of the papers cited in Tables 1 and 2 when searched under flight experiments by the key words “muscle” and “rat”. Most recently, Fitton and Moore’31 tabulated European space life sciences research on a mission-by-mission basis from I980 to 1993. They discuss the focus and goals of each ESA member nation, but their survey includes neither the scientific literature nor information on the biological conclusions derived from those spaceflight missions. The majority (56%) of the opportunities to examine rodent tissues exposed to microgravity have been provided by the Soviet Union through its long and successful Biocosmos series of unmanned spaceflight. As noted by Souza and coll e a g u e ~scientists ,~ in many countries outside Russia, including the USA, profjted from these USSR flight opportunities. For the first ten years that rodents were placed on orbital platforms, the USSR “owned” the field. In the last ten years, the combination of economic problems of the ex-Soviet Union and the improved reliability of the U.S Space Shuttle program shifted the balance to U.S.A. flights. The shift can easily be seen in the second column of Table 2. Timewise, the regularity of Biocosmos launches-averaging one every two years-began to break down in the 1990s as the Soviet Union began to experience both financial and political stress. This was coincidental with the U.S. accelerating its space shuttle launch schedule to make up for the backlog formed in the wake of the 1987 Challenger Space Shuttle disaster. Despite this shift, the U.S.S.R. (now Russia) and the U.S.A. still remain the only countries with histories of launching rodents into space. The initial question we asked concerns the demography and political geography of the rodent muscle research community within space biology. Although the majority of rodent spaceflight experiments have been initiated by the former Soviet Union, the majority of published studies have come from the U.S.A. (Table 2). France placed third, Hungary and Poland tied for fourth place, and they were followed by Canada, Australia, and Japan. The number of investigators that have published on rat muscles from spaceflight follow in the same rank order by country. We can next determine whether space biologists who study rat skeletal muscle form a large open community or a small, relatively interrelated group. Figures in Table 2 suggest that the biologists who have had access to rodent tissue from space flight experiments are relatively few, numbering in the order of 40 to 50 over a 20to 25-year period. Those 50 or so scientists almost always published as teams rather than singularly. This is not surprising considering how procedurally complicated and labor intensive research in this area can be. We have made an effort to identify these research groups, recognizing that all such groups are dynamic, with members coming and going over the years. It is also true that the leadership of these groups can similarly evolve through time. Thus, although we have attached a person’s name to each group, these names are meant solely as labels of convenience. They should not be interpreted as implying that a particular person

Spaceflight Effects on Muscle

21

is more essential, central, or enduring than any other member to a particular research team. When the number of investigators is considered in terms of these research teams, then the community of biologists that have studied rat muscle from orbital experiments is reduced to about a dozen key groups. That estimate is imprecise and undoubtedly low. But even if the correct number is twice that, one must conclude that very few research groups have had access to rat muscle tissue from space. That small number of experimenters has undoubtedly helped fuel the belief that access to material from spaceflight experiments is a once-in-a-lifetime event. Indeed, as an example of this belief, we note that the book Fundamentals of Space Biology’32 is dedicated “to the corps of Space Biologists who toil so diligently for so many years for their once or twice in a lifetime opportunity to perform an authentic spaceflight experiment”. Surprisingly, when we examine by investigator group who has had multiple access to rodent tissue exposed to microgravity, this belief is not well supported. The five most productive research groups in the United States have all had access to tissue from two or more spaceflights. The three most successful groups have had access to tissue from five different flights. The international space biology community has in recent years established a formal peer review process for spaceflight access. Whether this process will change the demographic patterns discussed above remains to be seen. A positive sign is the increased publication of spaceflight results in internationally recognized peer-reviewed journals such as the Journal of Applied Physiology, the American Journal of Physiology, and Muscle and Newe (see Table 1). This increased exposure of results from space biology research in the mainline literature should lead to greater acceptance and growth of space biology as a legitimate, on-going scientific discipline.

V.

CONCLUSIONS

The repetition of results noted above is perhaps most marked between the earlier Russian-based Cosmos missions and later US.-based Space Shuttle missions, a fact that may reflect the competition of the Cold War era. As we make the transition from a “cold war” to a “cooperative” mindset for space exploration, this redundancy should be minimized. Additionally, space biologists are publishing increasingly in international peer-reviewed journals, which make their results better known to the scientific community at large. The greater exposure of results from spaceflight missions helps identify and define questions that remain to be answered. To improve the consistency and reduce the redundancy of results in studies on rodent myology, future research needs to address several additional issues. A greater number of different muscles should be examined to further relate the effects of spaceflight with the functional properties of various muscles and muscle

22

MONIKA B. FEJTEK and RICHARD J. WASSERSUG

groups. Myological properties need to be compared where, for example, either flight duration or postflight data collection time are varied, to elucidate the critical periods and time course of the effects of microgravity on muscle. Following from this, it is essential to discriminate between unloading in orbit and reloading effects postflight,a task requiring tissue collection in orbit. Such experimental procedures have already begun with the advent of inflight dissections aboard the Space Shutt1e.96 Space research to date with rodents has helped us understand the functional and morphological effects of unweighting on muscle in ways that would not be possible with ground-based research alone. Future work, however, needs to focus on the time course of deconditioning during flight, as well as on the postflight reconditioning, of muscle. With astronauts spending longer periods of time in space upon international orbiting platforms, we should be able to perform more inflight experimental manipulations. The data collected inflight will be important in determining where, when, and how to intervene with countermeasures to help maintain astronaut health and welfare. In addition to being instructive for planning future spaceflight experiments, some simple conclusions can also be drawn from our survey’s demographic data on the research community that studies rodent tissue exposed to microgravity. This has, indeed, been a relatively small community assembled into a few identifiable research groups. Access to tissue from orbital flights may be difficult for some, but the successful research groups have been very prosperous in this regard.

VI.

SUMMARY

Rodent muscles have been examined in more than 89 spaceflight studies over the last 25 years with much variation in the procedures and results. Mission duration ranged from four days to three weeks, postflight data collection ranged from a few hours to two days after landing, and there is great diversity in the number, size, and age of the rats that have flown. Several different types and sizes of animal enclosures have also been used-a significant factor because cage design affects animal activity and muscle loading. Only a small percentage (-1 6%) of the total number of striated muscles in the rat have been examined. We have identified both substantial redundancy and inconsistencies in the results from studies to date. However, many of these appear unavoidable due to the great variation in experimental protocol of the different missions. Nevertheless these studies repeatedly confirm that exposure to spaceflight decreases the mass of limb muscles and leads to muscle atrophy. The majority of missions were flown by the former Soviet Union, but the majority of papers have been published by U.S. researchers. A relatively small number of investigators (about 50) clustered into fewer than 15 identifiable research groups worldwide account for most of the results to date. These groups have had

Spaceflight Effects on Muscle

23

access to rodent muscle tissue from two to seven spaceflights each. International cooperation in the post-cold war era and the publication of future work in peer-reviewed international journals should help greatly in reducing redundancy and enriching our knowledge of how gravity affects biological systems.

ACKNOWLEDGEMENTS We would like to thank Ken Souza for introducing us to space biology, and Richard Mains for encouraging us with this project. Support for this research has been provided by the Canadian Space Agency and the Natural Sciences and Engineering Research Council of Canada.

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14. Belitskaya, R.A. Changes in amount and composition of phospholipids in rat skeletal muscle microsomal fraction under the influence of a flight aboard the Kosmos-690 biosatellite. Kosmicheskayci Biologiya i Aviukosmicheskaya Meditsina,13( 1 ): 19-23, 1979. 15. Gayevskaya, M.S., Belitskaya, R.A., Kolganova, N.S., Kolchina, Y.V., Kurkina, L.M., Nosova, Y.A. Tissular metabolism in mixed type fibers of rat skeletal muscles after flight aboard Cosmos-690 biosatellite. Space Biology and Aerospace Medicine, 1 3 : 3 8 4 2 , 1979. 16. Gaycvskaya, M.S., Veresotskaya, N.A., Kolganova, N.S., Kolchina, Y.V., Kurkina, L.M., Nosova, Y.A. Changes in metabolism of soleus muscle tissues in rats following flight aboard the Kosmos-690 biosatellite. Space Biology and Aerospace Medicine, 13:18-22, 1979. 17. Nesterov, V.P., Tigranyan, R.A. Electrolyte composition of rat blood plasma and skeletal muscles after flight aboard the Cosmos-690 biosatellite. Kosmicheskaya Biologiya i Aviakusmicheskuyu Meditsinu, 13(4):26-30, 1979. 18. Ilyina-Kakueva, E.I., Portugalov, V.V. Combined effect of space flight and radiation on skeletal muscles of rats. Aviation, Space, and Environmental Medicine, 48(2):115-119, 1977. 19. Baranski, S., Baranska, W., Marciniak, M., Ilyina-Kakueva, E.I. Ultrasonic investigations ofthe soleus muscle after space flight on the Biosputnik 936. Aviation, Space, und Environmental Medicine, 50(9):930-934, 1979. 20. Marciniak, M. Comparison of morphometrical ultrastructural changes in axonal endings of neuromuscular junctions in the diaphragm, quadriceps muscle and in the soleus muscle of rats after space flights on Biosputniks 782 and 936. Fulia Morphologica Warszawa, 4:449-459, 1979. 21, Ushakov, A.S., Vlasova, T.F., Miroshnikova, E.B. Studies of amino acid metabolism in the muscles of rats flown aboard the biosatellite Cosmos 782. In: Proceedings qf the Symposium on Gravitational Physiology. pp. 23 1-234, Permagon Press, Oxford, 1979. 22. Vlasova, T.F., Miroshnikova, Ye.B., Polyakov, V.V., Murugova, T.P. Amino acids of femoral quadriceps of rats following flight aboard the Cosmos-936 biosatellite. Kosmicheskaya Biologiyu i Aviukosmicheskaya Meditsinu, 16(2):53-56, 1982. 23. Oganov, V.S., Skuratova, S.A., Shirvinskaya, M.A. Effect of flight aboard Cosmos-936 biosatellite on contractile properties of rat muscle fibers. Kosmicheskaya Biologiya i Aviakosmicheskayu Meditsina, 15(4):58-61, 1981. 24. Castleman, K.R., Chui, L.A., Van Der Meulen, J.P. Spaceflight effects on muscle fibers. NASA Technical Memorandum, 78526274-289, 1978. 25. Chui, L.A., Castleman, K.R. Morphometric analysis of rat muscle fibers following space flight and hypogravity. The Physiologist, 23(6):S76-S78, 1980. 26. Nesterov, V.P., Veresotskaya, N.A., Tigranyan, R.A. Activity of some enzymes of carbohydrate metabolism in rat skeletal muscles after space flight. Kosmicheskaya Biologiya i Aviakosmic.heskava Meditsina, 15(5):75-78, 198 1. 27. Nosova, Ye.A., Veresotskaya, N.A., Kolchina, Ye.V., Kurkina, L.M., Belitskaya, R.A., Tigranyan, R.A. Metabolic processes in rat skeletal muscles after flight aboard Cosmos-936. Kosmicheskuyu Biologiyu i Aviakosmicheskaya Meditsina, 15(5):7 1-75, 1981 , 28. Oganov. V.S., Skuratova, S.A., Murashko, L.M., Shirvinskaya, M.A., Sziligyi, T., Szoor, A,, Kapcaik, M., Takacs, O., Oganeayan, S.S., Davtyan. 2h.S. Change in the composition and properties of contractile proteins after space flight. Biophysics, 27( 1):25-30, 1982. 29. Sziligyi, T., Sziiiir, A., Takics, O., RapcsLk, M., Oganov, V.S., Skuratova, S.A., Oganesyan, S.S., Murashko, L.M., Eloyan, M.A. Study of contractile properties and composition of myofibrillar proteins of skeletal muscles in the Cosmos-1 129 experiment. The Physiologist, 23:S67S70, 1980. 30. Mailyan, E.S., Buravkova, L.B., Kokoreva, L.V. Energetic reactions in rat skeletal muscles after flight in Cosmos- 1 129 biosatellite. Kosmicheskaya Biologiya i Aviukosmicheskaya Meditsina, 17(3):32-36, 1983.

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3 I . Takics, O., Rapcsik, M., Szoor, A,, Oganov, V.S., Sziligyi, T., Oganesyan, S.S., Guba, F. Effect of weightlessness on myofibrillar proteins of rat skeletal muscles with different lunctions in experiment OfbiOYdtdlite “Cosmos-1 129.’’Actu Physiologicu Hungarica, 62:228-233, 1983. 32. Rapcsik, M., Oganov, V.S., Szoor, A., Skuratova, S.A., SrilBgyi, T., Takics, 0. Effect of weightlessness on the function of rat skeletal muscles on the biosatellite “Cosmos-1 129.” Acta Physiologica Hungaricu, 62:225-228, 1983. 33. Castleman, K.R., Chui, L.A., Van Der Meulen, J.P. Automatic analysis of muscle fibers from rats subjected to spaceflight. NASA Technical Memorundum, 81289(2):267-278, 1981. 34. Rapcsik, M., Oganov, V.S., Murashko, L.M., Szilhgyi, T., Szoor, A. Effect of short-term spaceflight on the contractile properties of rat skeletal muscles with different functions. Acta Physiologica Hungarica, 76( I): 13-20, 1990. 35. Mailyan, E.S., Chabdarova, R.N., Korzun, E.I. Energy reactions in the skeletal muscles of rats after short-term space flight on Kosrnos- 1.5 14. Kosmicheskaya Riologiya i Aviukosmicheskuya Meditsinn, 22(3):.55-58, 1988. 36. Holy, X., Mounier, Y. Effects of short spaceflights on mechanical characteristics of rat muscles, Muscle and Nerve, 14:70-78, 1991. 37. Holy, X., Mounier, Y., Goblet, C. Microgravity effects on the contractile proteins of rat muscles. In: Space Physiology. (J.J. Hunt, Ed.), pp. 61-65. ESA Publication Division, Noordwijk, The Netherlands, 1986. 38. Holy, X., Oganov, V., Mounier, Y., Skuratova, S. Contractile protein behaviour of rat muscle fibres in microgravity conditions. C. R. Acadrmie des Sciences Paris, 303(6):229-234, 1986. 39. Babakova, L.L., Demorzhi, M.S., Pozdnyakov, O.M. Dynamics of structural changes in skeletal muscle neuromuscular junctions of rats under the influence of space flight factors. The Physiologist, 35(1): S224-S225, 1992. 40. Desplanches, D., Mayet, M.H., Ilyina-Kakueva, E.I., Sempore, B., Flandrois, R. Skeletal muscle adaptation in rats flown on Cosmos 1667. Journal ofApplied Physiology, 68( 1):48-52, 1990. 41. Gazenko, O.G., Ilyin, Ye.A., Savina, Y.A., Serova, L.V., Kaplanskiy, A.S., Oganov, V.S., Popova, LA., Smirnov, K.V., Konstantinova, I.V. Experiments with rats flown aboard Cosmos- 1667 biosatellite (Main objectives, conditions and results). Kosmicheskuya Biologiyu i Aviakosmicheskaya Meditsina, 21(4):8-16, 1987. 42. Riley, D.A., Ellis, S., Slocum, G.R., Satyanarayana, T., Bain, J.L.W., Sedlak, F.R. Morphological and biochemical changes in soleus and extensor digitorum longus muscles of rats orbited in Spacelab 3. The Physiologist, 28(6):S207-S208, 1985. 43. Riley, D.A., Ellis, S., Slocum, G.R., Satyanamyana, T., Bain, J.L.W., Sedlak, F.R. Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles. Muscle and Nerve, 10:560-568, 1987. 44. Martin, T.P., Edgerton, V.R., Grinde!and, R.E. Influence of spaceflight on rat skeletal muscle. Journal ofApplied Physiology, 65(5):23 18-2325, 1988. 45. Musacchia, X.J., Steffen, J.M., Fell, R.D., Dombrowski, J. Physiological comparison of rat muscle in body suspension and weightlessness, The Physiologist, 30(1):S102-S 105, 1987. 46. Musacchia, X.J., Steffen, J.M., Fell, R.D., Dombrowski, M.J. Comparative morphometry of fibers and capillaries in soleus following weightlessness (SL-3) and suspension. The Physiologist, 31(1):S28-S29, 1988. 47. Musacchia, X.J., Steffen, J.M., Fell, R.D., Dombrowski, M.J. Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats. Journal ofApplied Physiology, 69(6):22482253, 1990. 48. Steffen, J.M., Musacchia, X.J. Effect of seven days of spaceflight on hindlimb muscle protein, RNA, and DNA in adult rats. The Physiologist, 28(6):S379-S380, 1985. 49. Steffen, J.M., Musacchia, X.J. Spaceflight effects on adult rat muscle protein, nucleic acids, and amino acids. American Journal of Physiology, 251:R1059-R1063, 1986.

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50. Henriksen, E.J., Tischler, M.E., Jacob, S., Cook, P.E. Muscle protein and glycogen responses to recovery from hypogravity and unloading by tail-cast suspension. The Physiologist, 28(6):S 193,5194, 1985. 5 1 . Tischler, M.E., Henriksen, E.J., Jacob, S., Cook, P.E. Responses of amino acids in hindlimb muscles to recovery from hypogravity and unloading by tail-cast suspension. The Physiologist, 28(6):S376-S377, 1985. 52. Martin, T.P. Protein and collagen content of rat skeletal muscle following spaceflight. Cell and TisJw Research, 254:25 1-253, 1988. 53. Mancheater, J.K., Chi, M.M.Y., Norris, B., Ferrier, B., Krasnov, I., Nemeth, P.M., McDougal, D.B., Lowry, O.H. Effect of microgravity on metabolic enzymes of individual muscle fibers. FASEB Journal, 455-63, 1990. 54. Edgerton, V.R., Miu, B., Martin, T.P., Roy, R., Marini, J.. Leger, J.J., Oganov, V., Ilyina-Kakueva, E. Metabolic and morphologic properties of muscle fibers after spaceflight. NASA Technical Memorandum, 102254: 183-205, 1990. 55. Miu, B., Martin, T.P., Roy, R.R., Oganov, V., Ilyina-Kakueva, E., Marini, J.F., Leger, J.J., Bodine-Fowler, S.C., Edgerton, V.R. Metabolic and morphologic properties of single muscle fibers in the rat after spacetlight, Cosmos 1887. FASEB Journal, 4:64-72, 1990. 56. Ellis, S., Riley, D.A., Bain, J., Sedlak, F., Slocum, G., Oganov, V. Morphological and biochemical investigation of microgravity-induced nerve and muscle breakdown: Biochemical analysis of EDL and PLT muscles. NASA Technical Memorandum, 102254:259-261, 1990. 57. Riley, D.A., Bain, J., Sedlak, F., Slocum, G. Morphological and biochemical investigation of microgravity-induced nerve and muscle breakdown: Investigation of nerve and muscle breakdown during spaceflight. NASA Technical Memorandum, 102254:217-257, 1990. 58. Riley, D.A., Ilyina-Kakueva, EJ . , Ellis, S., Bain, J.L.W., Slocum, G.R., Sedlak, F.R. Skeletal muscle fiber, nerve, and blood vessel breakdown in space-flown rats. FASEB Journal, 4:84-91, 1990. 59. Baldwin, K.M., Herrick, R.E., Ilyina-Kakueva, E., Oganov, V.S. Effects of zero gravity on myofibril content and isomyosin distribution in rodent skeletal muscle. FASEB Journal, 4:79-83, 1990. 60. Baldwin, K., Herrick, R., Oganov, V. Effects of zero gravity on myofibril protein content and isomyosin distribution in rodent skeletal muscle. NASA Technical Memorandum, 102254:263273, 1990. 61. Musacchia, X.J., Stcffen, J.M., Fell, R. Biochemical and histochemical observations of vastus medialis from rats flown in Cosmos 1887. The Physiologist, 32(1):S2lPS22, 1989. 62. Musacchia, X.J., Steffen, J.M., Fell, R.D., Oganov, V.S. Biochemical and histochemical observations of vastus medialis. NASA Technical Memorandum, 102254:207-214, 1990. 63. Holy, X., Stevens, L., Mounier, Y. Compared effects of a 13 day spaceflight on the contractile proteins of soleus and plantaris rat muscles. The Physiologist, 33(1):S80-S81, 1990. 64. Desplanches, D., Mayet, M.H., Ilyina-Kakueva, E.I., Frutoso, J., Flandrois, R. Structural and metabolic properties of rat muscle exposed to weightlessness aboard Cosmos 1887. European Journal cfApplied Physiology, 63:288-292, 1991. 6 5 . Bell, G.J., Martin, T.P., Ilyina-Kakueva, E.I., Oganov, V.S., Edgerton, V.R. Altered distribution of mitochondria in rat soleus muscle fibers after spaceflight. Journal of Applied Physiology, 73(2):493497, 1992. 66. Oganov, V.S., Skuratova, S.A., Murashko, L.M. Contractile properties of skeletal muscles of rats after flight on “Kosmos- 1887.” Kosmicheskaya Biologiya i Aviakosmicheskaya Meditsina, 25(2):44-47, 1991. 67. Ilyina-Kakueva, E.I. Morphohistochemical study of skeletal muscles in rats after experimental flight on “Kosmos- 1887.” Kosmicheskaya Biologiya i Aviakosmicheskaya Meclitsina, 24(4):2225, 1990.

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68. Booth, F.W., Morrison, P.R., Thomason, D.B., Oganov, V.S. Actin mRNA and cytochrome c mRNA concentrations in the triceps brachia muscle of rats. NASA Technical Memorandum, 102254:275-278, 1990. 69. Musacchia. X.J., Steffen, J.M., Fell, R.D., Dombrowski, M.J., Oganov, V.S., Ilyina-Kakueva, E.I. Skeletal muscle atrophy in response to 14 days of weightlessness: Vastus medialis. Journal ojApplied Physiology, 73(2):448-50S, 1992. 70. Musacchia, X.J., Steffen, J.M., Fell, R.D., Oganov, V.S., Ilyina-Kakueva, E.I. Skeletal muscle atrophy in response to 14 days of weightlessness: vastus medialis. NASA Technical Memorandum, 108802(1):273-287, 1994. 71. Edgerton, V.R., Jiang, B., Leger, J.J., Marini, J.F., Ohira, Y.. Roy, R. Metabolic and morphologic properties of muscle fibers after spaceflight, Cosmos 2044. NASA Technicul Memorandum, 108802(1):255-269, 1994. 72. Ohira, Y., Jiang, B., Roy, R.R., Oganov, V., Ilyina-Kakueva, E., Marini, J.F., Edgerton, V.R. Rat soleus muscle fiber responses to 14 days of spaceflight and hindlimb suspension. Journal of Applied Plzysiology, 73(2):5 1S-57S, 1992. 73. Jiang, B., Ohira, Y . , Roy, R.R., Nguyen, Q., Ilyina-Kakueva, E L , Oganov, V., Edgerton, V.R. Adaptations of fibers in fast-twitch muscles of rats to spaceflight and hindlimb suspension. Journal ofApplied Physiology, 73(2):588-658, 1992. 74. Talmadge, R.J., Roy, R.R., Edgerton, V.R. Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. Journal of Applied Physiology, 81(6):2540-2546, 1996. 75. Chi, M.M.Y., Choksi, R., Nemeth, P., Krasnov, I., Ilyina-Kakueva, E., Manchester, J.K.. Lowry, O.H. Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers. Journal GfApplied Physiology, 73(2):668-733. 1992. 76. Ellis, S., Riley, D.A., Giometti, C.S. Morphological, histochemical, immunocytochemical, and biochemical investigation of microgravity-induced nerve and muscle breakdown: Muscle studies. NASA Technic.al Memorandum, 108802(1):327-337, 1994. 77. Riley, D.A., Ellis, S.. Giometti, C.S., Hoh, J.F.Y., Ilyina-Kakueva, E.I., Oganov, V.S., Slocum, G.R., Bain, J.L.W., Sedlak, F.R. Muscle sarcomere lesions and thrombosis after spaceflight and suspension unloading. Journal ($Applied Physiology, 73(2):333438, 1992. 78. Riley, D.A., Ellis, S., Haas, A.L., Slocum, G.R., Bain, J.L.W., Sedlak, F.R., Hoh, J.F.Y., Ilyina-Kakueva, E.I., Oganov, V.S. Morphological, histochemical, immunocytochemical, and biochemical investigation of microgravity-induced nerve and muscle breakdown: Muscle biochemistry. NASA Technical Memorandum, 108802( 1):291-326, 1994. 79. Booth, F.W., Thomason, D.B., Morrison, P.R., Oganov, V.S., Ilyina-Kakueva, E.I., Smirnov, K.V. mRNA levels in skeletal and smooth muscle: Some mRNA’s decrease in skeletal muscle during spaceflight. NASA Technical Memorandum, lOSSO2( 1):359-362, 1994. 80. Thomason, D.B., Morrison, P.R., Oganov, V., Ilyina-Kakueva, E., Booth, F.W., Baldwin, K.M. Altered actin and myosin expression in muscle during exposure to microgravity. Journal of Applied Physiology, 73(2):90S-938, 1992. 8 I . D’Amelio, F., Daunton, N.G., Ilyina-Kakueva, E.I. Effects of spaceflight in the muscle adductor longus of rats flown in the Soviet biosatellite Cosmos 2044: A study employing neural cell adhesion molecules (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy). NASA Technical Memorandum, 108802(2):33-71, 1994. 82. Stevens, L., Mounier, Y. Functional properties of soleus and EDL muscles after weightlessness (Cosmos 2044). The Physiologist, 34(1): S172-SI73, 1991. 83. Stevens, L., Mounier, Y., Holy, X. Functional adaptation of different rat skeletal muscles to weightlessness. American Journal of Physiology, 264:R770-R776, 1993. 84. Lowry, O.H., Krasnov, I., Ilyina-Kakueva, E.I., Nemeth, P.M., McDougal, D.B., Choksi, K., Carter, J.G., Chi, M.M-Y., Manchester, J.K., Pusateri, M.E. Effect of microgravity on metabolic enzymes of individual muscle fibers. NASA TechnicalMemorandum, 108802(2): 1 11-135, 1994.

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85. Oganov, V.S., Murashko, L.M., Kabitskaya, O.E., Szilagyi, T., Rapcsak, M. Physiological characteristics of rat skeletal muscles after the flight on board "Cosmos-2044" biosatellite. The Physiologist, 34( 1):s174-S 176, 1991, 86. Ilyina-Kakueva, E l . , Burkovskaya, T.E. The microgravity effect on a repair process in m. soleus of the rats flown on Cosmos-2044. The Physiologist, 34(1):S141-S143, 1991. 87. Stauber, W.T., Fritz, V.K., Burkovskaya, T.E., Ilyina-Kakueva, E.I. Effect of spaceflight on the extracellular matrix of skeletal muscle after a cmsh injury. Journal of Applied Physiology, 73(2):74S-8 IS, 1992. 88. Stauber, W.T., Fritz, V.K., Burkovskaya, T.E., Ilyina-Kdkueva, E.I. Effect of injury on mast cells of rat gastrocnemius muscle with respect to gravitational exposure. Experimental Molecular Pathology, 59:87-94, 1993. 89. Stauber, W.T., Fritz, V.K., Burkovskaya, T.E., Ilyina-Kakueva, E.I. Connective tissue studies. Part 111: Rodent tissue repair: Skeletal muscle. NASA Technical Memorandum, 108802(2):255269, 1994. 90. Baldwin, K.M., Herrick, R.E., Ilyina-Kakueva, E., Oganov, V.S. Effect of zero gravity on contractiie protein content and isomyosin distribution in fast and slow quadriceps muscles of rodents flown on Cosmos 2044. NASA Technical Memorandum, 108802(1):341-356, 1994. 91. Jiang, B., Roy, R.R., Navarro, C., Edgerton, V.R. Absence of a growth hormone effect on rat soleus atrophy during a 4-day spaceflight. Journal ofApplied Physiology, 74(2):527-531, 1993. 92. Tidball, J.G., Quan, D.M. Reduction in myotendinous junction surface area of rats subjected to 4-day spaceflight. Journal of Applied Physiology, 73( 1):59-64, 1992. 93. Backup, P., Westerland, K., Harris, S., Spelsberg, T., Kline, B., Turner, R. Spaceflight results in reduced mRNA levels for tissue-specific proteins in the musculoskeletal system. American Journal of Physiology, 266(29):E567-E573, 1994. 94. Baldwin, K.M., Herrick, R.E., McCue, S.A. Substrate oxidation capacity in rodent skeletal muscle: Effects of exposure to zero gravity. Journai ofApplied Physiology, 75(6):2466-2470, 1993. 95. Haddad, F., Herrick, R.E., Adams, G.R., Baldwin, K.M. Myosin heavy chain expression in rodent skeletal muscle: Effects of exposure to zero gravity. Journal of Applied Physiology, 75(6):2471-2477, 1993. 96. Riley, D.A., Ellis, S., Slocum, G.R., Sedlak, F.R., Bain, J.L.W., Krippendorf, B.B., Lehman, C.T., Macias, M.Y., Thompson, J.L., Vijayan, K.,DeBmin, J.A. In-flight andpostflight changes in skeletal muscles of SLS-1 and SLS-2 spaceflown rats. Journal of Applied Physiology, 81( 1):133-144, 1996. 97. Riley, D.A., Ellis, S., Slocum, G.R., Sedlak, F.R., Bain, J.L., Krippendorf, B.B., Macias, M.Y., Thompson, J.L. Postflight changes in muscles of rats flown 9 days aboard SLS- I . ASGSB Bulletin, 6(1):99A, 1992. 98. Riley, D.A., Ellis, S., Slocum, G.R., Sedlak, F.R., Bain, J.L., Krippendorf, B.B., Macias, M.Y., Thompson, J.L. Spaceflight and reloading effects on rat hindlimb skeletal muscles. ASGSB Bulletin, 7(1):81A, 1993. 99. Esser, K.A., Hardeman, E.C. Changes in contractile protein mRNA accumulation in response to spaceflight. American Journal of Physiology, 268:C466-C4711 1995. 100. Tischler, M.E., Henriksen, E.J., Munoz, K.A., Stump, C.S., Woodman, C., Kirby, C.R. Spaceflight and earth-based unweighting produce similar effects on muscle of young rats. ASGSB Bulletin, 6( I):57A, 1992. 101. Tischler, M.E., Henriksen, E.J., Munoz, K.A., Stump, C.S., Woodman, C., Kirby, C.R. Spaceflight on STS-48 and Earth-based unweighting produce similar effects on skeletal muscle of young rats. Journal ofApplied Physiology, 74(5):2161-2165, 1993. 102. Henriksen, E.J., Tischler, M.E., Woodman, C.R., Munoz, K.A., Stump, C.S., Kirby, C.R. Elevated interstitial fluid volume in soleus muscles unweighted by spaceflight or suspension. Journal of Applied Physiology, 75(4): 1650-1653, 1993.

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103. Lee, M.D., Tuttle, R., Girten, B. Effect of spaceflight on oxidative and antioxidant enzyme activity in rat diaphragm and intercostal muscles. Journal of Gravitational Physiology, 2( 1):68-69, 1995. 104. Baldwin, K.M., Caiozzo, V.J., Haddad, F., Baker, M.J., Herrick, R.E. The effects of space flight on the contractile apparatus of antigravity muscles: Implications for aging and deconditioning. Journal ojGravitational Physiology, 1( l):8-1 1, 1994. 105. Caiozzo, V.J., Baker, M.J., Herrick, R.E., Baldwin, K.M. Contractile properties of slow skeletal muscle following a 6-day spaceflight mission. ASGSB Bulletin, 7(1):99A, 1993. 106. Caiozzo, V.J., Baker, M.J., Herrick, R.E., Tao, M., Baldwin, K.M. Effect of spaceflight on skeletal muscle: Mechanical properties and myosin isoform content of a slow muscle. Journal of Applied Physiology, 76(4):1764-1773, 1994. 107. Caiozzo, V.J., Haddad, F., Baker, M.J., Herrick, R.E., Baldwin, K.M. Altered protein and mRNA expression of myosin heavy chain isoforms following spaceflight. ASGSB Bulletin, 7(l):79A3.1993. 108. Allen, D.L., Yasui, W., Tanaka, T., Ohira, Y., Nagaoka, S., Sekiguchi, C., Hinds, W.E., Roy, R.R., Edgerton, V.R. Myonuclear number and myosin heavy chain expression in rat soleus single muscle fibers after spaceflight. Journal ofApplied Physiology, 81(1):145-15 1, 1996. 109. O h m , Y., Yasui, W., Kariya, F., Tanaka, T., Kitajima, I., Maruyama, I., Nagaoka, S., Sekiguchi, C., Hinds, W.E. Spaceflight effects on b-adrenoceptor and metabolic properties in rat plantaris. Journal of Applied Physiology, Sl(1):152-155, 1996. 110. Caiozzo, V.J., Haddad, F., Baker, M.J., Herrick, R.E., Prietto, N., Baldwin, K.M. Microgravity-induced transformations of myosin isoforms and contractile properties of skeletal muscle. Journal of Applied Physiology, 81(1):123-132, 1996. 1 I I . Riley, D.A., Ellis, S . , Slocum, G.R., Sedlak, F.R., Bain, J.L.W., Lehman, C.T., Macias, M.Y., Thompson, J.L., Vijayan, K., De Bruin, J.A. SLS2 inflight and postflight changes in skeletal muscles of 14-day spaceflown rats. ASGSB Bulletin, 8( 1):85A, 1994. 112. Ilyina-Kakueva, E.I. Examination of skeletal muscles of rats after a short-term flight on Cosmos- 1667. Kosmicheskaya Biologiya i Aviakosmicheskayu Meditsina, 21(6):31-35, 1987. 113. Ilyina-Kakueva, E.I., Babakova, L.L., Demorzhi, M.S., Pozdniakov, O.M. A morphological study of skeletal muscles of rats flown aboard ihe space laboratory SLS-2. Aviakosmicheskaya Ekologiyu i Meditsina, 29(6):12-18, 1995. 114. Oganov, V.S., Rakhmanov, A S . , Skuratova, S.A., Shirvinskaya, M.A., Magedov, V.S. Functions of skeletal muscles of rats and monkeys after 5-day space flight (on Cosmos-1514). In: Space Physiology. (J.J. Hunt, Ed.), pp. 89-93. ESA Publication Division, Noordwijk, The Netherlands, 1986. 115. Oganov,V.S., Skuratova, S.A., Murashko,L.M., Guha, F., Takach, 0. Effect ofshort-term space tlights on physiological properties and composition of myofibrillar proteins of the skeletal muscles of rats. Kosmicheska.ya Biologiya i Aviakosmicheskaya Meditsinu, 22(4):50-54, 1988. 116. Baldwin, K.M. Effects of altered loading states on muscle plasticity: What have we learned from rodents'! Medicine and Science in Sports and Exercise, 28(10):S101-S106, 1996. 117. Edgerton, V.R., Roy, R.R. Neuromuacular adaptations to actual and simulated spaceflight. In: Hundhook qf Physiology-Environmental Physiology. (J. Fregly and C.M. Blatteis, Eds.), Vol. 1, pp. 721-763. Oxford University Press, New York, 1996. 118. Talmadge, R.J., Roy, R.R., Edgerton, V.R. Adaptations in myosin heavy chain profile in chronically unloaded muscles. Basic and Applied Myology, 5(2):117-137, 1995. 119. Fejtek, M., Alberts, J., Ronca, A.E., Wassersug, R.J. The effects of spaceflight on the abdominal musculature of the rat. Journal ofMorphology. 232:254, 1997. 120. Caiozzo, V.J., Haddad, F., Baker, M.J., Baldwin, K.M. Functional and cellular adaptations of rodent skeletal muscle to weightlessness. Journal of Gravitational Physiology, 2( 1):39-42, 1995.

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121. Edgerton, V.R., Roy, R.R. Neuromuscular adaptation to actual and simulated weightlessness. Advnnces in Space Biology und Medicine, 4:33-67, 1994. 122. Ilyina-Kakueva, E.I., Portugalov, V.V. Structural changes in the soleus muscle of rats on the Kosmos-series hiosatellites and in hypokinesia. Kosmicheskaya Biologiya i A viakosmicheskaya Medirsina, 15(3):37-40, 1981. 123. Ohira, Y., Edgerton, V.R. Neuromuscular adaptation to gravitational unloading or decreased contractile activity. Advances in Exercise and Sports Physiology, l(1): 1-12, 1994. 124. Riley, D.A., Ellis, S. Research on the adaptation of skeletal muscle to hypogravity: Past and future directions. Advances in Space Research, 3: 191-197, 1983. 125. Riley, D.A., Thompson, J.L., Krippendorf, B.B., Slocum, G.R. Review of spaceflight and hindlimb suspeusion unloading induced barcomere damage and repair. Basic and Applied Myology, 5(2): 139-14.5, 199.5. 126. Roy, R.R., Baldwin, K.M., Edgerton, V.R. The plasticity of skeletal muscle: Effects of neuromuscular activity. Exercise and Sports Science Reviews, 19:269-3 12, 1991. 127. Garenko, O.G., Ilyin, Ye.A., Cenin, A.M., Kotovskaya, A.R., Korolykov, V.I., Tigranyan, R.A., Portugalov, V.V. Principal results of physiological experiments with mammals aboard the Cosmos-936 biosatellite. Kosmicheskava Biologiyu i Aviukosmicheskayu Meditsina, 14(2):22-25, 1980. 128. Oganov, V.S. Results of biosatellite studies of gravity-dependent changes in the musculo-skeletal system of mammals. The Physiologist, 24(6):SSS-S58, 1981. 129. Oganov, V.S., Popatov, A.N. On the mechanisms of changes in skeletal muscles in the weightless environment. Life Sciences and Space Reseurch, 14: 137-143, 1976. 130. Rapcsik, M., Oganov, V.S., Sziligyi, T., Szoor, A. Effect of short- and long-term spaceflight on the contractile properties of rat skeletal muscles with different functions. The Physiologist, 36( 1):s143-S 146, 1993. 13 I . Fitton, B., Moore, D. National and international space life sciences research programmes 1980 to 1993-and beyond. In: Biological and Medical Research in Spuce: An Overview UfLife Sciences Research in Microgravity. (D. Moore, P. Bie, and H. Oser, Eda.), pp. 432-541. Springer-Verlag, Berlin, 1996. 132. Asashima, M., Malacinski, G.M. Fundamentals qf Space Biology. Japan Scientific Societies Press, Tokyo, 1990. 133. Baldwin. K.M., White, T.P., Arnaud, S.B., Edgerton, V.R., Kraemer, W.J., Kram, R., Raab-Cullen, D., Snow, C.M. Musculoskeletal adaptations to weightlessness and development of effective countermeasures. Medicine and Science in Sports and Exercise, 10:1247-1253, 1996.

Chapter 2

IS SKELETAL MUSCLE READY FOR LONGTERM SPACEFLIGHT A N D RETURN TO GRAVITY?

Danny A. Riley I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Primary lnflight Changes ..................................... 33 A. Simple Deconditioning and Adaptation . . . . . . . . . . . . . . . . 33 €3. Pathological Changes and Metabolic Adaptation . . . . . . . . . . . . . . . . . . . . . 35 C. Contractile Physiology, Contractile Proteins, and Myofilaments D. Preservation of Function Ill. Secondary Changes Induced by Reentry and Reloading. . . . . . . A. Movement in Space and Upon Return to Earth. . . . . . . . . . . . . . . . . . . . . . . B. Compromised Microcirc . . . . . . . . . . . . 40 C. Increased Susceptibility IV. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . 44 Acknowledgment.. .................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 11.

Advances in Space Biology and Medicine, Volume 7, pages 31-48. Copyright 0 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

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1. INTRODUCTION With the advent of an international space station in the 21st century and the maintenance of a permanent presence of humans in space, astronauts will experience longer periods in microgravity prior to returning to terrestrial gravity. Before this lifestyle can be accomplished with impunity, the deleterious effects on skeletal muscle of spaceflight and reloading upon return to Earth have to be better understood in order to maintain performance and prevent injury. The fact that many humans have successfully sojourned into space and returned in apparently good health does not fully vindicate space travel.'-'0 The assessment of health status has been largely carried out by noninvasive means. A recent NASA panel on countermeasures concluded that many questions remain to be investigated to identify efficacious countermeasure protocols. I The extensive compensatory and regenerative capacity of skeletal muscle could have repaired and masked pathological changes during the recovery period. If true, drawing on reserves too often may exceed their capacity and eventually result in permanent disabilities. To date, there have only been three studies of astronauts involved in relatively short missions ( 5 , 1 1, and 17 days) in which pre- and post-flight muscle biopsies were obtained. Physiological, biochemical, and structural changes were studied at the cellular and molecular level^.^,^^*^'^ Interpretation of the results from these investigations is complicated by a number of factors: the small number of subjects, possible sex differences, different levels of previous exercise conditioning, and different inflight exercise-type activities that were not adequately controlled. All of these factors could have significantly influenced the outcomes. Our present understanding of the cellular and molecular changes in skeletal muscles is derived largely from more than two decades of pre- and postflight studies of rodents orbited 1 to 3 weeks in Russian biosatellites and American Space Shuttles"-24. The first major inflight tissue acquisition from adult rats was a milestone accomplishment of the 1993 Spacelab Life Sciences mission (SLS-2).I9 A second in orbit procurement of tissues from adult and developing rats occurred in 1998 as part of the Spacelab Neurosciences mission (Neurolab). Unfortunately, this was the last scheduled Spacelab mission, so until the International Space Station is fully operative, no inflight tissue procurement and processing will be possible. The purpose of this review is to discuss primary changes in skeletal muscle induced by unloading during microgravity and secondary alterations induced by reentry and reloading. The findings surveyed are from representative human spaceflight studies, ground-based simulations of spaceflight (including prolonged bedrest), and complementary investigations of rodents subjected to spaceflight or simulated microgravity by hindlimb suspension unloading (HSU). HSU involves harnessing rats to elevate the hindquarters and remove weightbearing (loading) from the muscles of the hindlimbs.'8~'9~24-30

Spaceflight and Gravity Loading Effects on Skeletal Muscle

II. A.

33

PRIMARY INFLIGHT CHANGES Simple Deconditioning and Adaptation

The primary effects of spaceflight and HSU on skeletal muscles have been revealed by inflight dissection and by taking tissues in ground-based models before the affected muscles reexperienced weightbearing.19320,29These effects are distinguished from secondary alterations appearing in muscle tissue obtained hours to days after return to Earth or release from HSU.3,4,s,10,12-2",24,26-33 There are extensor muscles and flexor muscles. Extensor muscles such as soleus and adductor longus lift the body against gravity (antigravity muscles). These antigravity muscles show the greatest deterioration following spaceflight and HSU (Figure 1). They have slowly contracting (slow- twitch) fibers and utilize oxidative metabolism to provide resistance against fatigue. In contrast, flexor muscles such as tibialis anterior and extensor digitorum longus (EDL) contract rapidly (fast-twitch fibers) and are rich in enzymes for anaerobic glycolysis. In general, the primary changes represent simple deconditioning without pathology. These changes can be considered an appropriate adaptation for efficient functioning at a low workload, such as in a microgravity environment (Table 1). Transformation of muscle fibers from slow- to fast-type and decreased fiber size would be tolerable if astronauts did not have to return, rather abruptly, to gravity and use the microgravity-weakened muscles to deal with heavy workloads. This explains the importance of inflight countermeasures that maintain the Table 7. Primary Changes in Slow Skeletal Muscles of Adult Rats During 1 to 2 Weeks of Spaceflight Shift from quadrupedal to bipedal forelimb locomotion Loss of reaching reflex when lowered toward floor Reductions in contractile activity and rension output Progressive loss of muscle wet weight Decreased ratio of muscle weighthody weight Diminished muscle fiber cross sectional area Relative increase in the muscle cell membrane Relative elevation of macrophage concentration Lowered density of contractile filaments More fibers expressing fast myosin heavy chains More fibers expressing fast myosin light chains Reduced concentration of subsarcolemmal mitochondria Conservation of intramyofihrillar mitochondria1 content Preservation of oxidative enzyme concentrations Reduced capdbility to oxidize long chain fatty acids Elevation of glycogen and enzymes of glycolysis Decreased intramuscular blood flow and pressure Accumulation of blood proteins in the interstitiuni Necrosis of a small number of FOG fibers

34

Figure 7.

D A N N Y A. RILEY

Upper: Light microscope cross section of the slow-twitch, antigravity adductor longus muscle reacted histochemically for actomyosin ATPase activity shows normal muscle fiber types in a ground control rat from the 12.5 day Cosmos biosatellite 1887 mission." Slow-twitch oxidative (SO) fibers are lightly stained (low ATPase activity), and fast-twitch, oxidative glycolytic (FOG) fibers are darkly reactive (high ATPase activity). The small dark circular structures (arrows) at the margins of the fibers are capillaries in this highly vascularized muscle. Lower: Actomyosin ATPase reacted section of an adductor longus muscle from a Cosmos 1887 flight rat illustrates muscle fiber atrophy (36% fiber shrinkage) and slow fiber acquisition of fast fiber type properties. There is increased expression of fast actomyosin ATPase activity (18% more fibers stained moderately). Myosin heavy chain antibody staining on adjacent sections demonstrated that the moderately reactive fibers contained both slow and fast myosins. Expression of fast myosin is partly responsible for the increased velocity of contractile shortening. X270.

Spaceflight and Gravity Loading Effects on Skeletal Muscle

35

"readiness" of the skeletal muscle system to handle transition to earth gravity, which requires delivery of a high level of performance without undue injury.

B.

Pathological Changes and Metabolic Adaptation

There may also be pathological effects in the primary changes in the muscles of humans exposed to microgravity. Hindlimb suspension of adult rats for 10 days caused ischemiclike necrosis of fast-twitch, oxidative glycolytic fibers in the so leu^.^^ A possible interpretation is that the marked reduction in blood flow that occurs when a tonically active muscle becomes quiescent deprives the highly oxidative fibers of sufficient blood-borne metabolite^.^^ This metabolic vulnerability may be transient because, during unloading, soleus fibers gradually acquire an increased capacity for glycolytic metabolism. These fibers are then better equipped biochemically to derive energy anaerobically and tolerate i ~ c h e m i a . ' ~ 'It~ is ~ ' still ' ~ unexplained why this adaptation toward glycolysis is accompanied by a compromised ability to function oxidatively, which renders the muscle more fatiguable.2 The ability to oxidize long-chain fatty acids is reduced and the capacity to transport glucose is e n h a n ~ e d . ' The ~ , ~ shift ~ in metabolism is evident in the increased activity of glycolytic energy-deriving enzymes, elevated storage of glycogen, and disappearance of peripheral mitochondria.3~'5~'s~33 In normal slow fibers, mitochondria form clusters near the muscle cell membrane (Figure 2).33In the slow antigravity muscles of humans after 17 days of bedrest and in rats exposed to 7 to 14 days of HSU or spaceflight, mitochondria no longer encircle the I bands of myofibrils. They decrease in size and reorient themselves along the m y ~ f i b r i l s . ' ~ , ' After ~ , ~ ~bedrest, , ~ ~ there is an increased number of glycogen storage granules in the I bands, occupying spaces in the myofibrils vacated by thin filaments lost during fiber atrophy.36 Astronauts and rats returning after 1-2 weeks of spaceflight may experience muscle fatigue, weakness, dyscoordination and delayed onset muscle s ~ r e n e s s . ~The ' ~ ~reduced ,~~ endurance and increased fatiguability are probably due to the greater reliance of these muscles on glycolysis. C.

Contractile Physiology, Contractile Proteins, and Myofilaments

Muscle weakness following spaceflight and HSU is in agreement with a decrease of 20 to 50% in muscle fiber cross sectional area (CSA) and a preferential loss of contractile proteins relative to cytoplasmic proteins (Figures 1 and 3).2~5,7,13-15~'7-20 Surprisingly, significant atrophy was evident in human muscles after only 5 days in space.3 Muscle fiber force decreased, proportionally to or even more than the decrease in the CSA.27,31,34,35338The ratio between muscle fiber force and CSA is called the specific tension. After 17 days of human bedrest, specific tension was unchanged for soleus fibers, but after 6 weeks it had decreased by 40% for the quadriceps m ~ s c l e . ~ ~ , ~ ~

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DANNY A. RILEY

Figure 2. Upper: Electron microscope image of the edge of a crosssectioned, soleus muscle fiber of a normal rat reveals accumulations of mitochondria (M)in the cytoplasm subadjacent to the cell membrane. Deeper in the fiber, mitochondria (arrows) encircle myofibrils (bundles of thick and thin contractile filaments) in the lighter I band regions. Lower: After 14 days of suspension unloading, the soleus muscle fibers atrophy and lose the peripheral clusters of mitochondria. The mitochondria (arrows) between myofibrils are conserved, which is consistent with the retention of oxidative enzymes. Not visible at this magnification is an increase in glycogen granules indicative of a shift from slow fiber oxidative metabolism to fast fiber glycolytic metabolism. The metabolically-transformed fibers are more easily fatigued. XI 2,500.

Spaceflight and Gravity Loading Effects on Skeletal Muscle

37

Figure 3. Upper: Ultrastructural view of cross sectioned myofibrils in soleus muscle fiber from a normal rat shows that the thick and thin contractile filaments are densely packed within the myofibrils. Lower: In this atrophic fiber, the moth-eaten appearance of myofibrils (arrows) results from a loss of thick filaments and represents a decrease in the filament packing density after 13 days of suspension unloading.33 These fibers exhibit a decrease in contractile force output per cross sectional area (specific tension) as well as an increased velocity of contraction shortening. XI 9,000.

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DANNY A. RILEY

It has been suggested that a reduction in specific tension may be due to the transformation of slow to fast fibers. However, the alleged lower specific tension of fast fibers has been questioned for estimates made from cryostat- frozen sections.38 More direct measures of single fibertensions and diameters in isolatedphysiological preparations indicate no difference in specific tension between slow and fast fibers over a range of 1 I0 to 160kN/rn2. The physiological measurements may be distorted by significant swelling of skinned Nevertheless, longer duration bedrest may result in a more complete fiber-type transformation. Fast fibers have more sarcoplasmic reticulum and thinner myofibrils to permit expedient and uniform exchange of Ca2+ions during cycles of rapid c o n t r a c t i o n / r e l a ~ a t i o nThe . ~ ~ soleus ~~~ muscles ofrats, after7days ofHSUexhibitedadramatic (54%)increase in the velocity of shortening and a 17% decrease in specific tension.31 A possible explanation for the reduced tension is the disproportionate loss of thick (myosin containing) filaments visible in electronmicrographs (Figure 3).33 Accelerated loss of thick filaments is suggested to be the consequence of the foot-drop posture in the HSU rat, a behavior that chronically shortens the working range of the soleus by about 20%.33 Reorganization of sarcomeres in shortened muscles is necessary to reestablish optimal overlap (crossbridge interaction) of thick and thin filaments for the midpoint of the abbreviated working range. Adjustment in the number of sarcomeres in series in fibers operates throughout our lifetime. The process is especially important for the increase of fiber length along with the elongation of the growing skeleton. Another consequence of the reduced packing density of contractile filaments is an increased shortening velocity.39 This may account for the speeding up of slow fibers without an associated elevation of fast myosin (heavy and light chain) observed in single-fiber m e a s ~ r e r n e n t s . ~We ” ~ ~have morphological and physiological evidence that a 20% reduction in thin filaments after 17 days of bedrest accounts for the elevated shortening velocities of soleus fibers.35 A similar reduction was detected after a 17-day spaceflight! When floating in microgravity, humans are prone to foot-drop posture (ankle plantarflexion). This shortens the extensor compartment and appears to accelerate loss of thick filaments. The abolition of weightbearing (unloading) appears to diminish thin filament concentration. Astronauts that exercise on bicycle ergometers and treadmills to preserve muscle strength and endurance, also counteract the shortening adaptation. During these exercises, flexion of the ankle stretches the soleus through its full range. Even the strength testing sessions conducted during bedrest and in orbit, involving around 300 voluntary contractions of the foot against a strain gauge (dynamometer), may have reduced degenerative changes in soleus muscle fiber^.^,'^,^^ D. Preservation of Functions during Atrophy

It is truly amazing how muscle tissue compensates the loss of function due to atrophy. The elevated speed of shortening, resulting from decreased contractile

Spaceflight and Gravity Loading Effects on Skeletal Muscle

39

filament packing density and increased fast myosin expression, compensates for the reduced force by diminishing the loss in output of power (= product of velociiy times force).4326,27,3 Another example is the increased capillary density, which occurs when muscle fibers shrink in size more rapidly than the downsizing of the microvascular n e t ~ o r k .This ~ theoretically compensates for the lower blood flow, because the average diffusion distance from the capillary to the center of muscle fiber decreases (Figure 1). Muscle fatiguability is also forestalled by the slower reduction in mitochondria1 content relative to contractile protein loss. This conserves the normal concentration of intermyofibrillar mitochondria and associated oxidative enzyme capacity (Figure 2).22'0,16,'8333

111.

A.

SECONDARY CHANGES INDUCED BY REENTRY AND RELOADING Movement in Space and Upon Return to Earth

In weightlessness, both bipedal humans and quadrupedal rats move about by Because of the loss of proprioception in weightusing the upper or Table 2. R+2 hours

Postflight Secondary Changes in Slow Skeletal Muscles of Adult Rats

Overt weakness, fatigue, and dyscoordination Reduced quadrupedal walking speed Noninflammatory interstitial edema R+5 hours Sarcomere eccentriclike lesions Mast cell degranulation R+7 hours Scattered muscle fiber necrosis Elevated interstitial edema Increased muscle wet weight Increased muscle fiber necrosis R+10 hours Activation of' macrophage phagocytosis Extravasation of red blood cells Thrombosis in postcapillary venules Recovery of coordination, walking speed, and reaching reflex R+2 days Ischemic-anoxic-like tissue necrosis Inflammatory myopathy with mononuclear cell infiltration Neutrophil and monocyte invasion in damaged areas Continued macrophage phagocytosis of damaged fibers Sarcoinere lesions patched in intact fibers Extravasated red blood cells Activation, proliferation, and growth of satellite cells R+9 to 14 days Muscle weight/body weight recovered Muscle fiber areas not recovered Sarcoinere lesions fully repaired Regeneration of muscle fiber5 well established Enlarged interstitial area due to regeneration Note: R+n = Time after recovery (in n hours or days) when change occurs.

DANNY A. RILEY

40

lessness and the dependence on visualizing limbs for positional information, it is not surprising that the less visible hindlimbs or lower limbs are less frequently utilized. A novel pattern of locomotion evolves that is appropriate and sufficient for directed movements in microgravity. The weightless astronaut soon ceases the reflex of stepping out with a foot when moving forward. Rats pull themselves around with their forelimbs, and the hindlimbs trail outstretched behind.20 Upon return to Earth, astronauts must reactivate Earth-gravity motor skills, such as recalling to step out to prevent falling when moving forward. Immediately upon return they are very unstable from a combination of orthostatic intolerance, altered otolith-qpinal reflexes, relying on weakened atrophic muscles, and inappropriate motor patterns.2,20 In the first few hours after landing, spaceflown rats do not extend their limbs and reach for the ground when lowered to it. This reflex. beneficial in Earth-gravity, returns in one day.20Spaceflown rats walked significantly slower than normal during the first two days, but by the third day they ambulated as fast as ground controls. The jerky, stilted, stepping of the hindlimbs quickly evolved to the smooth walking pattern of a terrestrially adapted rat.2" Early during spaceflight, humans subjected to sudden "drop tests" ceased anticipatory contractile activity in the extensor muscles, as observed by electromyography. This reflex returned to normal within a day after landing. Thus, terrestrial motor skills are restored rapidly and strongly, well before muscle fiber regrowth (recovery of CSA) and still during slow muscle necrosis. The central nervous system appears to undergo significant reprogramming (plasticity) and provide compensatory activation of motor units, which masks the deteriorated state of the neuromuscular system.2,9,1720 B.

Compromised Microcirculation

The headward fluid shift and the reduced muscle contractions (musculovenous pumping) in microgravity result in a reduced blood flow in the lower (hind) limbs.'332,42This is associated with a movement of blood proteins, such as albumin, into the interstitium.6 In the absence of musculovenous pumping, the return of the extravasated proteins to the vascular system via postcapillary venules and lymphatic vessels proceeds less efficiently.4244 At the shortest time examined after Shuttle landing (2 hours after wheel stop), the slow adductor longus muscles of rats already showed simple (noninflammatory) interstitial edema, which was not evident inflight (Table 2).20 By 2 days postflight, the muscle condition has advanced to inflammatory myopathy with more severe edema (Figure 4). This scenario of reloading-induced edema is also presented by rats after 12.5 days of HSU and subsequent reloading of antigravity slow muscle^?^ The postflight pooling of blood in the lower limbs was not present in the legs of astronauts during quiet standing initiated about 4 hours after landing.' This indicates that the pull of gravity (hydrostatic pressure) alone is not sufficient in the

Spaceflight and Gravity Loading Effects on Skeletal Muscle

41

short term to cause fluid accumulation in relatively quiescent limbs. The onset and severity o f interstitial edema in rats appears to be linked to the intensity of postflight muscle contractile activity. 16-20329 The osmotic pressure of the extravascular proteins is thought to pull water into the interstitiurn when the microvascular network is reperfused with blood at high pressure and flow in response to resumption of strong muscle contractions. 18320 If the muscle activity is sufficiently strenuous (“sufficient” is undefined), interstitial edema increases and leads to ischemiclike tissue necrosis causing mast cell degranulation and greater vascular permeability (Table 2).2” At this stage, muscles exhibit inflammatorylike myopathy with infiltration of mononuclear cells (Figure 4). 17,2”,29 Since on Earth muscles can become edematous during intense e ~ e r c i s e : ~ , ~ it ~ appears that adaptation to microgravity lowers the threshold for the onset of edema. These results suggest that edema in returning astronauts may be minimized by avoiding strenuous muscle contractions during readapation to gravity and possibly by medication before reentry with drugs that block mast cell degran-

Figure 4.

Rat soleus muscle section, stained with hematoxylin and eosin after 12.5 days of spaceflight and 2 days of reloading in earth gravity, shows inflammatory myopathy.lb There is interstitial edema (expansion of the connective regions between fibers) with invasion of mononuclear cells (macrophages, monocytes and neutrophils) and ischemic-like muscle fiber necrosis (arrow). X220.

42

DANNY A. RILEY

ulation. It appears that under-used microvessels adapt to low flow and pressure during spaceflight and HSU and become inherently “more leaky” during the rapid onset of high blood flow and pressure upon resumption of gravity-loaded muscle contraction^.",^^,^^ Furthermore, the unloading-induced shift from oxidative to glycolytic metabolism results in a more robust stimulation of blood pressure during muscle contraction.46 These data indicate that flushing extravasated proteins from the interstitium by exercise-induced muscle contractions combined with microcirculation filling by the induction of lower body negative pressure (LBNP) is the type of multidirectional countermeasure needed for prolonged spaceflight to minimize reentry reloading-induced edema and ischemic tissue necrosis. Indeed, the LBNP regimen and the exercises performed routinely by Russian cosmonauts aided their successful readaptation to gravity after return from a year in space.5 This circulation-related problem indicates that muscle adaptation during spaceflight goes beyond changes restricted to muscle fibers. In fact, the nervous and cardiovascular system involvement in muscle performance reminds us that we are sending organisms, not isolated organ systems, into space. Effective countermeasures need to target multiple systems.

C . Increased Susceptibility to Structural Damage Atrophic muscle fibers resulting from spaceflight and HSU are structurally weakened. They are thus more susceptible to eccentric (lengthening) contraction tearing of contractile elements, fiber cell membrane (sarcolemma), and associated connective tissue (Figure 5).’6-’9347These tissue changes are reminiscent of those in Earth-gravity-adapted human muscles associated with delayed onset muscle soreness after unaccustomed strenuous exercise, especially muscle lengthening biased, and in rat muscles stimulated electrically to generate forceful eccentric contraction~.~~p~~ Some astronauts are aware that minimizing during the first days back on Earth activities that eccentrically load their leg muscles, such as walking down stairs, reduces the severity of delayed-onset soreness and stiffness.36 Adaptation to the lower workload during spaceflight or HSU appears to render muscle tissue more prone to structural failure when reloaded. This is particularly noticeable in the lengthening contractions. 6-20,47 This phenomenon is partly explained by the relatively greater workload on the antigravity muscles because of fiber atrophy.2320 A 50% decrease in soleus mass is equivalent to increasing muscle loading by doubling the body weight. However, there may also be a lowering of the threshold for structural failure so that muscle fibers are damaged at a specific tension level that was previously tolerated without injury. 8p20 A disproportionate decrease in structural proteins (which harness tension) relative to contractile proteins (which generate tension) would lower this threshold, but this remains to be demon~ t r a t e d . ~There ’ are structural similarities between postflight damage, sports-

Spaceflight and Gravity Loading Effects on Skeletal Muscle

43

Figure 5.

Toluidine blue-stained longitudinal section of adductor longus muscle fibers of a rat flown for 9 days on SLS-1 illustrates light areas of eccentric contraction-like sarcomere damage in which the contractile filaments have been disrupted and lost about 5 hours after landing.18 The muscle fibers atrophied during the 9-day spaceflight and became susceptible to earth gravity reloading injury. X950.

related injuries, and degeneration in muscular d y ~ t r o p h y . As ~ ~ President -~~ Clinton demonstrated by the hyperexertion injury of his quadriceps, skeletal muscles are capable of generating more force than the connective tissue (interstitium and myotendinous junctions) can tolerate without structural failure.55 The atrophic degenerative changes at the myotendinous junctions and along the length of the fibers that have been noted after spaceflight and HSU are consistent with a reduced safety margin for structural integrity during weightbearing contractions, 1 8 ~ 2 0 ~ 2 3 Structural proteins can fail within the sarcomeres. This process has been thoroughly reviewed for Earth-gravity adapted muscles.50 The sarcolemma (cell membrane and basal lamina) is another potentially weakened component. Cytoskeletal actin and intermediate filaments normally transmit contractile force through

DANNY A. RILEY

44

linking proteins to integral membrane glycoproteins that bind to extracellular matrix (integrins to fibronectin and dystrophin/dystroglycan to lamir1in-2).~~53,56,57 Absence of a single component protein of the dystrophin-glycoprotein complex can result in greater susceptibility to contraction-induced tearing of the ~ a r c o l e m m a . ~This ~ ' ~ is ~ , seen ~ ~ in human dystrophies and mouse dystrophy mutants.57 Muscles of the mdx dystrophic mouse are more easily torn during contraction than the corresponding muscles in normal animals.54 HSU renders the atrophic soleus muscle more susceptible to contraction-induced muscle tearing but not the nonatrophic extensor digitorum longus muscle.47 Investigations are underway to determine whether the ratio of sarcolemmal to contractile proteins is lowered to shift the balance toward increased susceptibility. Fortunately, genetically normal muscle fibers rapidly restore sarcomere lesions by Z line-like patching and segmental necrosis by membrane sealing and satellite cell regeneration.'7,19920~3035x This restorative ability is decreased in dystrophic muscles, which therefore undergo more extensive and persistent degeneration. This may also be the case for spaceflight-induced muscle a t r ~ p h y . ~There ' is a need for studies of the possibility to promote repair processes with growth factors and pharmacological agents and thus to minimize the negative impact of muscle injury on astronaut p e r f o r m a n ~ eExtravehicular .~~ activity (space walks) of the astronauts during the Hubble telescope repair mission apparently produced muscle soreness. This problem needs to be abolished because space walks will have to be extensively used during construction of the space station.

IV.

CONCLUSIONS AND SUMMARY

It is now clear that prevention of muscle debilitation during spaceflight will require a broader approach than simple exercise aimed at strengthening of the muscle fibers. The levels of several hormones and receptors are altered by unloadPharmacotherapy and gene ing and must be returned to transfer strategies to raise the relative level of structural proteins may minimize the problems faced by astronauts in readapting to Earth-gravity.' ',2239,6'-64 Up to now, we have only minimally exploited microgravity for advancing our understanding of muscle biology. A research laboratory in the space station with a centrifuge facility (gravity control) is essential for conducting basic research in this field. Microgravity has proven an excellent tool for noninvasively perturbing the synthesis of muscle proteins in the search for molecular signals and gene regulatory factors influencing differentiation, growth, maintenance and atrophy of muscle. Understanding the relation between blood flow and interstitial edema and between workload and subsequent structural failure are but two important problems that require serious attention. The roles of hormones and growth factors in regulating gene expression and their microgravity-induced altered production are other urgent issues to pursue.

Spaceflight and Gravity Loading Effects on Skeletal Muscle

45

These types of studies will yield information that advances basic knowledge of muscle biology and offers insights into countermeasure design. This knowledge is likely to assist rehabilitation of diseased or injured muscles in humans on Earth, especially individuals in the more vulnerab!e aging population and persons participating in strenuous sports. Will the skeletal muscle system be prepared for the increased exposure to microgravity and the return to gravity loading without injury when space station is operational? The answer depends in large part on continued access to space and funding of ground-based models and flight experiments. The previous two decades of spaceflight research have described the effects of microgravity on multiple systems. The next generation of experiments promises to be even more exciting as we are challenged to define the cellular and molecular mechanisms of microgravity-induced changes.

ACKNOWLEDGMENT Preparation of this manuscript was supported in part by NASA grant NAG-2-956 and NIH grant 5UO 1-NS33472.

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Spaceflight arid Gravity Loading Effectson Skeletal Muscle 27

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63.

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Chapter 3

NUTRITION AND MUSCLE LOSS IN HUMANS DURING SPACEFLIGHT

T.P. Stein I. Introduction. . . . . . . . . . . . . . . . . . . . ...................... 50 A. spaceflight Effects and Recovery from Spaceflight . . . . . . . . . . . . . . . . . . . 50 52 B. Importance of Protein Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 11. Muscle Loss in Spaceflight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Muscle Loss in Rats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 ................................. 56 B. Muscle Loss in Humans C. Comparison of Human a esponses . . . . . . . . . . . . . . . . 111. Protein Loss in Spaceflight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 A. Ground-Based Models: Need and Problems . . . . . . . . . . . . . . 59 . . . . . . . . . . . . . . . . . 60 B. Possible Causes of Protein Loss . . . . . C. Metabolic Stress Response D. Pathological Breakdow

B.

................................... ..................... Sampling Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

E.

Cortisol . . . . . . . . . . . . . . . . . . . . . . .

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

Advances in Space Biology and Medicine, Volume 7, pages 49-97. Copyright 0 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

49

. . . . . . . . . . . . . . . . . . 73

T.P. STEIN

50

F. Prostaglandins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 V. Energy Deficit and Nutrition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77 A. Energy Deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 B. Nutritional Needs of Astronauts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 References 89

1. A.

INTRODUCTION

Spaceflight Effects and Recovery from Spaceflight

Spaceflight is associated with chronic losses of protein from muscle and calcium from bone. The major sites of these losses are the muscles and bones with anti-gravity function, which are located mostly in the trunk and legs (Table 1). 1-4 Even though these changes leave the body poorly adapted for a return to Earth gravity, most interest has focused on the inflight period because of its novelty. Eventually more attention will have to be directed to the recovery process-or as it is probably better described, the readaptation process. Once humans start adventuring forth to the Moon and Mars and beyond, the ability to function and stay healthy will be crucial and this means that humans have to be able to successfully adapt to different levels of gravity. Currently it is estimated that a round trip to Mars will last about 30 months and there will be four transitions to different levels of gravity: from 1 G to 0 G for the journey to Mars, from 0 G to 0.3 G on Mars, from 0.3 G to 0 G for the return trip, and finally from 0 G to 1 G after landing back on earth. The decrease in muscle mass has been a consistent finding in humans and animals after short- and long-duration space missions. Although the changes in muscle mass and muscle functional capacity are usually described as “muscle Table 1.

Mean Changes in Muscle Volume after an 8-Day Spaceflight

Muscle Calf Anterior Soleus + Gastrocnemius Thigh Quadriceps Hamstrings Lumbar Intrinsic Psoas Notes:

R+I‘

R+15‘

-3.9 ? 0.5b -6.3 f 0.6b

-3.3 f l . l C -4.4 f 2.2=

-6.0 f 1.7‘ -8.0 ? 0.9b

-3.1 f 2.3 -4.8 * 1.3h

-10.3 ? 2.4b -3.1 f 1.5

-5.9 ? 1.5b -2.4 ? 1.6

Data for four astronauts from 1 and 15 days posttlight (from ref. 4) bp<0.05 versus preflight; ‘p<0.07 versus preflight

51

Nutrition and Muscle Loss

Table 2.

Comparison of Muscle Loss after Spaceflight and Bed Rest Muscle

Spaceflight

Calf -15 & 3 Anterior -18 + 4 SoleudGastrocnemius Thigh -11 + 3 Quadriceps -13 ? 4 Hamstrings Lumbar Intrinsic -18 4 Psoas -6 ? 6 Nute: After 4 months of spaceflight and bed rest; adapted from ref. 7.

*

Bed Rest

-20 ? 4 -31 2 5

+3 -14 * 3 -18

*

5 0+6

10

atrophy", this is not strictly true; what is being observed is a remodeling of the muscle in adaptation to the changed environment. The losses have occurred on both U S . and Russian missions despite attempts to ensure an adequate diet and a vigorous exercise regimen.23536 Analysis by magnetic resonance imaging (MRI) after the Shuttle-MIR (1 995) missions by LeBlanc and colleagues showed that after 115 days in space the protein loss from the various muscle groups ranged from 16 to Comparison of these spaceflight data with those from a similar period of bed rest shows that the trend is very similar, although there are some differences (Table 2 ) . When astronauts and rats return from spaceflights lasting only 1 to 2 weeks, they often experience muscle fatigue and weakness, lack of coordination in movement, and in the case of humans, muscle Isometric, concentric, and eccentric force development declines by as much as 30%. The loss of muscle mass is at least partly responsible for the decrease in muscle strength and the increased fatigability observed after Neural pathways from the central nervous system to the affected muscles and the recruitment of muscle fibers are also impacted because coordination of movement after flight is poor. In the case of rats, the weakness and poor limb coordination are striking: the animals have difficulty in raising their bodies above the cage floor and their pattern of movement does not return to normal for several days. l 2 This postflight muscle weakness is paralleled by a considerable (20-50%) decrease in cross sectional area of the muscle fibers and a preferential loss of contractile proteins relative to cytoplasmic proteins.6,8,'2-'6 Movement and the force that can be applied by muscle depend on the level of motor pool activation, on coordination of the motor pools, and on the amount of muscle protein available to effect the movement. This review focuses on the protein content and the role of nutrition, with emphasis on data obtained on humans. On Skylab, most of the protein loss occurred in the first month, but it continued into the third Grigoriev reported that during a 1-year flight on MIR, the calf volume of the two cosmonauts steadily decreased and that by the end of

T.P. STEIN

52

the flight it was 2 0 8 below the preflight baseline." This occurred in spite of two daily exercise sessions. If the loss of body protein is chronic, this is a serious concern calling for counter-measures. Death inevitably results when body protein loss approaches a 30 to 40% level. 19320Intermediate protein losses are associated with progressive loss of strength and endurance, decreased immune competence, impaired wound healing, and increased susceptibility to disease. 21-23 It is not yet known whether the observed protein loss reflects a final (adapted) state or a continuing chronic loss of body protein. Although most, if not all astronauts lose protein, there is some variability in the amount of protein lost. If the protein loss is part of an adaptive response of the body's musculature to the new environment that is, once attained, is stable, then the problem is finite and attention needs to be focused on maintaining functional capacity inflight and facilitating recovery postflight. It would seem likely that the process is an adaptation to a new steady state, but there is an important caveat: If there is an energy deficiency, as has occurred on two of the three missions for which energy balance data are available, 24,25,'62 true adaptation may never occur. Rather there will be an accommodation to limit the rate of loss of body protein. The ability of humans to remain in microgravity will be limited and missions lasting more than 1.5 years, such as a round trip to Mars will not be feasible. A chronic, uncontrolled loss of protein, either from a failure to completely adjust to microgravity or from an energy deficit, is therefore potentially very serious. In order to understand the reason for this statement, it is necessary to appreciate the role of protein in the body.

B.

Importance of Protein Metabolism

Quantitatively, the two major proteins in the body are the structural proteins, collagen and elastin, which together make up about half of the total protein in an adult. Normally, collagen and elastin turn over very slowly with half-lives of months or even years and so contribute little to the body's protein metabolism except after injury. Of the remainder, half consists of other muscle proteins and the rest is distributed among the various visceral organs and blood proteins. These proteins are in a dynamic state; they are continually being broken down to their constituent amino acids, which are then reincorporated into new protein (Figure 1). The process is called protein turnover. Humans make and break down about 300 g of protein per day. In general, visceral protein synthesis rates are much higher than muscle protein synthesis rates. Tissue proteins have half-lives of the order of 3 to 6 days. Muscle proteins turn over much slower, their half lives ranging from 20 days and longer depending on the muscle. Even though the actual rate of muscle protein synthesis is low compared to that of visceral protein, the total amount of muscle protein made per day is large because there is so much muscle in the body. A dynamic protein metabolism provides some major benefits to an organism:

53

Nutrition and Muscle loss Muscle 75 9

-

I

Skin

3

SYNTHESIS 300 g

INTAKE

____3

(Food) 80 9

PROTEIN 12000 g

BREAKDOWN 300 g

I \ Secreted intogut 79 9

1 1

Viscera (liver, lung, gut, brain etc.) 120 g

39 Collagen & Elastin, 5 g

--+

Plasma proteins. 12 9 Albumin Fibrinogen 2g tinmunoproteins 5 g Hemoglobin 8g WBCS 20 9

Urine 70 9 -1lgN

I -

EXCRETION

Figure 1. Protein metabolism and synthesis in various tissues. Units are g/d, except total body protein in g. Compiled from various sources.

Protein turnover makes maximal use of a minimal amount of amino acidsan example of economy of function. The body has no readily identifiable reserve amino acid stores, as is the case for carbohydrate (glycogen) and fat (adipose tissue). A continuing protein turnover allows unused enzymes to be replaced by needed enzymes. A continuous cycle of synthesis and breakdown allows for the removal of defective proteins. Most importantly, protein turnover is a major means of metabolic regulation.26 For a terrestrial vertebrate, gravity is a serious handicap; the metabolic cost of gravity increases with increasing size. This puts a constraint on space utilization within the body and precludes the maintenance of optimal levels of all proteins in optimal concentration at all times. Many proteins are needed at high concentrations only intermittently and at unpredictable times, for example, immunoglobulins and host defense proteins. In general, the more important a protein is in intermediary metabolism, the higher its turnover rate. This means that substrate fluxes through these pathways can be regulated by the amount of enzyme present as well as by the levels of hormones, activators and inhibitors. Regulation of the amount of an enzyme present

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is a very specific process. Thus, enzymes, particularly those at the branch points of metabolic pathways, tend to have very short half-lives, whereas structural proteins such as collagen hardly turnover at all. Protein turnover is an example of a multistep substrate cycle.27 A substrate cycle exists when opposing, nonequilibrium reactions catalyzed by different enzymes are active simultaneously. Substrate cycles are important for metabolic regulation and exist for all three of the macro nutrient^.^^'^^ Substrate cycling allows small changes in the concentrations of effector molecules, such as nutrients, hormones, activators, and inhibitors, to rapidly effect large changes in flux through metabolic pathways. For multistep substrate cycles such as protein turnover, the advantages are the same, but there are more potential control points. Substrate cycling allows the body to respond rapidly to altered circumstances.28In the case of the protein cycle, it allows the body to maintain levels of many different proteins and rapidly increase the concentration of any given protein when needed. Protein turnover is not a factor involved in very short-term regulation, which may occur on a minute-to-minute basis, but rather occurs for periods of hours, and longterm adaptation protein turnover is probably even the dominant factor in metabolic regulation.26 This capacity for longterm adaptation is progressively lost as the protein cycle becomes impaired. The cause could be a shortage of either free amino acids or of energy for protein synthesis. The consequence is a progressive impairment of the ability to respond to a challenge, be it a new environment, the need for a fight or flight response, invasion by a pathogen, or an injury. A dynamic protein turnover is very expensive in energy costs, because protein synthesis and its associated processes require a large amount of energy. The incorporation of an amino acid into a protein uses 3 molecules of ATP and 1 molecule of GTP. In addition, the amino acids must be present in the right place and in correct amounts when needed, and an excess of amino acids must be disposed of. Together these processes account for about 20% of the basal metabolic rate. The expenditure of such a high proportion of the metabolic rate on protein turnover attests to its physiological i m p ~ r t a n c e . ~ ~

II.

MUSCLE LOSS IN SPACEFLIGHT A.

Muscle Loss in Rats

Most of the available information on the changes in muscle structure during spaceflight comes from studies on rats. Ilynia-Kakueva was the first to report the differential muscle atrophy after spaceflight in Table 3 shows how the loss of gravity affects the protein content of various rat leg muscles. l 4 Mammals have two classes of muscles that can be distinguished on the basis of their color and speed of contraction. Fast-twitch muscles (pale) depend on glycolytic mechanisms for their energy, whereas slow twitch muscles (red) are rich in myoglobin

Nutrition and Muscle

Loss

Table 3.

Loss of Muscle Mass in Rats during Spaceflight

MUsClr

Soleus Adductor longus Extensor digitorurn longus Plantaris Tibealis anterior Mcdial gastrocnemius Notes:

55

Change in c/o

Controlu

Flight'

104? 2 55 ? 3 116?3 281 + I 500 ? 13 1379? 34

66 f 3b 40 ? 2h

-37

98 ? 4h 220 ? 9 h 453 ? 17b 1070 ? 4 1'

-16 -22 -9 -22

-27

"Wet weight in mg in ground control and spaceflight animals. Valuer are mean f SEM for 6 animals per group bp
and depend on aerobic respiration. Muscles with anti-gravity functions such as the soleus and the exterior digitorum longus muscle suffer the highest protein losses. 13,14,31-34Slow-twitch muscles lose more protein than fast-twitch muscles (Table 3). 3,14,3",33335 Thus the soleus, a slow-twitch muscle, lost more protein than the extensor digitorum longus muscle, a fast-twitch muscle. Nonweightbearing muscles, such as the gastrocnemius and vastus medialis, lose less protein. The loss of muscle mass is due to a decrease in fiber cross-sectional area rather than to a loss of cells. After 7 days of spaceflight, the cross sectional area of the soleus slow- and fast-twitch fibers have decreased by 40% and 3096, respectively.31The protein loss is mainly due to a decrease in myosin content,36but the ATPase activity of the myosin is u n a f f e ~ t e dThe . ~ ~ decrease in size and strength of the muscle is due not only to a loss of selective proteins but also to a loss of sarcolemmal mitochondria, downsizing of the microvasculature, degeneration of the motor innervation, and loss of ~ a t e r . ' ~ There ,'~,~ is ~also some loss of regulatory proteins.39 In spite of this loss of protein, the oxidative metabolic potential is r n a i r ~ t a i n e d . The ~ ~ . ~decrease ~ in muscle fiber cross section area is paralleled by an increase in the capillary density, which is greatest in the muscles that show the largest losses of water and p r ~ t e i n . ' . ' ~The increased capillary density reflects the compacting of the atrophied fibers. In tail-suspended rats the blood flow to the atrophied muscles is decreased, even though the number of capillaries is not.44 A decrease in blood flow will result in decreased substrate delivery to the affected muscles. This could be an important contributing factor to the muscle atrophy,15,31,32 A pivotal experiment was the inflight tissue acquisition from adult rats achieved during the 1993 Spacelab Life Sciences mission (SLS-2).I2 Hitherto all information had been derived from rats sacrificed at varying times after return to Earth, with the time elapsing between landing and tissue procurement ranging from 2 hours to 2 days. This made it difficult to separate changes due to spaceflight and subsequent recovery on Earth. It is clear that the greater the time elapsed after landing, the more likely it is that the observed histological changes occurred during the recovery period. Compounding the problem with some of the

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Russian studies were the rough landings. The inflight dissections on SLS-2 showed that all of the damage observed on previous studies occurred after landing. While the muscles from the rats sacrificed inflight showed similar atrophy to that found with the postflight dissections, there was no actual damage. After a spaceflight, the muscles of rodents are extremely susceptible to damage. l 2 In other words, the damage found in postflight investigations occurred after landing and not inflight.

B. Muscle Loss in Humans Because of the obvious limits for experiments on humans, the data available on humans are of a different and much less detailed nature than those for rodents. The human data consist primarily of inflight nitrogen balance determinations, postflight body composition measurements, and a few muscle biopsies taken after landing. Body composition analyses have shown that the main site of muscle loss is, as in rats, in the anti-gravity muscles of the lower back and legs. Table 2 compares the losses found after long duration spaceflight ( I 1.5 days) on MIR with those observed after four months of bed rest. Although they are similar in many respects, there are differences that can be attributed mostly to the different type of unloading in spaceflight and bed rest. Muscle biopsy studies have confirmed the magnetic resonance imaging (MRI) findings. The biopsies were taken pre- and post-flight from astronauts after relatively short Shuttle missions ( 5 , 1 I , and 17 days).38 Significant atrophy was evident in the quadriceps after only 5 days in space. Unfortunately, detailed interpretation of the muscle biopsy data is problematic because of the small number of subjects, differences in the amount of inflight exercise, and most importantly, differences in dietary intake during flight. Nevertheless the findings are similar to those for rodents, namely that the spaceflight-induced atrophy of fasttype fibers appears to be greater than that of slow-type fibers in the same mus~ l e These . ~ ~findings suggest that human muscle responds qualitatively in the same manner as rat muscle to ~ p a c e f l i g h t .There ~ ~ is a difference between the effects of spaceflight and, in one of the ground models, single-leg unloading: in the latter case, there was no change in the ratio of fast myosin fibers to slow myosin fibers.45 In humans a daily record of any changes in body protein content can be obtained by monitoring nitrogen balance. Nitrogen balance is the most sensitive and reliable method for detecting small changes in body protein ~ o n t e n t . It~ ~ - ~ ~ requires accurate measurements of both intake and excretion of nitrogen. A problem is that errors, although small, tend to be unidirectional leading to a slightly more positive balance than existing in real it^.^^,^^ Three nitrogen balance measurements have been performed to date, the first being the historic Skylab study The Skylab team measured both urine and fecal nitrogen from the mid 1970~.',~' output, the two later studies in the Space Shuttle were restricted to urine measure-

Nutrition and Muscle loss

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Nitrogen balance of the SLS-1 and SLS-2 crew members. Data for the two missions have been combined (ref. 52).

Figure 2.

ments. There is an extensive literature on how to derive the nitrogen balance from urine-based data, so the lack of inflight fecal collections is not a Fecal and insensible nitrogen losses are small and relatively constant; fecal losses on Skylab were about 20 mg N kg-'.d-' and were unchanged from those on E a ~ t h . ~ "All . ~ ' three studies showed a decreased nitrogen retention, although the magnitude varied with the mission and time in space. Combination of the SLS-I and SLS-2 data (short and intermediate term) with the Skylab data (longterm) gives a good picture of the changes in nitrogen balance during spaceflight (Figure 2). As with other parameters of protein metabolism, the observed changes in nitrogen balance are a composite of the responses to microgravity and changes in energy balance. This is particularly true in the early part of spaceflight. The initial response is best illustrated by the data from SLS-1 and SLS-2, where the nitrogen balance data were not complicated by a chronic negative energy balance.52

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1

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DAYS

Figure 3. Energy intake by the SLS-1 and SLS-2 crew members. Data for the two missions have been combined (from ref. 52). Energy intake for the flight period was reasonably constant, although somewhat less than preflight (Figure 3; see also section V). A plausible explanation for the complex pattern is that muscle begins adapting immediately to the reduced workload by losing nitrogen, but that this loss is increased due to the negative energy balance (decreased food intake). When the energy balance is partly restored on days 3 to 6, there is a transiently more positive nitrogen balance reflecting a catch-up for the intake deficiency on flight day 1 by tissues such as liver, overlying the muscle protein loss. After about 9 to 10 days in space the early losses of visceral protein have been compensated for, and the nitrogen balance reflects the loss of protein from muscle. For astronauts in energy balance, the slow loss of protein at that time reflects the natural downsizing of muscles with anti-gravity function in response to the decreased load.52Data from the long duration Skylab missions (28-84 days) showed that the rate of protein loss declined with increasing time in orbit.',5

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The only comprehensive longterm data available are those from the three Skylab missions, which lasted 28, 56, and 84 days. Dietary intake and the amount of csercise increased from the 28-day mission to the 84-day mission. There appears to be a trend for the nitrogen losses to decrease with increasing energy intake. The negative nitrogen balance persisted for the duration of the mission but declined with increasing time in space.25Such a reduction is not unexpected; it either indicates the approach of equilibrium or, more likely, the functioning of natural mechanisms to limit the rate of protein loss in the face of the energy deficit existing during the Skylab missions.

C.

Comparison of Human and Rat Responses

Overall, the human response to spaceflight appears to be much less severe than for rodents. It is not clear why this should be so. If anything, the muscle loss in humans should be greater because the larger the animal, the greater the fraction of the body represented by supporting structure^.^^ Some possible explanations for the differences between the responses of rats and humans follow: ( 1 ) in assessing the effects of weightlessness on humans during spaceflight, it is not, as Dietlein pointed out, “the absolute responses that are being measured”, because humans do not vegetate in space;54 humans make a conscious effort to maintain a healthy state (e.g., they exercise); ( 2 ) the rats in some missions may have suffered from under-nourishment as well as the effects of microgravity; (3) space vehicles are designed to minimize changes from the normal lifestyle for astronauts, whereas the housing conditions for the animals are confining and restricting movement, and the noise of the life support systems may disturb the rats; (4) whereas astronauts are mature adults cognizant of their novel environment, growing rats were used in some missions (allowing more animals to be fitted into the cages), introducing a “rate of growth” component in the data; ( 5 ) the psychological effect of spaceflight is an “emotional high” for humans but not for rats.

111. A.

PROTEIN LOSS IN SPACEFLIGHT Ground Based Models: Need and Problems

An understanding of the causes of the muscle protein losses inflight is a prerequisite for designing and optimizing countermeasures. As yet there are no effective measures for preventing the muscle atrophy associated with spaceflight. Exercise provides some protection, but it is not clear whether exercise alone can be the solution. Evaluation of the effectiveness of exercise would require that the subject be in energy balance or close to it, as it is physically impossible to maintain muscle mass in the face of an energy deficit. Further development of inflight measures

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requires a detailed knowledge of the biochemical and physiological changes involved. There are three aspects to be considered.

1. Since the opportunities and facilities for detailed studies in space are severely limited and will continue to be so for the foreseeable future, there is a definite need for appropriate ground-based models of weightlessness. Thus if we are ever to understand the mechanisms of the microgravity-induced changes in skeletal muscle, it is crucial that an appropriate ground-based model be found. 2. Once such a model has been defined, a question needs to be faced: Just how appropriate is the model? This question can only be answered by continually comparing the ground-based results against flight data. It is not enough to argue that the model mimics some o f the spaceflight-induced changes. There is an important distinction between the conditions of spaceflight and of bed rest or some other form of inactivity. The response to spaceflight is the compound effect of microgravity, conditions inside the spacecraft, dietary factors, activity levels, and work-sleep cycles. It will be very difficult to eliminate the nonmicrogravity effects from the spaceflight response. 3. The third aspect is the need for elucidating the actual mechanism at the cellular and molecular levels by which the muscles respond to spaceflight. At least four causes of muscle loss on the ground can be distinguished, most of which are reasonably well understood, and much is known about the cellular and molecular mechanisms involved. In the next sections, these mechanisms will be discussed with consideration of their relevance for the muscle changes observed in spaceflight. B.

Possible Causes of Protein Loss

Ground-based studies have identified four plausible mechanisms that could cause the inflight loss of muscle protein (1) atrophy (or better: reductive remodelling) due to a decreased workload on the weight-bearing anti-gravity muscles in space, (2) existence of an energy deficit, (3) actual pathological damage to the muscles leading to muscle breakdown, and (4) a metabolic stress response. An energy deficit, either due to decreased food intake or increased activity, could lead to protein loss when insufficient energy is available to support the required rate of protein synthesis. Pathological damage could occur if muscles weakened by atrophy are subjected to extreme forces, as might happen when a subject overexercises. It is likely that the overall response is a combination of two or more of these factors and that their relative importance will vary with the stage of the pacef flight.'^

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C . Metabolic Stress Response While a potential role of the first three causes for protein loss (inactivity, muscle damage, and an energy shortage) i s obvious, the possible involvement of a metabolic stress response is not intuitively obvious. However, entry into a gravitationless environment and living in it is a novel situation for humans that the body may perceive as a stress to which it will respond. A common response to stress consists of the activation of the hypothalmic-pituitary-adrenal (HPA) axis with accompanying increases in the whole body protein turnover rate, gluconeogenesis, substrate cycling, proinflammatory cytokine activity, basal energy expenditure and a loss of body p r ~ t e i n . There ~ ~ . ~is~ also synthesis of so-called “acute-phase proteins”, which are plasma proteins produced in the liver in response to stress, an example being fibrin0gen.6~Collectively, these reactions serve several functions: (1) they limit the extent of the injury or stress, (2) they protect the rest of the organism against any further stresses by mobilizing host defense mechanisms, and (3) they initiate various restorative processes aimed at returning the organism to homeostatic balance. The process is regulated by the combined effects of the central nervous, neuroendocrine, and immune systems.64 What is the evidence for the involvement of a metabolic stress response in spaceflight-induced muscle losses? Characteristic of such a response are increases in whole-body protein turnover, acute-phase protein synthesis, and stress hormone levels, as noted above. In contrast, ~ n d e r n u t r i t i o n ~and ~ - ~hind ~ limb unloading in rats68969result in a decrease in the whole-body protein synthesis rate. Measurement of whole body and acute-phase protein synthesis would thus provide an unambiguous test for the occurrence of a metabolic stress response. This was investigated during the SLS-l(1991) and SLS-2 (1993) missions, where the synthesis rates of whole-body protein and fibrinogen were measured by the single pulse I5N glycine method before, during and after pacef flight.^' Subsequently, additional data on two three subjects were obtained on the German D-2 mission75. The use of I5N glycine gives two measurements of the whole-body protein synthesis rate depending on whether ammonia (PSRA) or urea (PSRU) is used as the end point. The protein synthesis rate found with urea is higher than with the ammonia end-product method. The values obtained with the two methods do not correlate with each other. Why this is so is not known for certain, although a number of hypotheses have been proposed. The most plausible is that the urea end-product method gives values that are biased toward hepatic protein metabolism and the ammonia end-product method biased results favor muscle protein metabolism. The argument is that the liver is the site of urea synthesis and muscle is the major source of the glutamine that reaches the kidneys. Plasma glutamine is the major precursor of urinary a m m ~ n i a . ~Because ~ . ~ ~ of , ~this ~ problem, results are often reported as the mean of the urea-and ammonia-based values (PSRM). The interpretation of whole body protein kinetics, irrespective of the isotope used can be complicated by the fact that what is actually measured is

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Table 4. Whole Body Protein Synthesis Rates Time Pre-Launch 1 Pre-Launch 2 Prc-Launch 3 Flight day 2 F l i g h t day 8 Flight day I2

+0 +6 + 14 R e t u r n + 45 Return Return Keturn Notes;

PSMU (g prot. k g - ' . d ' )

PSRU"

PSRM'

(g prot. k g - ' d ' j

( g prot. k g - ' - d ' )

2.37 ? 0.30 (9) 2.26 f 0.22 (9) 1.98 f 0.19 (10) 2.28 0.30 (8) 3.01 f 0.44 ( l o ) * 2.08 f 0.55 (5) 3.00 f 0.43 ( I 0)* 2.55 ? 0.3X (10) 2.69 ? 0.41 (10) 2.1 I f 0.26 (8)

3.95 + 0.32 (7) 4.15 + 0.46 (7) 4.43 + 0.50 (7) 5.78 + 0.47 (6)* 3.97 + 0.44 (7) 3.50 + 0.62 (3) 4.14 + 0.48 (7) 4.23 + 0.48 (6) 3.64 + 0.53 (6) 4.33 + 0.30 (5)

3.01 f 0.22 (6) 3.21 f 0.32 (6) 3.13 k 0.27 (7) 3.87 f 0.26 (6)* 3.20 2 0.36 (7) 2.63 0.35 (3) 3.29 f 0.33 (7) 3.15 f 0.37 (6) 2.98 f 0.33 (6) 2.46 f 0.36 (7)

*

*

PSKA = rate based on ammonia, PSRU = rate based on urea, PSRM = means of both. Combined means for SLS- 1 and SLS-2 ? SEM and number of subject5 in parentheses, Prelaunch measurements: in respective order for SLS-1 56, 26, and 18 days before launch; for SLS-2 88, 10, and 12 days before launch. *:p
either the nonoxidative amino acid disposal rate or the amino acid flux. In either case it is an assumption that the nonoxidative amino acid disposal rate or the flux can be equated with or related to the whole-body protein synthesis rate. Usually, assumption is reasonable and justified. Table 4 shows the protein synthesis rates based on ammonia (PSRA), urea (PSRU). and the means of these rates (PSRM) for the combined SLS- 1and SLS-2 missions.52 The data in Table 4 show that the whole-body protein synthesis rate was increased during the first part of the flight. The important point is that there was no evidence for a decrease on flight days 2 and 8, which is the predicted response from bed rest studies. The overall rate of protein synthesis and breakdown are the composite sums of many different tissue and protein rates. For a net loss of body protein, the sum of the individual protein breakdown rates must be greater than the sum of the individual protein synthesis rates. Thus any inflight increase in protein synthesis must have been associated with a greater increase in the protein breakdown rate. Since both protein synthesis and protein breakdown were increased, it follows that protein turnover was increased. The increased protein turnover reflects the increased synthesis of proteins involved in host defenses. Even though there was no actual injury, fibrinogen synthesis is increased (Figure 4).52 Thus there is a metabolic response associated with entry into Earth orbit. During the first day or two of spaceflight there is also an increased secretion of cortisol (Figure 5 ) and of the proinflammatory cytokines IL-6 and ILIO (Figure 6).70All of these findings indicate that entry into orbit is associated with a metabolic stress response. Since the increases in fibrinogen synthesis, cortisol, and IL- 10 did not persist beyond flight day 2, it follows that the major part of the stress response was an acute reaction to the entry into Earth orbit.

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PREFLIGHT

INFLIGHT

POSTFLIGHT

* peO.05 vs preflight T

L

L T

I

~

L-45 L-15 L-8 FD-2 FD-8 FD-12 R+O R+6 R+14 R+45

MISSION DAY Figure 4. Fibrinogen synthesis in four SLS-2 crew members. L-45 = 45 days before launch, FD-2 = after 2 days inflight, R + 6 = 6 days after return (from ref. 52).

Exactly how long the metabolic stress response lasts is not clear. The SLS-1 and SLS-2 cortisol and cytokine data indicate that the level of metabolic stress is low after the first few days. This conclusion receives support from the consideration that it is likely that muscle protein synthesis is decreased early in the mission, whereas a trend toward a decrease in the whole body rate was not found until the 12th day in orbit. In apparent conflict with this conclusion is the prolonged elevation of 3-methylhistidine excretion during the Skylab mission. This phenomenon suggests that the duration of the metabolic stress response may be mission-dependent (Table 5, see also section I11 d). During the German D-2 mission, protein synthesis rates were determined on two subjects inflight on flight days 6 and 7.75The mean value (PSRM), which is the composite of a decreased urea value (PSRU) and an unchanged ammonia value (PSRA), was decreased. Comparison of these findings with the SLS-1 and SLS-2 data leads to the following two points:

T.P. STEIN

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350

3 a

-0- Preflight --t lnflight ---A- Post flight

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ELAPSED TIME (DAYS) Figure 5. Urinary cortisol excretion before, during, and after spaceflight. Data for SLS-1 and SLS-2 missions. lnflight values are higher than preflight values (p<0.06 vs. mean preflight value).

1. PSRA: A plot of the combined data for PSRA together with some preliminary data from longterm missions gathered as part of the ShuttleMIR program16' is shown in Figure 7. On D-2 PSRA was unchanged inflight. For SLS-I and SLS-2, there was an increase for 8 out of 10 subjects and no change for the other two on flight day 8. On flight day 12, PSRA was increased for 2 subjects, decreased for two, and unchanged for a fifth. By 3 months (longterm data from MIR)16', there was clear evidence for a decrease in PSRA. On the whole, it appears that the short-term trend for PSRA is either toward an increase or no change for most of the subjects. 2. PSRU was decreased on flight days 6 and 7 for both D-2 subjects (Figure 8). For SLS-1 and SLS-2 no change was found on flight day 8. On flight day 12,

Nutrition and Muscle Loss

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350 - 11-6

+3- PREFLIGHT

+ INFLIGHT

300

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I (3 5

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Figure 6. Proinflammatory cytokines (IL-6 and I L - I 0) excretion before, during, and after spaceflight. Data for SLS-1 and SLS-2 missions. Significant elevation during first two flight days ( ~ ~ 0 . vs. 0 5mean preflight value).

however, for which there were only four subjects, one showed an 11% increase, two showed decreases of about 696, and the fourth showed a 20% decrease. Taking these data together, there is a trend towards a decrease during the first 12 days, that is statistically significant (p<0.05). In a 3-day bed rest study with 6' head-down tilt PSRA was increased at 48 h while PSRU was unchanged.76 A 6' head-down tilt is not a benign procedure. Unlike normal bed rest, the tilt is sufficient to induce similar fluid shifts to those found after entry into orbit and is often associated with a feeling of malaise and headaches. There was also an increased energy expenditure. These findings suggest the occurrence of a metabolic stress response in bed rest with head-down tilt, as was the case in the SLS-1 and SLS-2 missions.

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Urinary Excretion of 3-Methylhistidine during Spaceflight and Bed Rest

Table 5. Condition Shuttle Skylab Bed rest Notes:

Bed Rest/Flight (,uMol-'.kg-'&') 4.59 t 0.39 (9) 1 I .20 f 0.57 (9)* 5.71 f 0.30 (7)

Control (,uMol-'. kg-'.d-') 4.98 f 0.37 (9) 7.92 t 0.45 (9) 5.30 * 0.29 (7)

Recovery (,uMol-'.kg-'.d-')

4.53 f 0.50 (7) 6.38 f 0.28 (9) 5.88 f 0.41 (6)*

Shuttle: SLS-1 and SLS-2 missions 9 and 12 days, respectively; Skylab: first 28 days of mission; Bed Rest 16 days. Means k SEM with number of Subjects in parenthesea. *p<0.05 versus pretlighthed rest mean (from ref. 133)

250 0

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FLIGHT DAY Figure 7. Chan es in whole body protein synthesis rate during spaceflight. Measured with "N glycine and ammonia as end product (PSRA). Data from SLS-1, SLS-2, D-2, MIR missions (refs. 52 and 75; Stein, unpublished observations).

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180

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FLIGHT DAY Changes in whole body protein synthesis rate during spaceflight. Measured with 5N glycine and urea as end product (PSRU). Data from SLS-1, 515-2, and D-2 missions. Regression is significant (p
Figure 8.

The combined findings from the spaceflight missions and the head-down tilt bed rest study suggest the occurrence of a metabolic stress response associated with entry into space orbit. The duration of the response may vary between missions and between subjects. The consistent absence of a decrease in protein synthesis rate during the first 12 days and the 45% decrease seen after 120 days on MIR suggest an ending of the stress response sometime after flight day 12.

D.

Pathological Breakdown of Muscle Protein

On some spaceflight missions, actual muscle protein breakdown may have occurred as suggested by measurements of the urinary excretion of 3-methylhisti-

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dine (3-MeH). Monitoring the urinary 3-MeH is a standard assay for assessing myofibrillar protein b r e a k d ~ w n . A ~ ~limitation .~~ of the technique is that changes in the 3-MeH excretion rate cannot be unambiguously attributed to changes in muscle protein breakdown rate, since there are various actin pools in the body turning over at different rates8' In humans skeletal muscle contains about 90% of the total 3-MeH, with the remainder in gastrointestinal tissue and skin.*' The latter two pools have faster turnover rates than muscle, so they account for about 25% of the excreted 3-MeH. Since there is no reason to suspect any chronic changes in skin or gastrointestinal tissue protein metabolism during spaceflight, any changes in the 3-MeH excretion rated can safely be attributed to skeletal muscle changes.

v)

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MONTHS IN SPACE Figure 9. Decline in urinary 3-methylhistidine excretion with time in orbit on Skylab (adapted from ref. 158).

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Numerous ground-based studies have shown that 3-methylhistidine excretion is unchanged during bed rest and mild-to-moderate u n d e r - n ~ t r i t i o n It . ~ would ~~~~ therefore be expected that 3-MeH excretion be unchanged during longterm (adapted) spaceflight. However, on Skylab, 3-MeH excretion was increased inflight (Table 5 ; Figure 9) indicating myofibrillar protein breakdown. This was not the case during the SLS- 1 and SLS-2 missions (Table 5), suggesting that muscle protein breakdown is not an inevitable consequence of pacef flight.^^ An increased 3-MeH excretion would be expected as the result of a metabolic stress response, because this causes a simultaneous increase in protein synthesis and breakdown. In Skylab, the increased 3-MeH excretion and resulting muscle (myofibrillar) protein breakdown diminished with time in space (Figure 9), although it failed to reach the preflight value. The whole body protein synthesis rate would be expected to decrease on the basis of bed rest studies65~67,*3~85 and flight rodent data86,87. The Skylab findings suggest a strong initial metabolic stress response, which diminishes with time of adaptation to microgravity as would be expected. The question is, does the remaining 20% elevation of the 3-MeH excretion after 75 days represent a continuing metabolic stress response or does it represent actual muscle damage from the effect of vigorous exercise on the weakened muscles? Such damage could in turn induce a metabolic stress response. Significantly, on Skylab (but not on the Shuttle), there was a strong correlation between the increase in cortisol and urinary 3-MeH e ~ c r e t i o n .This ’ ~ suggests that there was a low-grade chronic metabolic stress response persisting for the duration of the Skylab mission. The exercise regimen was probably suboptimal, being useful for some muscles but counterproductive for others. Several NASA sponsored panels have reviewed the Skylab-findings and made recommendations for the proper type and extent of inflight exercise.’* However, there has been no inflight controlled testing of the effectiveness of exercise. No comparisons of aerobic versus resistance exercise or of different exercise regimens have been carried out. It is unlikely that the amount of exercise alone on Skylab could have caused muscle damage. On the ground the exercise has to be very vigorous to lead to such damage. However, the existence of an energy deficit in space could have a synergistic effect, thus lowering the threshold for damage. Such a deficit did exist in the Skylab astronaut^.^^

IV.

ENDOCRINE ASPECTS A.

Bed Rest Studies

There is a general belief that “bed rest” and “disuse” are the most appropriate ground-based models for the response of the human musculoskeletal system to spaceflight. Bed rest is associated with a decrease in the whole-body protein syn-

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thesis rate that is due to decreased skeletal muscle protein synthesis. Such effect was not found on the short duration SLS-I and SLS-2 missions, but it was present after a three-month stay on MIR. Preliminary data on six subjects during a longterm mission, conducted as part of the Shuttle-MIR program, show that the wholebody protein synthesis rate was reduced by about than 45% after three months in orbit. Even if the long term spaceflight effect resembles a “bedrest” or “disuse” type of response, this does not completely identify which of the various ground-based inactivity models best reflect the spaceflight situation. At 1 G, there are many ways in which changes in human muscle protein content can be effected. Decreased growth hormone activity, hypoinsulinemia, decreased prostaglandin activity, elevated cortisol, and various neuroendocrine effects such as degradation of the neuromuscular function may all lead to loss of muscle protein. Each proceeds via a different mechanism, and there are different ground based models for each. Investigation of the hormonal profile during spaceflight can help to distinguish between the various possibilities.

B. Sampling Methods Plasma hormone measurements have been made on many missions, beginning with Skylab, but the results have not been particularly informative. The reason is that plasma hormone levels are single point determinations of rapidly turning over systems. To be useful, plasma levels need to be measured repeatedly under carefully controlled conditions. Because of many other demands on the astronauts it has so far proven to be very difficult to collect enough blood samples under carefully controlled conditions. The use of 24-h urines instead of blood to assess total hormone production has some distinct advantages: ( 1 ) Changes can be detected in urine when none are present in the plasma; (2) Plasma samples represent single spot values and plasma hormone levels can fluctuate very rapidly with time, while urine measurements give an integrated value over time depending on the biological half life of the molecule. This is particularly advantageous in situations where anticipated changes are small, or the hormone is released in a pulsatile manner and sampling opportunities are limited. Disadvantages of urine analysis have also been cited: (1) Urine-derived data could be impacted by abnormal renal function; however, there is no evidence that renal function is generally altered by spaceflight; on SLS-2, there was an early increase in the glomerular filtration rate, but this did not persist beyond the first week in ~ r b i t ; * ~(2) . ~ ’urine is further removed from the sites of synthesis and action of a hormone than plasma; however, neither are actually at the site of tissue hormone production and action. C. Hypothalamic-Pituitary-Adrenal Axis

The hypothalamic-pituitary-adrenal (HPA) axis is assumed to be responsible for stress responses. Given the importance of the HPA axis in regulating interme-

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diary metabolism, it is very likely that it is involved in the metabolic stress response, but so far it has proven to be difficult to identify its precise role beyond the initial metabolic stress response. There is little evidence from human studies for the involvement of some of the key HPA axis hormones, specifically ACTH and growth hormone." On Skylab, growth hormone was unchanged except for a transient increase on flight days 3 and 4.92 Figure 10 shows that it was also unchanged on the SLS-I and SLS-2 missions.93 These growth hormone findings agree with the analyses on blood samples collected during the Skylab This conclusion is supported by animal experiments. Inflight replacement of growth hormone in rats was without any effect on skeletal muscle mass.94 However, secretion of the hormone was decreased in rats during spaceflight, while it was sequestered in the pituitary gIand.95,96There is, however, good evidence for

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the involvement of thyrotropic hormone (TSH), as determined from reduced triSince iodothyronine (T3) activity inflight on the SLS-1 and SLS-2 tood intake was reduced, this finding is not u n e ~ p e c t e d . ~ ~ D.

Insulin

Insulin is an important factor in the regulation of muscle protein synthesis and breakdown, and therefore insulin resistance is often associated with the muscle protein loss. Many studies have shown that there is a correlation between insulin levels. insulin resistance, and a decreased nitrogen balance.66398399 Insulin could

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potentially be a major factor in the spaceflight induced protein loss because insulin resistance does occur in stress states and u n d e r n ~ t r i t i o n . ~Measurements ~,'~~ by Russia scientists showed that the postflight plasma insulin levels were iricreased relative to preflight levels, and the increase persisted for as long as two weeks after 1anding.I" A study on a single subject suggested that glucose tolerance map be impaired during spaceflight.lo2 The Skylab data are conflicting. After an initial drop, there appears to be a trend towards an increase followed by a very sharp drop and then a spike between the third and fourth weeks.17 For the remaining two months, plasma insulin was below the preflight level. Insulin is secreted from the pancreas as proinsulin, which is cleaved in the plasma to give insulin and a remnant peptide called C-peptide. The C-Peptide is fully excreted in the urine, so its excretion can serve as a measure of the rate of insulin production. Insulin resistance is likely to be common to most pathways for muscle downsizing. It can serve as a means of decreasing glucose uptake by muscle, thereby sparing the glucose for tissues that are obligate glucose users and forcing a reduction in muscle protein synthesis by reducing nutrient availability. Measurement of C-peptide excretion on the SLS-1 and SLS-2 missions showed a steady increase with increasing time in space (Figure 1 I).''' On these missions, IGF- 1 was slightly lower initially and then remained unchanged.93

E.

Cortisol

Although increased urinary cortisol has been a frequent finding during spaceflight (e.g., during the SLS-I and SLS-2 missions; see Figure 5), there are several arguments against assuming it to be the primary agent i n the musculoskeletal losses: 1. On the ground, infusion of cortisol results only in a transient increase in protein degradation with the preferential degradation of myofibrillar proteins, as evidenced by increased 3-MeH e x c r e t i ~ n . ~ In ~ ~contrast, - ' ~ ~ there was no increase in myofibrillar protein breakdown on these two missions (Table 5). 3-MeH excretion was unchanged during ~ p a c e f l i g h t . ~ ~ 2. The spaceflight-induced increase in cortisol excretion differs from that found during a metabolic stress response, where an increase in plasma cortisol is associated with an increase in ACTH. This relationship is not found during human spaceflight, where ACTH is usually unchanged. Both U.S. and Russian investigators have observed and commented on this apparent a n ~ m a l y . ~ , 'Although ~ ~ ~ ' it has been suggested that this might be due to cortisol secretion lagging behind ACTH secretion, so the blood showing the high cortisol level would have been sampled after the increase in ACTH secretion had o c ~ u r r e d , ' ~ this ' * ~ explanation is inconsistent with the urine data, because these are integrated over 24 h. Moreover, the cortisol effect persisted for the duration of the mission. An alternative explanation is that the elevated cortisol is not due to a metabolic stress response but rather to the emotional stress associated with pacef flight.'^,'^^

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3. Increased cortisol has been found in some, but not all bed rest studies,91,1n6-1n9while bed rest is invariably associated with muscle atrophy. 4. In spaceflight, slow-twitch fibers (type I) atrophy more rapidly than fasttwitch fibers (type II), whereas cortisol affects predominantly the fast-twitch fibers.' 5. An increased cortisol production is a systemic response, which cannot by itself account for the specificity of the observed muscle and bone losses. 6. Interestingly, a one-week bed rest study by Vernikos showed cortisol to be increased only in males and not in females.lo6 7. A recent 42-day bed rest study by Blanc and colleagues found cortisol to be elevated for the first three weeks of bed rest after which it declined to the pre-

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bed rest level even though longer duration bed rest studies have shown that the . ~ ~ implies cortisol is associated with the loss of muscle protein ~ o n t i n u e dThis metabolic stress of adjusting to the new environment but not thereafter.I6* This suggests that there is no close coupling of cortisol and the muscle loss in the absence of a metabolic stress response. F.

Prostaglandins

Observations on the SLS- I and SLS-2 missions suggest that prostaglandins play a key role in the human response to spaceflight. The plasma prostaglandins PGE, and PGF,, are unstable, so they are usually measured as their metabolites

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PGE-M and PGF-M, which are the major metabolites of the E and F prostaglandins, respectively."', PGE-M and PGF-M are excreted unchanged in the urine and are generally considered to reflect whole body as opposed to renal plus systemic prostaglandin production."',' 13-' l 5 During spaceflight the secretion of PGE, and its metabolite PGE-M are markedly decreased (Figure 12), suggesting that the systemic production of PGE, is reduced during spaceflight. Marginal inflight decreases are found for PGF,, and its metabolite PGF-M, but the latter showed a dramatical postflight increase (Figure 13).93 The magnitude (-30%) and symmetry of the inflight PGE-M decrease and the large postflight increase in PGF-M suggest that these changes are spaceflight-specific responses. As with any whole body study, an unambiguous assignation of the source of the observed effects cannot be made. However, it is reasonable to assume that the sites are ones that must be affected by spaceflight, namely, muscle and bone. Both muscle and bone release prostaglandins in response to mechanical stress.l'6-119Muscle is likely to be the major site of the decreased prostaglandin production during flight because there is much more muscle than bone, and muscle is a more metabolically active tissue. PGE, stimulates muscle protein degradation, while PGF,, stimulates its synthesis.'2""21 Particularly relevant to spaceflight-induced muscle atrophy is that in vitro PGE, and PGF,, function as autocrine second messengers in regulating stretch-induced changes in muscle protein synthesis and breakdown.l18,122-124 Furthermore, cell culture studies have shown that muscle cells release PGE, and PGF into the medium in amounts varying with the tension applied to the cell. 'f88,125 Prostaglandin release decreases as tension is lowered. This suggests that prostaglandins can also act in a paracrine manner. In rats, the PGE, secreted by muscle acts synergistically with nitrogen oxide (NO) to dilate the microcapillaries in the muscle.'26 Conversely, a decrease in PGE, release leads to constriction of the blood vessels. Histological examination of human quadriceps muscle biopsies taken immediately after landing showed a decreased capillary size.38 A likely consequence of decreased blood flow is a decrease in nutrient availability thereby initiating a localized starvation response in the muscle (or bone). Without adequate nutrition, cells will adapt by conserving energy, decreasing protein synthesis, and remodeling. The process is one of adaptation; the decrease in cell protein content and distribution can be selective with the possibility of strength being conserved at the expense of some other property (e.g., increased fatigabilty).9'38 And as soon as the resting muscles experience tension, prostaglandin release occurs opening up the capillaries and making nutrients available for the regenerating muscle. Even though the urine prostaglandin measurements relate primarily to the muscle loss, a similar mechanism might also account for the bone calcium loss. Both PGE, and PGF,, are powerful stimulators of bone resorption. The inflight prostaglandin measurements are consistent with well established ground models for both muscle and bone. 23,125,127-129 The prostaglandins produced by osteo-

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blasts and osteoclasts in response to mechanical stress are involved in the local regulation of bone metabolism. 173129The resulting prostaglandins promote angiogenesis by stimulating the release of vascular endothelial factor by osteob 1 a ~ t s . Conversely, I~~ decreased prostaglandin release will lead to microcapillary constriction. These observations provide strong evidence for the conclusion that the coupling between tension and localized changes is mediated by prostaglandins. This being a localized process can account for the specificity of the response by muscle and possibly bone. The inflight prostaglandin measurements are consistent with well established ground models for both muscle and bone.50,58,6',63,'30,131 Therefore, it would appear that the dominant response of skeletal muscle in the absence of an energy deficit during spaceflight resembles a bed rest-style muscle remodeling response mediated by prostaglandins.

'

V.

ENERGY DEFICIT AND NUTRITION A.

Energy deficit

There is some evidence that there may be a problem in maintaining energy balance during spaceflight. The first energy expenditure determination was done during the 1973 Skylab missions using a combination of dietary intake and body composition measurement^.^^' 25 On this longterm mission, the deficit was about 3 kcal kg-'.d-' with the deficit being greatest during the first m ~ n t h . ~The ~,'~~ second energy expenditure determination was on the 1996 Life and Microgravity Sciences (LMS) Shuttle mission, where the doubly labeled water method was used.16' Here again astronauts were in negative energy balance. During the SLS-1 and SLS-2 missions, the astronauts were in approximate energy balance52 The doubly labeled water (*H2"0) expenditure method for measuring energy expenditure is simple, noninvasive, and highly accurate. The method was originally described by Lifson in 1955 and applied to humans by Schoeller in 1982.1332'34If 2H2'80 is given orally, it mixes with the body water in about 3 h. The two isotopes then leave the body at different rates. 2H leaves as water, mainly in the urine, whereas "0 leaves both as water and exhaled C"02. Thus the turnover rate of isotopic hydrogen and oxygen differ, and this difference is proportional to the rate of CO2 production. The method has been validated in humans, normal animals, and in animals in metabolically perturbed states. Energy balance on the LMS mission was determined by both the intake body composition method and the doubly labeled water method. Dietary intake for the four astronauts was reduced inflight (35.5 t 2.1 vs. 24.6 t 3.3 kcal. k g - ' d ' , p<0.05, Figure 14). Energy expenditure inflight was 40.8 f 0.6 kcal. kg-'.d-' resulting in a negative energy balance of -15.7 f 1.0 kcal kg-'.d-', p<0.05, Figure 15). This corresponded to a loss of 2.1 t 0.4 kg body fat, which is equal within the

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experimental error to the fat loss determined by "0 dilution (1.4 f 0.5 kg) and DEXA (-2.4 ? 0.4 kg).16* This low level of energy intake during space flight is not unique. On the recent US-Russian Shuttle-MIR missions, energy intake for some of the astronauts was low; for the six subjects for whom we have data, the mean inflight energy intake was 26.3 ? 2.2 kcal. k g - ' d ' . The short fall in energy intake on MIR was enough to negatively impact protein turnover (Figure 16). Bed rest studies have shown that the whole body protein synthesis rate is reduced by about 15%, which can be accounted for by a 50% reduction in the protein synthesis by the antigravity muscles. lo7 The reduction in whole body protein synthesis found during space flight on MIR was much greater, 46 _+ 6%.16" Figure 16 shows a plot of the estimated deficit in energy expenditure-as defined by the difference between energy intake on the day of measurement and the mean energy intake for the preflight period. The plot shows there to be a good correlation between the estimated energy deficit and the protein synthesis rate (? = 0.70, p <0.04, Figure 16). The inflight decrease in energy intake also correlated with weight loss (r2 = 0.53, p = 0.10). The implication is that the reduction in protein synthesis found with the Shuttle-MIR astronauts/cosmonauts was due to a combination of an atrophy and undernutrition response acting in synergy.16' The reduction in protein synthesis is an adaptive response to conserve energy. Protein turnover by itself accounts for about 20% of the BMR.23 Comparison of energy balance during the LMS mission against SLS 1/2 and Skylab, the other two missions for which energy balance data are available, suggests a reason for the inflight energy deficits on some missions. Figure 17 compares energy expenditure, energy intake, energy balance, and nitrogen balance during flight for comparable periods during the three missions for which data are available. The periods for comparison are the first 12 days for Skylab, LMS, and SLS-2, and the first 9 days on SLS-2. Skylab consisted of three similar missions of 28, 56, and 84 days with three astronauts on each mission. The data from the three missions have been combined into a single data set. Likewise the data from SLS-I and SLS-2 have been combined, since SLS-2 was a reflight of SLS-I but with a longer flight time ( 1 6 days versus 9 days). LMS was a single Shuttle mission flown in 1996 with the objective of investigating the musculoskeletal system; as such there were heavy exercise requirements for the crew. The measured nitrogen balance values are based on the excretion of nitrogen in the urine, but have been corrected for the estimated fecal nitrogen losses. Fecal nitrogen losses were only determined on Skylab and amounted to about 20 mg N. kg-'.d-'. Numerous studies on the ground have shown that the sum of fecal plus other insensible losses ranges from 15 to 25 mg N kg-1.d-1.',47,49 For Skylab the energy expenditure and energy balance were determined by the intake balance method.24 For the SLS-1 and SLS-2 missions these values were estimated from the combination of dietary and nitrogen balance data.52For the LMS mission they were measured by the doubly labeled water method.16'

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Preflight there was no difference in either energy intake (Figure IS) or nitrogen balance between the three missions (Skylab, 53.9 +. 4.0 mg N k g - ' d ' ; SLS-I and SLS-2, 37.5 f 9.1 mg Nkg-'.d-'; and LMS, 42.3 k 11.8 mg Nkg-'.d-'). However, there were substantial and statistically significant differences during flight. Figure I4 shows that as energy expenditure increased across the missions, the nitrogen balance decreased. Intake failed to meet energy needs on Skylab and LMS. The negative energy and nitrogen balance on the Skylab and LMS missions was a consequence of an imbalance between activity, energy intake, and energy expendit ~ r eOn . ~these ~ missions, the astronauts were required to exercise. On the SLS-I and SLS-2 missions, exercise was not required and was not performed by most of the The SLS-I and SLS-2 astronauts were in approximate energy bala n ~ ewhereas , ~ ~ the nine Skylab astronauts and four LMS astronauts were in negative energy balance (Figure 17). The Skylab crew lost 1.2 + 0.3 kg body fat during the I to 3 months of the three missions5 and the LMS astronauts 1.5 & 0.6 k&. A high rate of aerobic exercising is very costly in energy needs.'35 The energy costs of the two daily inflight exercise periods on the Russian Salyut-7 mission were estimated at around 20 kcal kg-'d'.'36-138Neither the Skylab nor the LMS astronauts were able to meet these superimposed energy costsJ2 On LMS, the imbalance between energy intake and expenditure was greater than that on Skylab. This is probably due to the fact that dietary intake of the Skylab crews was monitored from the ground and they were encouraged to eat whereas no such monitoring was done for the LMS crew with the consequences shown in Figures 17 and 18. Since a negative energy balance does not occur on all missions, it appears that the negative energy balance and some of the nitrogen losses found on Skylab and LMS are mission-specific responses. Typical for these missions is that they had a heavy exercise component, whereas SLS-1 and SLS-2 did not.33139,52 The latter were very busy missions: the crew did not exercise, because this was not required and was not placed into the flight time line. There was no spare time, as all available time was spent in carrying out experiments. The occurrence of a negative energy balance on spaceflight missions with high exercise components raises some important questions: Is it possible to maintain energy balance with a high exercise regimen? Some exercise studies on the ground have reported a cachectic effect from intensive e ~ e r c i s e . ~ ~ " - ' ~ ~ 2. Is there a confluence of events that decrease appetite? One possible factor is altered taste of food in orbit. Another is delayed transit of the chyle through the digestive tract. In the absence of gravity the progression of food through the GI tract is slower. 3. Is there a problem with the food or the time allowed for eating? The latter seems improbable; energy intake was also low (26 kcal.kg-'d') on the 1.

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long-duration MIR missions where there was adequate time available for eating." 4. What is the contribution of the negative energy balance to the muscle protein loss? If energy intake is inadequate, exercise will exacerbate the loss of body protein during 5 . Does a negative energy balance also affect bone homeostasis? Irrespective of the reasons for the poor food intake, the consequences of a chronic energy deficit are serious. On the ground, chronic depressed protein synthesis and the associated protein wasting has serious consequences. It leads to decreased physical p e r f o r m a n ~ e , ~ ,and ' ~ ~to~ a' ~progressively ~ increased suscep-

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tibility to i n f e ~ t i o n . ~ ' ,Decreased ~ ~ , ' ~ ~ immunocompetence during spaceflight has been reported. Wound healing is also compromised, which could present a ? ' ~ ~though - ' ~ ~ humans problem if injury ever occurs during s p a ~ e f l i g h t . ~ ~Even can and do adapt to chronic energy deficits, longterm adaptation is not without consequences, particularly as body fat stores become increasingly depleted. A small shortfall in energy intake is not serious, but chronic deficits such as occurred on the LMS and MIR missions are not indefinitely sustainable even with some adaptation. It is essential for the long term health and safety of astronauts on very longterm missions (e.g., a mission to Mars) that they remain in energy balance. Undernutrition is treatable by increasing food intake. The questions are therefore as follows: ( I ) how much food should an individual astronaut eat and (2) how do we identify the individual crew members who are in negative energy balance so that steps can be taken to increase dietary intake?

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Nutritional Needs of Astronauts

Energy expenditure rates during spaceflight are highly variable. In a study of 16 astronauts, a range from 28 to 47 kcal kg-'.d-' was f 0 ~ n d . l ~Although ' the

mean value corresponded well with the value predicted by the WHO equation for moderate activity, the individual values did not correlate at all with the WHO-predicted values (Figure 19). This indicates that it will not be possible to formulate a single recommendation to suit all astronauts on all missions. Dietary recommendations must be customized to meet individual needs. With current technology, it is feasible to make a reasonably accurate estimate of the energy requirements for an individual astronaut on a particular mission and

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Figure 17. Energy intake, energy expenditure, energy balance, and nitrogen balance during the first 1 2 days of spaceflight on the Skylab, SLS-1, SLS-2, and LMS missions. The SLS-1 and SLS-2 data have been combined into a single data set. Means k SEM. Differences in energy intake and expenditure and nitrogen balance are statistically significant (p<0.05) (ref. 52 and unpublished observations).

to monitor food intake to see if it remains close to the estimated energy requirements. The technology for dietary monitoring food intake is available and has been used on numerous missions including SLS-1, SLS-2, LMS, and the recent long duration MIR missions. A simple bar coding system for the prepackaged food has proven to be adequate for this purpose. When the astronauts remove a food item for consumption, they scan the bar code of the package with a portable scanner. After the meal, they record with a voice recorder the amount of the item consumed. The recorded information can be downlinked to the ground at convenient intervals, and crew members who show indication of a seriously negative energy balance can be alerted to increase their food intake.

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Figure 18. Energy intake for the 10 days before flight on the Skylab, SLSl, SLS-2 (SLS/2), and LMS missions. The SLS-1 and SLS-2 data have been combined into a single data set. Data are means ? SEM. Significant differences inflight between missions ( p<0.05) (ref. 52 and unpublished observations).

Astronauts do not have to be constantly in precise energy balance, but they need to be close to it and certainly closer than they were on the recent LMS mission. In the way described in the preceding paragraphs, it is possible to derive an equation for estimating individual requirements for spaceflight. The astronauts on the SLS-I and SLS-2 missions did not exercise and, as a result they were in approximate energy balance with an intake of about 31 kcal kg-'.d-'.52 The measured basal metabolic rate (BMR) for these subjects was about 23 kcal kg-'.d-',giving an activity coefficient of I .4. All available evidence suggests that the energy costs

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of the exercises are about the same as on the ground. For EVA, the energy costs run from 180 to 200 kcal kg-',h-1.'5' Thus, the energy costs for a proposed mission can be estimated in advance from the relationship (all in kcal kg'.d-'):

Energy need = 1.4 x BMR + cost of exercise + any EVA activity So far we have focused on the energy balance, but the protein balance must also be considered. Here protein intake and synthesis are of crucial importance. There is some evidence that suggests that amino acids can become limiting in the postflight period rather than during flight. The evidence is based on the examination of the distribution pattern of amino acids in the plasma. Determination of the

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plasma amino acids has proven to be a useful tool for investigating human amino acid metabolism and can provide an index of substrate availability for protein synthesis. There have been several attempts to use the plasma amino acid distribution to evaluate the adequacy of protein intake during and after flight by Russian investig a t o r ~ . " ~ ~A' ' systematic ~ inflight analysis of the changes in the plasma amino acid levels was carried out for the SLS-2 mission. Blood was collected from four SLS-2 astronauts before, during, and after the flight. Examination of the plasma amino acid profile showed some interesting features. Firstly, after the initial adjustment period, the plasma levels of the essential amino acids (EAA) and the branched chain amino acids (BCAA) in particular were increased inflight. In Fig-

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ure 20, the results for leucine are shown, indicating a significant increase on days 8 and 12, both with regard to preflight and postflight levels. This increase occurred in spite of a 20% reduction in protein intake and suggests that aniino acids are not limiting during the flight. The observations do not support the inflight supplementation of the diet with amino acids to attenuate the protein loss in microgravity, provided that food intake remains safely above habitual intake. Amino acid supplementation might lead to an excess with unforeseen metabolic consequences. The unexpected increase in the BCAAs and the EAAs inflight even when dietary intake is reduced suggests an explanation for the following observation. In space flown rats there was a decrease in the enzymes of the lipid oxidation pathway toward the glycolytic pathway together with glycogen accumulation. '51 The shift toward glycolysis accompanied by a compromised ability to oxidize longchain fatty acids renders the muscle more fatigable, even though the capacity to transport glucose is e n h a n ~ e d . * ~The > ' ~shift ~ in metabolism is evident in the increased content of glycolytic energy-deriving enzymes, elevated storage of glycogen, and disappearance of peripheral mitochondria. 14-16,38244Muscle strength is not seriously impacted in humans, but the ability to maintain output is compromised. Excess amino acids promote gluconeogenesis, which is substrate-driven. In humans, most excess acids are converted to alanine and glutamine and then to glucose prior to disposal.'56 Disposal can be by oxidation, conversion to glycogen, or conversion to fat. Increased glucose availability would lead to an increase in the enzymes associated with the glycolytic pathways, an increase in glycogen, and a decrease in the enzymes associated with lipid metabolism indeed has been found. x7,'55 Another interesting feature is the difference between the last inflight measurement (day 12) and the sample collected immediately after landing (Figure 20). In fact, most of the essential amino acids are increased during flight and decreased postflight, notwithstanding the variability in the values due to the fact that the plasma amino acids are a small, rapidly turning over pool interconnected to different protein pools of varying turnover rate. To reduce the need for inflight blood collection, Russian investigators have collected samples as soon as possible after landing, assuming that these would still represent the inflight s i t ~ a t i o n . ' The ~~~'~~ SLS-2 data show that this assumption is not warranted, probably because even immediately after landing, tension has already been applied to the atrophied muscles and the postflight stress response is operative. Russian investigators have found instances of plasma amino acids being reduced after flight, most consistently for r n e t h i ~ n i n e . ' ~ A ~ , similar '~~ observation was made for methionine in the four astronauts of the SLS-2 mission. The immediately postflight lowering of the plasma levels of methionine and other amino acids appears to be a common phenomenon. This decrease persists for the first week of recovery. These findings are consistent with the assumption that the rate at which amino acids are removed from the free amino acid pools in tissues

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and plasma is increased in order to support an increased rate of protein synthesis by the regenerating muscles. There are some rodent data consistent with the assumption that amino acids are limiting in the postflight period. In rat hind limb suspension studies, Tucker found that after release of the animals, protein synthesis in the gastrocnemius muscle returned to the preimmobilization baseline within 6 h, remained unchanged for the next 2 days, and doubled on the fourth day.'57 The lag period during the first 2 days of release may have been due to a shortage of amino acids. The Russian3 investigators have interpreted their 7-day postflight findings of decreased plasma protein levels as indicating a deficit in hepatic protein synthesis. 13' It is thus possible that some of the essential amino acids may be a limiting factor in supporting optimal protein synthesis during the period immediately after landing. The first two days after a period of spaceflight are likely to constitute the most critical part of the recovery phase. A competition for scarce resources may occur between the needs for increased muscle protein synthesis and other systems such as an acute phase protein response. Thus, there might be some advantage to amino acid supplementation before landing and in the early postflight period after a long-duration mission. The data on which to base a specific recommendation for amino acids are not as clear as for energy. However, the available information does show two things: (1) there is no need for inflight amino acid supplementation, provided that the energy intake is within the prescribed limits; ( 2 ) amino acid supplementation immediately before or after landing may be of benefit in increasing the rate at which lost protein is regained. The available information is not strong enough to make the second suggestion into a recommendation, since we do not know at this time whether only one or two essential amino acids become limiting or whether this is a general phenomenon. If a need for postflight amino acid supplementation can be convincingly demonstrated, this will in all likelihood be done with a balanced mixture of amino acids (or protein) rather than with specific amino acids. The reason is that individual amino acids in excess can be toxic, although the chance is remote.

VI.

SUMMARY

The protein loss in humans during spaceflight is partly due to a normal adaptive response to a decreased work load on the muscles involved in weight bearing. The process is mediated by changes in prostaglandin release, secondary to the decrease in tension on the affected muscles. On missions, where there is a high level of physical demands on the astronauts, there tends to be an energy deficit, which adds to the muscle protein loss and depletes the body fat reserves. While the adaptive response is a normal part of homeostasis, the additional protein loss

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from an energy deficit can, in the long run, have a negative effect on health and capability of humans to live and work in space and afterward return to Earth.

ACKNOWLEDGMENT This study was supported by NASA contracts NAS9-18755, NAS9-I9409 and NASA-N65934.

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94. Jiang, B., Roy, R.R., Navarro, C., Edgerton, V.R. Absence of a growth hormone effect on rat soleus atrophy during a 4-day spaceflight, Journal of Applied Physiology, 74527-53 I , 1993. 95. Hymer, W.C., Grindeland, R.E., Salada, T., Nye, P., Grossman, E.J., Lane, P.K. Experimental modification of rat pituitary growth hormone cell function during and after spaceflight. Journal ofApplied Physiology, 80:955-970, 1996. 96. Sawchenko, P.E., Arias, C., Krasnov, I., Grindeland, R.E., Vale, W. Effects of spaceflight on hypothalamic peptide systems controlling pituitary growth hormone dynamics. Journal of Applied Physiology, 73:S158-8165, 1992. 97. Huntoon, C.L., Cintron, N.M., Whitson, P.A. Endocrine and biochemical functions. In: Space Physiology and Medicine. (A.E. Nicogossian, C.L. Huntoon, S.L. Pool, Eds.), pp. 334-351. Lea 2nd Febiger, Philadelphia, PA, 1994. 98. Fukdgawa, N.K., Minaker, K.L., Rowe, J.W., Goodman, M.N., Matthews, D.E., Bier, D.M., Young, V.R. Insulin-mediated reduction of whole body protein breakdown. Dose- response effects on leucine metabolism in postabsorptive men. Journal o j Clinical Investigation, 76:2306-2311, 1985. 99. Tessari, P., Trevisan, R., Inchiostro, S., Biolo, G., Nosadini, R., De Kreutzenberg, S. V., Duner, E., Tiengo, A,, Crepaldi, G. Dose-response curves of effects of insulin on leucine kinetics in humans. American Journal uf Physiology, (Endocrinol. and Metab.), 251:E334-E342, 1986. 100. Stein, T.P., Schulter, M.D., Boden, G. Development of insulin resistance by astronauts during spaceflight. Aviation, Space and Environmental Medicine, 65:1091-1096, 1994. 101. Grigoriev, A.I., Popova, LA., Ushakov, A.S. Metabolic and hormonal status of crewmembers in short-term spaceflights. Aviation, Space and Environmental Medicine, 58:A121-A125, 1987. 102. Alexandrov, A., Gharib, C., Grigoriev, A.I., et al. Tests d’hyperglycemie provoque par voie orale chez I’homme au cours d’un vol spatial de 150jours (Salyut 7-Soyuz T9). Comptes Rendue Sociefi Biologique, 179: 192-195, 1985. 103. Darmaun, D., Matthews, D.E., Bier, D.M. Physiological hypercortisolemia increases proteolysis, glulamine, and alanine production. American Journal of Physiology (Endocrinol. and Metab.),25S:E366-E373, 1988. 104. Kayali, A.G., Young, V.R., Goodman, M.N. Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis. American Journal of Physiology, (Endocrinol. and Metah.), 252:E621-E626, 1987. 105. Odedra, B.R., Bates, P.C., Millward, D.J. Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver. Biochemical Journal, 214:617627, 1983. 106. Vernikos, J., Dallman, M.F., Keil, L.C., O’Hara, D., Convertino, V.A. Gender differences in endocrine responses to posture and 7 days of -6 degrees head-down bed rest. American Journal of Physiology (Reg. Integ. and Comp.), 265:E153-E161, 1993. 107. Ferrando, A.A., Lane, H.W., Stuart, C.A., Davis-Street, J., Wolfe, R.R. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. American Journal of Physiology (Endocrinol. and Metah), 270:E627-8633, 1996. 108. Gmunder, F.K., Baisch, F., Bechler, B., Cogoli, A,, Cogoli, M., Joller, P.W., Maass, H., Muller, J., Ziegler, W.H. Effect of head-down tilt bedrest (10 days) on lymphocyte reactivity. Acta Ph.ysio/ogim Scandinavicu, 604 (Suppl.):131-141, 1992. 109. Vernikos-Danellis, J., Leach, C.S., Winget, C.M., Rambaut, P.C., Mack, P.B. Thyroid and adrenal cortical rhythmicity during bed rest. Journal ofAppZied Physiology, 33544-648, 1972. I 10. Gardiner, P.F., Montanaro, G., Simpson, D.R., Edgerton, V.R. Effects of glucocorticoid treatment and food restriction on rat hindlimb muscles. American Journal ofPhysiology (Endocrinol. and Metah.), 238:E124-E130, 1980. 11 1. Ferretti, A,, Judd, J.T., Ballard-Barbash, R., Nair, P.P., Taylor, P.R., Clevidence, B.A. Effect of fish oil supplementation on the excretion of the major metabolite of prostaglandin E in healthy male subjects. Lipids, 26:500-503, 1991.

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I 12. Naray-Frejes-Toth, A,, Rosenkranz, B., Frolicli, J.C., Fejes-Toth, G. Glucocorticoid effect on arachidonic acid metabolism i n vivo. Journal of Steroid Biochemistry, 30: 155-159, 1988. 1 1 3. Fert-etti, A., Judd, J.T., Taylor, P.R., Schatzkin, A,, Brown, C. Modulating influence of dietary lipid intake on the prostaglandin system in adult men. Lipids, 24:419422, 1989. I 14. Murphy, R.C., FitzGerald, G.A. Current approaches to estimation of eicosanoid formation in vivo. Advances in Prostaglaizdin, Thromboxane, and Leukotriene Research, 22~341-348, 1994. 1 I S . Seyberth, H.W., Tulassay, T., Kuhl, P.G., Soeding, K., Rascher, W., Schweer, H. Excretion of primary prostanoids and their metabolites during acute volume expansion. Prostaglandins 3 5 2 2 1-232, 1988. 116. Klein, D.C., Raisz, L.G. Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology, 86: 1436-1440, 1970. 117. Murray, D.W., Rushton, N. The effect of strain on bone cell prostaglandin E2 release: A new experimental method. Cakffied Tissue International, 47:35-39, 1990. I 18. Vandenburgh, H.H., Hatfaludy, S., Sohar, I., Shansky, J. Stretch-induced prostaglandins and protein turnover in cultured skeletal muscle. American Journal of Physiology (Endocrinol. and Metah.), 259:C232-(3240, 1990. 119. Wennmalm, A., Fitagerald, G.A. Excretion of prostacyclin and thromboxane A2 metabolites during Icg exercise in humans. American Journal of Physiology (Endocrinol. and Metab.), 255:H15-H18,1988. 120. Rodcmann, H.P., Goldberg, A.L. Arachidonic acid, prostaglandin E2 and F2 alpha influence rates of protein turnover in skeletal and cardiac muscle. .lournu1 qf Biological Chemistry, 257:1632-1638, 1982. 121, Turinsky, J. Phospholipids, prostaglandin E2, and proteolysis in denervated muscle. American Journal ofPhysiology (Endocrinol. and Metah.), 251:R165-R173, 1986. 122. Palmer, R.M., Reeds, P.J., Atkinson, T., Smith, R.H. The influence of changes in tension on protein synthesis and prostaglandin release in isolated rabbit muscles. Biochemistry Journal, 214: 101 1-101 4, 1983. 123. Palmer, R.M. Prostaglandins and the control of muscle protein synthesis and degradation. Prostaglandins Leukotrienes and Essential Fatty Acids, 39:95- 104, 1990. 124. Smith, R.H., Palmer, R.M., Reeds, P.J. Protein synthesis in isolated rabbit forelimb muscles. The possible role of metabolites of arachidonic acid in the response to intermittent stretching. Biocheniic.al Journd, 214:153-161, 1983. 125. Vandenburgh, H.H., Shansky, J., Karlisch, P., Solerssi, R.L. Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation. Journal of Cellular Physiology, 155:63-71, 1993. 126. Koller, A., Sun, D., Huang, A,, Kaley, G. Corelease of nitric oxide and prostaglandins mediates tlow-dependent dilation of rat gracilis muscle arterioles. American Journal ofPhysiology (ffeurt and Circ,),267:H326-H332, 1994. 127. Harada, S . , Nagy, J.A., Sullivan, K.A., Thomas, K.A., Endo, N., Rodan, G.A., Rodan, S.B. Induction of vaacular endothelial growth factor expression by prostaglandin E2 and E l in osteoblasts. Journal of Clinical Investigation, 93:2490-2496, 1994. 128. Marks, Jr.. S.C., Miller, S. Local infusion of prostaglandin E l stimulates mandibular bone formation in vivo. Journal o f O m l Pathology, 17500-505, 1988. 129. Raisa, L.G., Alander, C.R., Fall, P.M., Simmons, H.A. Effects of prostaglandin F2 alpha on bone formation and resorption in cultured neonatal mouse calvariae: Role of prostaglandin E2 production. Endocrinology, 126: 1076-1079, 1990. 130. Legenko, V.J., Balschovsky, I.S., Beregovkin, O.V., Moshkalo, Z.S., Sorokina, G.V. Variation in the composition of the peripheral blood of cosmonauts during 18 and 24 day spaceflights. Kosmicheskaia Biologiin i Aviakosinicheskaia Meditsina, 1:39-45, 1973. 131. Gauldie, J., Richards, C., Baumann, H. IL6 and the acute phase reaction. Research in Immunology. 143:755-759, 1992.

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132. Schoeller, D.A., Ravussin, E., Schutz, Y., Acheson, K.J., Baertschi, P., Jequier, E. Energy expenditure by doubly labeled water: Validation in humans and proposed calculation. American Journal of Phvsiology (Endocrinol. and Metab.), 250:R823-R830, 1986. 133. Schoeller, D.A., van Santen, E. Measurement of energy expenditure in humans by doubly labeled water method. Jonrnal ofApplied Physiology, 53:955-959, 1982. 134. Lifson, N., McClintock, R. Theory of use of turnover rates of body water for measuring energy and material balance. Journal of Theoretical Biology, 12:46-74, 1966. 135. Convertino, V.A. Physiological adaptations to weightlessness: Effects on exercise and work performance. Exercise and Sport Sciences Reviews, 18:119-1 66, 1990. 136. Bychko, V.P., Ushakov, A.S., Kalandarov, S., Markarian, M.V., Sedova, E.A. Crew nutrition on the Saliut-6 orbital station. Kosmicheskuia Biologiia i Aviakosmicheskaiu Meditsina, 1 6 10-1 3, 1982. 137. Gazenko, O.G., Grigor’ev, A.I., Bugrov, S.A., Egorov, A.D., Bogomolov, V.V., Kozlovskaia, I.B., Tarasov, I.K. Results of medical studies in relation to the program of the second space flight on the orbital complex “Mir”. Kosmichrskaia Biologiia i Aviakosmicheskaia Meditsina, 24~31 1 , 1990. 138. Vorobyov, E.I., Gazenko, O.G., Genin, A.M., Egorov, A.D. Medical results of Salyut-6 manned space flights. Aviation, Space, and Environmental Medicine, 54:S3 I-S40, 1983. 139. Johnson, P.C., Rambaut, P.C., Leach, C.S. Apollo 16 bio-energetic considerations. Nutrition and Metabolism, 16:l 19-126, 1974. 140. Kissileft’, H.R., Pi-Sunyer, F.X., Segal, K., Meltzer, S., Foelsch, P.A. Acute effects of exercise on food intake in obeae and nonobese women. American Journal of Clinical Nutrition, 52:240245, 1990. 14 I , King, N.A., Burley, V.J., Blundell, J.E. Exercise-induced suppression of appetite: Effects on food intake and implications for energy balance. European Journal gf Clinical Nutrition, 48:715-724, 1994. 142. King, N.A., Lluch, A., Stubbs, R.J., Blundell, J.E. High dose exercise does not increase hunger or energy intake in free living males. European Journal ofClinical Nutrition, 51:478-483, 1997. 143. Friedl, K.E., Moore, R.J., Martinez-Lopez, L.E., Vogel, J.A., Askew, E.W., Marchitelli, L.J., Hoyt, R.W., Gordon, C.C. Lower limit of body fat in healthy active men. Journal of Applied Physiology, 77:933-940, 1994. 144. Iyengar, A., Narasinga Rao, B.S. Effect of varying energy and protein intake on nitrogen balance in adults engaged in heavy manual labour. British Journal UfNutrition, 41: 19-25, 1979. 145. Berg, H.E., Dudley, G.A., Haggmark, T., Ohlsen, H., Tesch, P.A. Effects of lower limb unloading on skeletal muscle mass and function in humans. Journal ofApplied Physiology, 70:18821885, 1991. 146. Askanazi, J., Weissman, C., Rosenbaum, S.H., Hyman, A.I., Milic-Emili, J., Kinney, J.M. Nutrition and the respiratory system. Critical Care Medicine, 10:163-172, 1982. 147. Kinney, J.M., Elwyn, D.H. Protein metabolism and injury. Annual Review ofNutrition, 3:433466, 1983. 148. Stein, T.P., Gaprindashvili, T. Spaceflight and protein metabolism, with special reference to humans. American J ~ ~ u r n of a l Clinical Nutrition, 60:S806-S819, 1994. 149. Kirkpatrick, A.W., Campbell, M.R., Novinkov, O.L., Goncharov, I.B., Kovachevich, I.V. Blunt trauma and operative care in microgravity: A review of microgravity physiology and surgical investigations with implications for critical care and operative treatment in space. Journal of American College of Surgery, 184:441-453, 1997. 150. Lane, H.W., Gretebeck, R.J., Schoeller, D.A., Davis-Street, J., Socki, R.A., Gibson, E.K. Comparison of ground-based and space flight energy expenditure and water turnover in middle-aged healthy male US astronauts. American Journal of Clinical Nutrition, 654-1 2, 1997.

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15 I . Powell, M.R., D.J. Horrigan, J., Waligora, J.M., Norfleet, W.T. Extravehicular Activities. In: Space Physiology and Medicine. (A.E. Nicogossian, C.L. Huntoon, S.L. Pool, Eds.), pp. 128140. Lea and Febiger, Philadelphia, PA, 1994. 152. Popov, I.G., Latakevich, A.A. Blood amino acids in astronauts before and after a 21 1 -day space flight. Kosn~iche.skaiuBiologiia i Aviakosmiche.skaia Meditsina, 18:10-15, 1984. 1.53. Vlaaova, T.F., Miroshnika, E.B., Ushakov, A.S. Various aspects of amino acid metabolism in humans exposed to 120-day anti-orthostatic hypokinesia. Kosmicheskaia Biologiia i Aviakosmiclzesk&~ Medirsina, 19:35-38, 1985. 154. Ushakov, A S . , Vlasova, T.F. Free amino acids in human blood plasma during space flights. Aviution, Space. and Environmental Medicine, 47: 1061-1 064, 1976. 155. Baldwin, K.M., Herrick, R.E., McCue, S.A. Substrate oxidation capacity in rodent skeletal muscle: Effects of exposure to zero gravity. Journal ujAyp1it.d Physiology, 75~2466-2470, 1993. 156. Nurjhan, N., Bucci, A,, Perriello, G., Stumvoll, M., Dailey, G., Bier, D.M., Toft, I., Jenssen, T.G., Gerich, J.E. GlUtdmine: A major gluconeogenic precursor and vehicle for interorgan c a bon transport in man. Journal of Clinical fnvesfigafion,95: 272-277, 1995. 157. Tucker, K.R., Seider, M.J., Booth, F.W. Protein synthesis rates in atrophied gastrocnemius musclcs after limb immobilization. Journal ojApplied Physiology, 51:73-77, 1981. 158. Leach, C.S., Rambaut, P.C., DiFcrrante, N. Amino aciduria in weightlessness. Actu Aeronuurica, 6:1323-1333, 1979. 159. Stein, T.P., Schluter, M.D. Plasma amino acids during human space flight. Aviarion, Space, and Environnzmtal Medicine, 70: 250-255, 1999. 160. Stein, T. P., Leskiw, M. J., Schluter, M. D., Donaldson, M. R., and Larina, I. Protein kinetics during and after long term spaceflight on MIR. Am. J. Physiol. (Endo. and Metub.). 276:E1014E1021, 1999. 161. Stein, T. P., Leskiw, M. J., Schluter, M. D., Hoyt, R. W., Lane, H. W., Gretebeck, R. E., and LeBlanc, A. D. Energy expenditure and balance during space flight on the Shuttle: The LMS mission. Am. J. Physiol (Reg. and Inreg). 276:R1139-R1748, 1999. 162. Blanc, L. R., Normand, S., Ritz, P. Pachiaudi, C., Vico, P., Gharib, C., and Gauquelin-Koch, G. Energy and water metabolism, body composition, and hormonal changes induced by 42 days of enforced inactivity and simulated weightlessness. J. Clin. Endocrind. Metah. 83:4289-4297,

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Chapter 4

HORMONAL CHANGES IN HUMANS DURING SPACEFLIGHT

Felice Strollo I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Feedback Mechanisms in Endocrine System Regulation 111. Hormonal Regulation of Bone Turnover. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. OntheGround . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. InSpace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Hormonal Regulation of Hypothalamic-Pituitary-Adrenal Axis. . . . . . . . . . . . . A. OntheGround . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. InSpace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Hormonal Regulation of Hypothalamic-Pituitary-Gonadal Axi A. OntheGround . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Space. ......................... VI. Hormonal Reg y-Somatomammotrophic Axis. A. OntheGround . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Space VII. Hormonal Regulatio A. On the Ground B. InSpace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Space Biology and Medicine, Volume 7, pages 99-129. Copyright 0 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

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100 101 102 102 103 I04 104

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VIIT. Renin-Angiotensin-AldosteroneSystem and

A. On the Ground . . .

. . . . . . . . . . 115

OntheGround . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 InSpace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 X. The Endocrine Pancreas . . . . . . . . . . . ,118 A. On the Ground ... . . . . . . . . . 118 B. In Space. . . . ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 XI. The Sympathetic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119 ,120 XII. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ 2 2 A.

B.

1.

INTRODUCTION

Many years have passed since the beginning of human space exploration, but most questions are still pending with respect to hormonal adaptation to wejghtlessness. However, the adoption of ever stricter protocol definition strategies-in spite of the many logistic difficulties to be overcome-has brought us closer to an understanding of endocrine space physiology during the last decade. On the other hand, only in 1989 a paper was published in a specialized journal concerning the design and space-qualification of equipment for on-board blood storage developed at NASA Johnson Space Center.’ This shows how difficult it has been to give endocrine space physiology the necessary tools to operate on an adequate scientific basis. After almost a decade of joint efforts by astronauts, engineers, space agencies and scientists, the international community may now have a good chance to obtain solid endocrine data from future research on the International Space Station and to utilize these on the Earth as well as for the benefit of future generations of astronauts spending months or years in microgravity. Hormones are very potent “internal” drugs that are able to induce rapid effects in the human at relatively low concentrations. In fact, any endocrine patient may have his or her own clinical picture dramatically reversed by proper, low dosage, hormonal therapy. Therefore even subtle changes detected in space are important to understand the adaptation mechanisms occurring in microgravity and also to identify suitable treatment strategies for the future. The present paper will focus upon general endocrine pathophysiology in humans. Animal studies will be referred to only where needed for a more complete picture of any of the topics discussed. Nevertheless, animal studies are very important to help in aiming human research protocols, to try and verify some hypotheses, and to confirm results obtained in the still too few astronauts available for experiments in flight. This chapter will deal only marginally with chronobiology and melatonin, a hormone produced by the pineal gland, which may be

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considered as an internal clock. The topic of chronobiology and biorhythms deserves proper attention per ~ e , *warranting ,~ a separate chapter in a future volume of Advances in Space Biology and Medicine. Another research field that will noi be considered in the present review is that of isolation-confinement, as two earlier volumes in this series have been dedicated to this subject (Volume 3 , 1993 and Volume 5, 1 995).4 Moreover, isolation-confinement is a complex condition implying the expertise not only of the endocrinologist, but also-and I should say above all-of the psychologist, and it is extremely difficult to separate psychological effects from endocrine reaction^.^ This presentation will be divided into sections dealing with single endocrine systems in order to make the discussion more readily understandable for nonspecialists in the field of endocrinology. Overlapping areas of interest between the individual functional systems or axes will, therefore, be ignored as far as possible. The analysis will be generally divided in two subsections, one devoted to our understanding o f the mechanism on the ground, the other to the results obtained so far in real or simulated microgravity. Unfortunately, I was unable to cite all interesting papers related to endocrinology in space, especially since data from the most recent MIR missions are rapidly accumulating. I therefore apologize for any omissions from the reference list and invite all colleagues interested in the field of space endocrinology to send me any papers they feel useful for a future review.

II.

FEEDBACK MECHANISMS IN ENDOCRINE SYSTEM REGULATION The endocrine system is characterized by the ability to produce, store, and secrete into the general circulation a number of molecules called hormones. These molecules mostly activate target cells by interacting with specific receptors located on their surface. Typical exceptions are the steroid hormones, which penetrate into the target cells, where they act directly on the nucleus. The endocrine system is invariably based upon feedback signals, which ensure autoregulation through self-dumping mechanisms. This means that a continuous flow of signals travels back and forth through each functional system or “axis” to maintain a constant output of hormone. A hierarchical structure allows a certain group of cells to regulate the activity of others, which are responsible for activating lower level glands and so on down to the most peripheral levels. In turn, the signals coming from each target organ progressively inhibit higher level glands in order to maintain hormonal circulating concentrations within a certain range, corresponding to the “set point” for activation or deactivation of the system. This set point may be shifted up or down depending upon physiological conditions. When the shift goes beyond a naturally occurring range, the system is deranged and pathological changes occur. A typical example is presented by the hypothalamic-pituitary-adrenal axis, but any other hormonal subsystem follows this pattern.

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111.

HORMONAL REGULATION OF BONE TURNOVER A.

On the Ground

The bone remodeling unit consists of the various cells involved in ( 1 ) the synthesis and ordered alignment of collagen fibers, (2) the deposition o f hydroxyapatite crystals upon these fibers (i.e., mineralization), and (3) the reabsorption of the same structures in the frame of a continuous, dynamically balanced process.6 The cells involved in bone remodeling are osteocytes, osteoclasts, and osteoblasts and their precursors. Osteoclasts are multinucleated cells, endowed with the task o f bone resorption. Osteoblasts are mononuclear cells that are in charge of bone formation. Several local signals, like those triggered by mechanical stress of the piezoelectric, paracrine, and autocrine types, and endocrine messages, like hormones brought to the bone by the blood circulation, modulate the activity of the bone remodeling unit. Thus, bone formation and resorption are alternatively enhanced, which serves to keep the bone resistance at the appropriate level with respect to changing weight-bearing needs. The three hormones, serving as the primary contributors to the balance of this complex and sophisticated system are parathyroid hormone (PTH), active vitamin D3 ( 1 -25-dihydroxycholecaIcifer01,calcitriol, or active D3), and calcitonin (CT). Other hormones acting upon bone are growth hormone (GH), insulin (IRI), insulinlike growth factor- 1 (IGF1) and sex hormones. PTH increases calcium absorption by the intestines and counteracts calcium loss via the kidney. However, PTH also activates osteoclasts to dissolve bone mineral, which then dissociates into calcium and phosphate ions that diffuse into the blood stream. The net result of these effects is an increase in circulating calcium levels and an activation of the bone remodeling unit. The latter activation proceeds by stimulation of the differentiation of precursor cells into osteoblasts, which then initiate the process of bone matrix deposition. Interestingly, the mechanism by which PTH enhances bone formation, acts through an initial and temporary resorption of bone. Active D3 is a strong calcium- and phosphate-sparing substance and a powerful promotor of calcium absorption. PTH stimulates the synthesis of active D3 in the kidney by activating a hydroxylase enzyme that attaches a hydroxyl group at the carbon- 1 position of 25-hydroxy- cholecalciferol (which derives through two earlier chemical changes from the vitamin D ingested from the food). The action of active D3 promotes PTH-induced osteoid (bone matrix) formation, followed by the deposition of mature bone through orderly mineralization. A continuing increase in calcium levels caused by PTH and active D3 , if not checked, would be harmful and could even cause death through cardiac arrest. However, two compensatory mechanisms come into play, namely, self-inhibition of PTH and enhanced secretion of calcitonin by high calcium levels. Calcitonin is a peptide hormone, produced by specific cells dispersed through the thyroid gland, that

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lowers circulating calcium levels by stimulating calcium excretion by the kidney and by inhibiting osteoclast-dependent bone matrix demineralization. Growth hormone enhances bone growth until sexual maturation is reached and bone trophism throughout the life span mainly through stimulation of collagen synthesis. It also increases muscle mass and tone, which in turn stimulates bone trophism. Growth hormone accomplishes these tasks by triggering the production of an “effector” in the liver called insulinlike growth factor-1 (IGFl). This substance, which is a peptide resembling insulin, promotes protein-anabolic effects. Sex hormones are also involved in bone physiology. In women, estrogens (mainly estradiol or E2), and in men, androgens (mainly testosterone, T), are responsible for inhibition of bone calcium loss and enhancement of intestinal calcium absorption and bone cartilage maturation. To summarize, active D.3, IGFl, insulin, and sex hormones stimulate bone formation, CT inhibits bone catabolism, and PTH exerts a dual effect, initially catabolic and finally anabolic (through active D3). An increase in circulating calcium levels is “sensed” as a signal of bone catabolism, which leads to inhibition of the production of PTH and active D3 and to stimulation of CT secretion. The opposite effects occur in the presence of low circulating calcium levels.

B.

In Space

A well known effect of spaceflight is bone demineralization with hypercalcemia (high calcium levels in the blood), an effect already noticed after the first few mission^.^.^ Bone atrophy results from the loss of anti-gravitary muscle tone and tendon tension, and especially from the loss of weight-bearing function in microgravity. Calcium loss through urine and stool is high in the beginning of flight, and if i t were allowed to continue it would add up to a 20 to 25% mineral loss after three months in space. So if these high levels of demineralization continued, then astronauts on long-duration flights would be at a severe risk of spontaneously occurring fractures! Fortunately, self-limiting mechanisms come into play, so that such peak losses have never been observed in space, even after a year or longer. This may in part be due to the use of regular treadmill exercise sessions against some degree of artificial gravity-load by means of elastic bungees during longterm missions. Bone demineralization in astronauts resembles osteoporosis occurring in the elderly because, in both conditions, osteoblast activity is decreased. There has also been noticed during the first years of space exploration an activation of osteoclasts, that has recently been confirmed. These two effects could contribute to the bone demineralization occurring in space. Studies of humans in head-down bed rest and of tail-suspended rats point to a decrease in the levels of PTH and active D3.”, After a 7-day head down-tilt bed rest, there was an early and constant increase in circulating levels of osteocalcin, a marker of bone formation, and in urinary excretion of deoxypyridinoline, a

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marker of bone resorption.’* However, on the basis of data available in the literature, the endocrine system is apparently far from being “turned off’ in space by the hypercalcemia caused by bone demineralization. Thus, active D3 levels have been found to increase and bioactive PTH to keep normal in four astronauts during the 8-day SLS-2 m i ~ s i o n . ’Actually ~ PTH has been more often reported to increase in space, both in humans during flightJ4 and in rats at reentry, possibly due to relative functional impairment of the kidney.’ Still some papers describe PTH There seems to be no general agreement about changes in circulating calcitonin levels. A general overview of space biomedical results, which should also be interesting for those working in the field of bone metabolism, was presented by Russian authors17 and a paper on the theoretical basis for physical activity as a countermeasure against bone demineralization by American scientists. Hypoandrogenism (low levels of testosterone) observed in space may increase bone demineralization, while the high IGFl levels found in the astronauts participating in the D2 mission might be somewhat protective. The scientific community has recently shown growing interest in this topic, so future missions are expected to focus on defining preventive strategies against muscle and bone deterioration in space. Hopefully, such studies may shed more light on the endocrine mechanisms influencing the system and eventually suggest suitable hormonal treatment protocols useful for both space purposes and for the treatment of osteoporosis on Earth.

’

’*

IV. HORMONAL REGULATION OF HYPOTHALAMIC-PI TUITARY-ADRENAL AXIS A.

On the Ground

The hypothalamic-pituitary-adrenal axis (HPAA) mediates the so-called “stress reaction”, the overall response of the body to any environmental, psychological, or inner (fever, etc.) factor sensed as potentially dangerous to the organism and able to upset its equilibrium status. Upon nerve signals from the brain cortex, the hypothalamus secretes, through the neurons of the paraventricular nucleus, corticotrophin-releasing hormone (CRH) and antidiuretic hormone (ADH, or arginin-vasopressin). These two hormones act on the pituitary where they synergistically induce the release of adrenocorticotropin (ACTH), a peptide hormone that in turn activates the production of cortisol (known for its anti-shock effects) and other corticoadrenal steroids, including a weak androgen called dehydroepiandrosterone (DHEA), which is known for its anti-aging effects. CRH also acts on the adrenal medulla, triggering the secretion of epinephrine and norepinephrine, the two catecholamines involved in the hyperacute neurogenic stress reaction (see section XI). The HPAA is regulated by a feedback mechanism in the

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sense that output from the target organs turns off the upper level endocrine organs. Thus high blood levels of ACTH inhibit CRH production, whereas high cortisol leveis inhibit both ACTH and CRH production. When a stressful event occurs, the secretion of ACTH and cortisol increases thus preparing the body to defend itself against the hostile element. When the stimulus persists over a long period, as in chronic stress, the HPAA is under a continuous challenge. After some time it adapts, thus often coming down again to prestress levels. Moreover, a paradoxical HPAA activity pattern may be found in the presence of chronic stress. In this case, circulating cortisol attains lower baseline levels and shows a less prominent response than usual. A typical chronic stress reaction is the one commonly observed in depressed subjects, as well as in many elderly people, who are exposed to adverse environmental, affective, and socioeconomic conditions. These two categories are characterized by moderately elevated circulating levels of ACTH and cortisol and are even less prone to physiological HPAA inhibition by administration of a corticosteroid. A similar condition might be expected to occur during the preparatory phase for a spaceflight in astronauts, who have the prospect of a very busy schedule involving the execution of highly specialized tasks, who will undergo severe physical training under the psychological pressure of approaching mission, and who will be subjected to the separation from family and friends. Yet, as will be seen in the following subsection, theoretical expectations have been contradicted by real flight results.

B.

In Space

Since the beginning of spaceflight, many papers have been dealing with cortisol and other adrenal steroids, such as the 17-hydroxycorticoids excreted in the urine. Generally, an increase was found during spaceflight suggesting that microgravity activates the adrenals. Actually, most of the studies were comparing only pre- to post-flight results so that the results were riot necessarily reflecting microgravity per se but also-and maybe mostly-the effects of reentry stress together with any pharmacological and nutritional countermeasures utilized before landing. 1c)-2’ As a matter of fact, when an astronaut was studied during the Spacelab-1 mission, cortisol excretion peaked just after launch in parallel with ADH excretion. Both hormones leveled off thereafter.” This time-dependent pattern might be interpreted as an indication of how stressful the launch maneuver was for the crew and, conversely, of how low was the impact of microgravity per se upon cortisol production.23 Subsequent reports were also in line with this interpretation suggesting that cortisol-related stress levels were not sensed as very high, at least when compared to the observed circulating concentrations of cortisol. This was even more true when nutritional and biorhythmic conditions could be monitored so that scientists were able to exclude the occurrence of any uncontrolled perturb-

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ing factors. Table 1 clearly shows that ACTH and cortisol did not increase during the D-2 flight. DHEA sulfate did not change either throughout the m i s ~ i o n . ~ ~ . ~ ~ An interesting finding was that reported during real or simulated longterm flights by Russian scientists, who showed some decline of cortisol secretion, starting about two months after launch or simulated mission start.29 This might also be interpreted as a chronic stress effect with exhausted response by the organism, but the psychological tests performed at the same time allowed the ruling out of such a conclusion. Therefore, the latter results once again seem to point to a reduced emotional impact of the mission upon the subject rather than to a stressed condition.

V. HORMONAL REGULATION OF HYPOTHALAMIC-PlTUITARY-GONADAL AX1S A.

On the Ground

The hypothalamic-pituitary-gonadal axis is the system by which the gonads are stimulated. As in the case of the HPAA, the hypothalamus represents the highest level in this system. Certain hypothalamic cells produce gonadotrophin-releasing hormone (GnRH), a decapeptide responsible for the secretion of two hormones by the pituitary: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH is necessary for follicular development and estrogen production in the ovaries as well as for spermatogenesis in the testes. LH triggers ovulation and causes production of progesterone by the corpus luteum in the ovaries and is necessary for androgen secretion by the testes. The ovaries produce three estrogens in response to FSH: the major estrogen is 17-P-estradiol and the others are estrone and estriol. The major androgen produced by the testis is testosterone (T), but weaker androgens are also secreted like androstenedione.Testosterone and 17-P-estradiol circulate in the blood largely bound to a high-affinity protein, the sex hormone-binding globulin. The two hormones are steadily and slowly released from the protein complex, providing stable free hormone concentrations and allowing them to reach their target organs. The blood concentration of the globulin is regulated by the two hormones: it is increased by 17-P-estradiol and decreased by testosterone. The action of the former protects against excessive peripheral activity of the hormones. Conversely, globulin levels decrease with increasing levels of testosterone, which thus turns out to enhance its own delivery to the periphery. Target organs finally convert testosterone into the even more potent androgens dihydrotestosterone (DHT) and 3-a-androstanediol. The latter mostly circulates in the blood as a glucuronide (3ADG). Finally, both are converted into inactive metabolite^.^'.^^ As is the case for HPAA, the hypothalamic-pituitary-gonadal axis is regulated by a feedback mechanism so that the secretion of gonadotrophin-releasing hor-

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mone is blunted by high levels of LH, while it’s production as well as that of LH are inhibited by high sex hormone levels. In addition, the gonadotrophin-releasing hormone neurons are under the influence of inhibiting or stimulating brain monoamines or peptides, like P-endorphin, acetylcholine, serotonin, dopamine, and norepinephrine. The dynamic equilibrium between these substances modulates the firing rate of these neurons into a pulsatile pattern so that LH production by the pituitary has an episodic and rhythmic attitude with bursts occurring every 90 to 100 minutes. The blood levels of many hormones fluctuate in like manner so that, in physiological studies on the ground, investigators preferably deal with the mean of three samples obtained within a short time frame in order to rule out uncertainties due to episodically high or low concentrations corresponding to peak or nadir secretion phases. Of course logistic and ethical constraints (e.g., shortage of time and health hazards) impose severe constraints on space scientists, who have to accept Table 7. Changes of Various Hormones lnflight and Postflight in Percent of Preflight Levels A

DUJ

ACTH

Cortisol

LH

MDC

pT

1

Mission R+O R+ 1 R+7/8 R+15

119.4 28.5 103.9 74.8 109.4

126.1 144.4 110.9 103.1 121.9

160.7 130.4 282.6 143.5 130.4

79.9 59.8 49.2 106.1 151.6

76.8 69.2 62.6 83.4 103.3

Mission R+O R+ 1 R+7/8 R+15

182.7 54.0 182.0 105.0 112.27

75.2 58.8 43.4 38.2 6.0

119.1 95.2 123.8 114.3 138.1

79.8 47.0 80.3 91.6 126.5

81.3 33.1 79.9 92.8 61.9

Mission R+O R+ 1 R+7/8 R+15

76.7 36.7 68.5 44.5 81.0

82.4 37.1 72.6 56.7 60.8

126.1 1 13.0 100.0 100.0 113.0

50.8 69.0 68.8 73.1 95.2

16.3 61.2 66.0 51.7 94.6

Mission R+O R+ 1 R+7/8 R+lS

129.9 345.1 103.0 89.4 138.6

131.9 136.2 50.4 62.9 107.3

161.1 94.4 127.8 105.6 150.0

89.9 54.7 71.6 56.7 110.1

34.6 88.5 33.9 45.5 77.0

2

3

4

Noter:

salT

5.8 -

urT

60.2

-

426.2

-

-

61.8

-

-

1.6 -

90.5 -

59.8

-

-

93.7

-

-

73.2

71.3

-

-

75.0

-

-

64.0

-

5.7 -

4.4

-

85.4

-

-

98.7

-

-

Data from Spacelab D2 mission, 1993. A=astrondut, LH=luteinidng hormone, 3ADG= 3-a-androstanediol glucuronide, T=testosterone, pT=plasma T, salT=salivary T, urT=urinary T. “Mission” indicates flight day 4 or 5 for blood samples salT: mean concentrations for flight days 0 and 1 . urT: mean concentrations for flight days 0 and 4 or 5 .

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A

LH

U/1

3 A D G mg/l 18

T nmol/l

-

18

-

12

Ur-T nrnoli 10 min

\

0 Sal-T T

12

pmol/dl

S.E.M.

- 6

6

*

- 0

0

n P

I

n P

I

n P

I

n P

I

n P

I

Figure 7. Changes in the HPCA-related hormone concentrations in four male astronauts on the D2 mission in 1993. Data points are means with S.E.M. *indicates statistical significance at 95% level of difference between preflight (P) and inflight levels ( I ) . LH= luteinizing hormone, 3ADC= 3-a-androstanediol glucuronide, T=testosterone, Sal-T = salivary T Ur-T = urinary T.

these limitations in the number of samples obtained in space in order to try and solve at least some of their original questions. This is why the microgravity studies conducted so far have had to ignore the problem of pulsatility and base their conclusions on single-point determinations. Unfortunately, this sometimes poses a problem in getting a paper on a space endocrinology experiment accepted for publication; the reviewers expect all papers to satisfy well established criteria for “good science”, in this case the use of multipoint hormone assays.

B.

In Space

Few studies on the behavior of the hypothalamic-pituitary-gonadal axis in space have been carried out so far. Russian scientists conducted the first ones. In their early studies, they suggested that rat spermatogenetic potential is not

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affected by microgravity per se but rather by radiation occurring during spaceTen years later, Serova and her group showed that reproduction was still possible in space-flown rats, but they found some spermatogenetic abnormalities and reported a lower viability of rats conceived in space.33 A recent paper reported on the absence of any corticoadrenal signs of stress in pregnant rats under simulated weightlessness conditions, although fetaI growth was adversely affected by the space simulation technique used by the authors.34 On the other hand, fertilization might be somewhat impaired in microgravity, as frozen bull spermatozoa showed less motility after being thawed in space than on the ground.35 In any case, Japanese investigators showed that frogs and fishes could copulate in space, despite some need for learning and overcoming “logistic diffi~ulties”.~ The ~-~ testicular ~ endocrine function in rats in space has also been investigated. On the whole, androgen production was decreased during real and simulated w e i g h t l e s ~ n e s sbut , ~ ~some ~ ~ ~ authors could not confirm this trend in three-week long simulation ~ t u d i e s . ~The ’ same impairment in androgen production has more recently been found in humans during the D-2 mission in 1993: testicular androgen levels decreased in various biological fluids, including saliva.42 A slight perhaps compensatory increase in LH blood levels was detected in all four astronauts participating in this study. The increase slowly reversed through the reentry period and normalized within two weeks after landing (see Table 1 and Figure I ).43-44 Even though women astronauts are no longer the exception, no studies are available on their HPGA function, unfortunately. Ethical considerations suggest that they should keep ovulation inhibited during exposure to the space environment. In view of the high radiation risk to the ovaries during spaceflight, there is a danger o f the occurrence of genetic abnormalities that could be transmitted in a subsequent pregnancy. However, on account of the future role of women in space, well designed studies on the effects of spaceflight in women are to be desired, not only in the field of reproduction but also with respect to the whole range of physiological mechanisms of adaptation to weightlessness since gender differences have already been found to exist at various levels including the cardiovascular ~ystern.~~.~~

VI. HORMONAL REGULATION OF HYPOTHALAMIC-PITUITARY-SOMATOMAMMOTROPHIC AXIS A.

On the Ground

The hypothalamic-pituitary-somato-mammotrophic axis is a complex endocrine system that starts with the hypothalamic neurons producing various regulatory peptides that either stimulate or inhibit hormone secretion by the pituitary.

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These hormones are growth hormone (GH, or somatotropin) and prolactin (PRL). GH and PRL were initially known as somato-mammotrophic hormones, because of their structural and functional analogies to human placental lactogen (HPL), a hormone that is produced by the placenta during pregnancy and enhances both somatic growth and mammary development. Stimulating secretion of growth hormone is the hypothalamic peptide called GH-releasing hormone, while another hypothalamic peptide called somatostatin (SS), or somatotropin-release inhibiting hormone, inhibits secretion of growth hormone. These peptides are secreted by specific hypothalamic neurons into the pituitary portal blood flow and thus affect GH release by the pituitary. Various brain biogenic amines are responsible for the regulation of the peptide production: the catecholamines norepinephrine and dopamine enhance GH-releasing hormone production, and acetylcholine inhibits somatostatin production. The balance between these two biogenic amine classes ensures proper GH secretion, which is pulsatile during the day and comes in huge bursts during the late nightearly morning period. This part of the day is characterized by a high parasympathetic activity and, therefore, by the withdrawal of somatostatin inhibition.I8 From the mechanisms explained above, it can be concluded that a sudden or sustained discharge of catecholamines into the hypothalamus results in an overproduction of GH-releasing hormone, which causes an increase in GH level. This makes GH a typical stress hormone and one that reacts promptly even to the painful insertion of a needle into the arm vein for blood chemistry (“needle stress”). This phenomenon is so well known to scientists that, in order to overcome the problem, 30 minutes are generally allowed after needle insertion when blood is drawn for a GH assay, because by then the wave of GH release has passed due to the relatively short half-life of the hormone. It is also important to note that similar precautions are necessary for other “stress peptides”, like prolactin, or catecholamines themselves. What does GH do at the peripheral level and how does it do this? GH is a typical anabolic agent, which means that it acts at any level by enhancingprotein synthesis, after mobilizing energy storage molecules like triglycerides and glycogen in order to yield high energy phosphate radicals (ATP) from fatty acids and glucose oxidation. The anabolic effect is directed towards all kinds of tissues, even if the main targets are believed to be bone and muscle, which become stronger and heavier in the presence of properly high GH levels. Paradoxically, pathologically high titers of the hormone have a deteriorating effect. For its anabolic action, it actually needs the liver to synthesize a peripheral effector called insulinlike growth factor- 1 (IGFI, also called somatomedin-C). The latter amplifies the GH signal, as the IGFl level remains rather steady for hours, and this accounts for the main GH effects. This feature makes IGFl a major marker of sustained GH release (which otherwise would have to be verified by repeated sampling throughout the day) and of proper GH biological activity. For instance, a clinical picture

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of high GH/low IGFl, known as Laron’s syndrome, results from a genetic defect, causing the synthesis of biologically inactive GH.47 What can be said about the regulation of prolactin secretion? This is stimulated by the releasing factor for thyroid-stimulating hormone. This factor is a tripeptide produced by the hypothalamus that activates certain pituitary cells to secrete the thyroid-stimulating hormone (TSH) and others to secrete prolactin. This dual role of the releasing factor explains why hypothyroid subjects, who have a chronic compensatory hypersecretion of both releasing factor and TSH, have high prolactin levels in the blood. The opposite effect, namely inhibition of prolactin secretion, is exerted by dopamine (DA). An increased level of dopamine in the hypothalamus has prolactin-inhibiting effects.48 Although it is still believed that dopamine itself is the prolactin-inhibiting factor, a specific peptide is still being investigated for this role. The target organ for prolactin is the mammary gland. The hormone acts directly on its peripheral tissue without any need for an intermediate endocrine gland to amplify its effect. When considering together the stimulating effect of dopamine on GH secretion and its inhibiting effect on prolactin secretion, one can understand why opposite changes in the circulating levels of these two hormones is commonly interpreted by neuroendocrinologists as a reflection of a changed DA level in the central nervous system (CNS), especially in the hypothalamus. Thus, an increasing GH level with a decreasing prolactin level suggests the possibility of an increase in CNS DA level, whereas increasing prolactin level with a decreasing GH level suggests the possibility of a decrease in CNS DA level. Conversely, a simultaneous sharp increase in GH and prolactin levels signals a “stress effect”, especially when accompanied by increased blood levels of norepinephrine, epinephrine, and cortisol.

B.

In Space

Some papers on spaceflight studies of GH and prolactin contain only pre- and post-flight data. These will not be discussed here as too many interfering factors may have affected the values in single blood samples obtained post-flight. Any changes observed may reflect post-flight stress from reentry and post-flight readaptation to Earth’s gravity. As mentioned before, stress may increase GH and prolactin levels. Few studies of the hypothalamic-pituitary-somato-mammotrophic axis have been carried out in space. These were mostly dealing with rats, which unfortunately are not the best species to be chosen for the study of GH adaptation mechanisms in astronauts as they do not necessarily reflect human hypothalamic-pituitary-GH response to various stimuli. Initially these studies pointed to the possibility of an impairment in the secretion of bioactive GH during spaceflight. This was concluded from the finding that pituitary cells isolated from rats returned from flights of biosatellites COSMOS 1887 and 2044 showed a greatly reduced GH secretion on the ground. The immuno histochemical and in

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<

I

350 -

n P

I

,&

P R L ~ U I ~

111)

GHng/l IGF-1 ngPml

1

5.E.M-

n P

I

*

i n P

I

figure 2. Changes in the HPSMTA-related hormone concentrations in four male astronauts on the D2 mission in 1993. Data points are means with S.E.M. *indicates statistical significance at 95% level of differences between preflight (P) and inflight (I) levels. PRL = prolactin GH = growth hormone, IGF = insulin1ike growth factor. situ hybridization localization of mRNA for GH-releasing hormone and somatostatin in the hypothalamus of rats flown on the same biosatellites further supported these findings. A more pronounced depletion of GH-releasing hormone than of SS was found.49 However, this was not the case in hind limb-suspended rats, a widely used simulation model, suggesting that the observed changes might not depend only on fluid ~ h i f t . ~An ~ . opposite ~' trend was found in humans. In the four astronauts tested during the D-2 mission in 1993, the circulating levels of GH and IGFl were increased (see Figure 2).44This was interpreted as a possible effect of increased blood flow to the brain and a consequent counteracting increase in catecholamine release in the CNS as an autoregulatory response of the microvascular system.

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On the other hand, a decrease in prolactin secretion by pituitary cells cultured in space seems to be a consistent finding.283s2-s3 In agreement with this is the fincling of decreased prolactin levels in blood from the four D-2 astronauts (see Figure 2). These results support the hypothesis of an increase in catecholamine levels in the CNS. Since so few space experiments have been carried out in this field, there will be an opportunity for further and more extensive studies on the International Space Station.

VII. H O R M O N A L REGULATION OF HYPOTHALAMIC-PITUITARY-THYROID AXIS A.

On the Ground

The hypothalamic-pi tuitary-thyroid axis involves the hypothalamic neurons producing releasing hormone for thyroid-stimulating hormone (TSH), the pituitary cells producing TSH, and the thyroid gland synthesizing the hormones thyroxine and triiodothyronine. The latter two hormones are present in blood mainly bound to a thyroxine-binding globulin. This helps by keeping their concentrations in the peripheral tissues at an adequate and steady level. There, most of the thyroxine is converted into its more active metabolite triiodothyronine. As with all other endocrine axes, the hypothalamic-pituitary-thyroidaxis is regulated by a feedback mechanism. High TSH levels reduce the activity ofthe neurons producing TSH releasing hormone, while TSH secretion is inhibited by high thyroid hormone levels. The axis is stimulated by exposure to a cold environment. This is explained by the fact that thyroid hormones uncouple oxidative phosphorylation so that part of the energy coming from glucose oxidation is not conveyed to the final metabolic steps yielding the maximal amount of ATP. This energy is then dissipated as heat, the so-called thermogenic effect. This explains why hyperthyroid patients are heat-intolerant and tend to lose weight. There are two borderline conditions in which the feedback mechanism is still active, even if in a less apparent fashion: ( 1 ) subclinical hypothyroidism, characterized by low-to-normal thyroid hormone levels and a moderately high TSH concentration, often associated with high anti-thyroid autoantibody titers and (2) nonthyroidal illness, which is typical of chronic stress, in which the high CRH and cortisol levels blunt both TSH and thyroid hormone release thus maintaining relatively low thyroxine and triiodothyronine levels without a concomitant increase in TSH. B.

In Space

Spaceflight seems to induce in rats a condition of mild hypothyroidism, as shown by circulating thyroxine and triiodothyronine determinations and by morphological and histochemical changes of the t h ~ r o i d . ' ~ . ' ~ This is in agreement

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with the observation of reduced thermoregulatory responses in rhesus monkeys flown aboard biosatellite COSMOS 1514.59 A similar trend was found in humans during the D-2 mission. Gunga and colleagues reported slightly increased TSH levels during flight, suggestive of mild hypothyroidism with compensatory pituitary hyperf~nction.~' If these findings can be confirmed during longer missions, then this would represent another case of primary endocrine failure, like the one found in the case of human testes.43 Hence, there is a need for further studies in this field.

VIII.

RENIN-ANCIOTENSIN-ALDOSTERONESYSTEM AND WATER-E LECTROLYTE-REGULATlNC PEPTIDES A.

On the Ground

The renin-angiotensin-aldosteronesystem (RAAS) is by far the most extensively investigated endocrine axis since the beginning of manned spaceflight. This is mainly due to the need to understand the detailed mechanisms underlying the phenomena related to headward fluid shift in microgravity. In fact, the engorgement of the central circulation, where the mechanoreceptors involved in plasma volume regulation are located, has been thought to cause the observed body mass loss due to dehydration and increased water-electrolyte excretion rate.60-6 When plasma volume decreases due to hemorrhage or dehydration, antidiuretic hormone (arginine-vasopressin, ADH) release by the posterior pituitary increases, allowing it to exert its antidiuretic action. ADH acts by acute inhibition of free water clearance through the renal collecting tubules, resulting in increased preurine concentration and restoration of the intravascular volume. At the same time the juxtaglomerular apparatus, consisting of groups of cells located near the kidney glomeruli, senses lowered tension levels in the afferent arteriolar walls. It therefore tries to restore intraglomerular pressure by secreting larger amounts of renin. This is a peptide hormone, that transforms angiotensinogen, a protein produced by the liver, into angiotensin I. The latter increases blood pressure and in turn generates angiotensin I1 through the intervention of the angiotensin-converting-enzyme, an event occurring mostly in the lung. Angiotensin I1 is endowed with an even higher hypertensive effect than angiotensin I. In addition, angiotensin I1 stimulates the adrenal cortex to produce aldosterone, a steroid which enhances retention of water and sodium and excretion of potassium at the tubular level. Associated with the renin-angiotensin-aldosteronesystem (RAAS) are three peptides with a function in water-electrolyte regulation. These are (1) the already mentioned antidiuretic hormone, (2) the atrial natriuretic peptide produced by the heart atrial wall, and (3) the recently discovered tubular natriuretic peptide (also

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called urodilatin) secreted by the renal tubules. The third one, assayed in urinary samples, is responsible for fast massive natriuresis and d i u r e s i ~ . ~ The ~ . atrial ~~ ndtriuretic peptide is produced in response to mechanical distention of the heart atrial wall. It promotes, like urodilatin, fast natriuresis and diuresis, and both are obviously inhibited in a condition of hypovolemia. This is why the fastest endocrine response to hypervolemic stress-as occurring during excess fluid administration or posture-dependent intravascular fluid redistribution-is represented by hypersecretion of the two natriuretic peptides with reduced RAAS activity (due to increased atrial natriuretic peptide secretion) and reduced firing rate of the ADHproducing neurons. It should be noted that ADH is rather slow-acting and is therefore unable to stop natriuresis immediately; it is typically aimed at ensuring a positive water balance in the long run by enhancing drinking and maintaining a low level of water-electrolyte loss. Hence, the lower ADH secretion leads to reduced water intake through thirst inhibition and to reduced tubular water reabsorption, thus reinforcing the diuretic effect produced by the two natriuretic peptides.

6.

In Space

The above-mentioned features of the renin-angiotensin-aldosterone axis and associated peptides explain why this system has aroused so much interest among space scientists. Actually, the onground mechanisms of water-electrolyte regulation let us all hypothesize a typical hypervolemic-type response of the axis in space, so mathematical models trying to anticipate changes to be found in flight experiments were already produced in the 1980’s. However, experimental results often contradict mathematical models based upon strict rules and overly schematic premises. This explains why many spaceflight results have often been in conflict with the theoretical expectations. Hence, trying to find out the adaptation mechanisms operating in space and the most useful countermeasures has challenged the intelligence of the investigators, but over time this has led to a better understanding of the whole system and to a search for clinically useful pharmacological drugs. Drug resistance is a typical feature of space science. It has been found, for instance, that orally administered drugs do not necessarily follow the same pharmacokinetic and pharmacodynamic rules in space as onground (see chapters 4 and 5 in volume 6 of this series). This is due to fluid redistribution and to loss of hydrostatic forces. The latter effect allows low-energy bonds unexpectedly to prevail and surface tension to become so strong as to cause conformational and functional changes in the membrane-linked receptors for hormones and drugs. An obvious consequence of this is that parenteral drug administration seems to be more predictable and efficient than the oral intake and that generally higher doses are required in space due to reduced r e c e ~ t o r - a f f i n i t y . ~The ~ . ~same ~ considerations hold true for “endogenous drugs” like hormones, which are led to the target organs by blood circulation and act by coupling to their specific receptors. Periph-

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era1 resistance to hormones has still to be established in space. However, the possibility should be taken into account in interpreting the results obtained in space 50 far. It would certainly be worthwhile to undertake specific studies in the future directed at clarifying this topic. Many of the published data derive from simulation studies. Bed rest is the most extensively used technique to simulate in humans the effects of weightlessness on the cardiovascular system and related endocrine systems.60. 68-69 It is preferable to combine bed rest with a head-down tilt at a 6" angle, which shifts the hydrostatic indifferent point of the venous system (low pressure) from the upper abdomen to the chest cage, and that of the arterial (high pressure) system from the heart level to the head. The hormonal changes appear to vary with the duration of bed rest. This may account for some of the discrepancies between the results reported in different publications as will be noticed from the following discussion. There is a need for standardization of experimental protocols. After four hours of bed rest with head-down tilt an increase in atrial natriuretic peptide and a decrease in angiotensin TI and aldosterone levels were o b ~ e r v e d . ~ " However, after four days in this condition there was a decrease in atrial natriuretic peptide level. When lower body negative pressure was applied as a countermeasure, the peptide level increased suggesting a better conservation of plasma volume in this case. After four days of bed rest, the plasma renin activity (determined by indirect bioassay) was increased, while after both shorter and longer periods of bed rest with and without head-down tilt, this activity was mostly found to d e c r e a ~ e . ~ADH ' - ~ ~and neurophysin-I, an ADH-related posterior pituitary hormone, were ~ n c h a n g e dIt. ~has ~ been suggested recently that the action of ADH, though certainly important, cannot explain the entire diuretic response to orthostatic maneuvers, and that other unidentified factors must be involved in the regulation of free water clearance during simulated w e i g h t l e s ~ n e s s . ~ "Water ~~ immersion has been used as a simulation of weightlessness by a few research groups, which managed to cope with the obviously more complicated logistics of this technique.78-80Water immersion causes, like bed rest, the headward shift of body fluids typical of the microgravity condition. It suppresses the renin-angiotensin-aldosterone system and the sympathetic nervous activity as indicated by a decreased plasma norepinephrine level. At the same time, there is an ability to increase cardiac output and decrease peripheral resistance. These changes are consistent with the markedly increased urine flow and sodium excretion together with raised plasma atrial natriuretic peptide and urinary urodilatin levels. The results of many of these studies are still somewhat contradictory, even though more than 25 years have passed since the first manned space mission and over 200 astronauts have been flown in space. Only a general trend is visible and can be outlined here. During spaceflight, ADH was found to increase up to sevenfold after 20 days of microgravity, a result that contradicts the theoretical expectations. More recent missions (SLS-I and D-2) could not confirm these changes. During a 7-month mission, the urinary excretion of ADH tended to increase,

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while the circulating levels of the hormone decreased.22,81-'73 ' 0 5 In rats after 14 days of spaceflight, the ADH content of the posterior pituitary was 20 to 33% lower than in the ground controls.88 This is a typical example of the difficulty in drawing definite conclusions from the available studies. The increased ADH output, observed in some flights may be due to one or more of the following factors: ( I ) a relative dehydration, eventually reinforced by a reduced central venous pressure and heart prefilling, ( 2 ) peripheral resistance to the hormonal action, (3) psychological stress, (4) unreported space motion sickness symptoms as shown in animals, and in humans, plasma ADH levels sharply increase during this condition.89-94The extensive review by Smith and colleaguess3 of the mechanisms regulating body fluid volume and electrolyte homeostasis in spaceflight emphasizes the need for stricter control of dietary intake of sodium and fluid in future studies in space to expand our understanding of the adaptability of human physiological processes as well as to establish suitable countermeasure strategies. The renin-angiotensin-aldosterone system seemed to be activated in the early phase of the Skylab missions. However, in later studies in space as well as in short-term bed rest with head-down tilt, a dissociation between renin and angiotensin I (both increasing) and aldosterone (decreasing) was ~ b s e r v e d . ~ The ~.~~ latter phenomenon might be caused by inhibition of the angiotensin-converting-enzyme in the lung after fluid shift. During a long term mission, urinary excretion of aldosterone increased, while 1 1-deoxycorticosterone, one of its precursors, decreased.' This may indicate an exhaustion of the aldosterone precursor store in the activated adrenal glands. During a MIR mission one astronaut was studied who showed increased levels of circulating renin and aldostero r ~ e . ~ These ' . ~ ~ results may be explained by the low sodium levels and low blood osmolality found in early missions. Actually it is still not possible to describe a definite direction of aldosterone changes during spaceflight because of the variety of conditions possibly interfering with single mission results. Based upon simulation studies, the atrial natriuretic peptide level was expected to be raised during spaceflight and thus to be responsible for hyponatremia and dehydration. However, the opposite effect was detected during the latest missions, even in the presence of increased urinary sodium excretion."' It appears that the tubular natriuretic peptide may actually be the most important factor in space-related diuresis and natriuresis. There is a correlation between the level of this peptide and sodium excretion in spaceflight, although the correlation is weaker than on Earth. This phenomenon might be due to a mechanism whereby the peptide attempts to counteract the decrease in sodium excretion observed in space, 101- 105 The picture for postflight changes is even more conflicting, possibly because of the alteration of the physiological response to the transition from microgravity to Earth's gravity by the operation of some interfering factors, namely, the salt-load and other countenneasures adopted before reentry, the stress experienced during the landing phase, and the postlanding discomfort from orthostatic intolerance.

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An interesting suggestion has recently been advanced: malnutrition during spaceflight might play a major role in fluid loss in microgravity.'"' This is an important issue, that probably will be addressed in future studies.

IX.

ERYTHROCYTE MASS REGULATION A.

On the Ground

Erythropoietin is a polypeptide enhancing recruitment of erythroblasts and their maturation first to reticulocytes and then to erythrocytes in the bone marrow. It is produced by the kidney in response to reduced erythrocyte mass. The initial signal responsible for its secretion is still poorly defined, but the central venous pressure is believed to be an important regulator of erythropoietin secretion.

6.

In Space

During space missions anemia is invariably observed. The initial explanation for this phenomenon was that the erythrocyte production was inhibited by high blood oxygen levels due to the pure oxygen atmosphere in which the astronauts had to live in the beginning of the spaceflight era. In all later flights, however, the cabin atmosphere composition was made similar to common air, so this explanation for space anemia could no longer be accepted. 13, I 5 - l 7 , 21, 87,'07~108It was then proposed that low erythropoietin levels might be invoked as a consequence of the higher dissociation constant of oxyhemoglobin in the presence of the increased phosphate ion concentrations as a result of the accelerated bone demineralization in space. Even if the latter explanation for the trigger mechanism in space anemia were to be correct, it must be recognized that the mechanism underlying lowered erythropoietin production is still far from being fully u n d e r s t ~ o d . ~ ' ~A possible explanation for lowered erythropoietin production is feedback to the lowered central venous pressure 1evel.This is suggested by results obtained by German investigators during their interesting studies of high altitude hypoxia as a unique simulation method for the haemopoietic and cardiovascular system changes in space,109-1 1 0 3 ' '

X.

THE ENDOCRINE PANCREAS A.

On the Ground

Insulin and glucagon are the main hormones produced by the endocrine pancreas in the islets of Langerhans. They regulate glucose metabolism. Insulin

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enhances the utilization of glucose in liver, muscle, and fat tissues and the storage of its energy in the form of glycogen and triglycerides. Thus insulin lowers blood glucose concentrations preventing postprandial hyperglycemia and glucose loss with the urine. Glucagon has opposite effects. Inactivity causes impaired insulin sensitivity so that higher amounts of secreted insulin are required to provide the same hypoglycemic effect after a meal. Obesity further increases the insulin resistance, eventually leading to noninsulin dependent diabetes mellitus. When sedentary and obese people become active, insulin sensitivity gradually recovers.' '-I I 2 6.

In Space

The effect of weightlessness on insulin and glucagon has so far mostly been studied by means of simulation through bed rest with head-down tilt. Invariably, a loss of insulin sensitivity over time was found. This was obviously the result of physical inactivity and, as a consequence, muscle atrophy. In space experiments, there was also a loss of insulin sensitivity as reflected by the excretion rate of the insulin precursor C-peptide and by the response of blood glucose and insulin to an oral glucose tolerance test in the humans.' 13-'17 In rats, the decreased insulin sensitivity was confirmed by postflight studies of insulin level in the blood, insulin receptors in tissues, and morphology of pancreas endocrine cells."*~' I 9 The decreased insulin sensitivity in microgravity is a very important issue for humans living in the future in space colonies as insulin resistance is known to be a cardiovascular risk on Earth, where it is often associated with increased abdominal adiposity, obesity, diabetes mellitus, hypertension, coronary heart disease, and stroke. It is therefore desirable to identify safe nutritional and behavioral strategies to prevent insulin resistance during longterm flight. Exercise, which is extensively utilized onboard the MIR station as a countermeasure against orthostatic intolerance, might well also be a suitable countermeasure against insulin resistance. Some research groups have already been addressing this issue during the last few years.

XI.

THE SYMPATHETIC SYSTEM

The sympathetic system acts by means of the catecholamines. These monoamines include epinephrine, norepinephrine, and dopamine. Epinephrine is released by the adrenal medulla and the adrenergic nerve terminals, while norepinephrine and dopamine are released by adrenergic nerve terminals only. The sympathetic system functionally interacts with the parasympathetic or vagal system. The latter system releases acetylcholine and can therefore be viewed mostly as a part of the autonomic nervous system. It is in fact better expressed by quickly occurring elec-

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trophysiological changes than by shortlived but slowly reacting hormonal changes. ‘2”-’25 The effect of spaceflight on the adrenal medulla has not been extensively studied. Some reports deal with catecholamine levels in blood and nervous tissue or urinary excretion of catecholamines-reflections, to a considerable extent, of the activity of the sympathetic system. The situation is complicated by the fact that an adaptation of the adrenergic system in space is expected through changes in the metabolism, anatomy, and neurotransmitter physiology of the central nervous system that eventually will result from exposure to microgravity.’26 In view of this fact, the results reviewed here are not dealing with the sympathetic system by itself. Nevertheless, it is worthwhile mentioning the sympathetic system in this review for two reasons: (1) indirect signs of an increased adrenergic activity may be inferred from two typical endocrine signals, namely GH and prolactin levels, the apparently and (2) based on this finding and on some other reports,28, 44, unopposed assumption in space physiology that microgravity is a “hyposympathetic condition”, is now being challenged. Preliminary data from the Neurolab experiment (still to be published) seem to reflect an increased rather than a decreased sympathetic activity in microgravity. This is, of course, in contradiction to the assumption of a hyposympathetic condition, which was mostly based upon theoretical considerations and reinforced by results from early missions and subsequent simulation studies. Nowadays experimental conditions are under stricter control by the investigators, who are trying to eliminate as many interfering factors as possible. This may explain why the early results are not being confirmed. Such paradoxical findings as the hyperadrenergic condition in space may be very important, in that they might disclose a new set of “physiological rules”, which may even help in the interpretation of cardiovascular regulation mechanisms on Earth. This is a challenging matter, of course, that illustrates the real magic of microgravity research. Because of such unexpected results that open the way to new interpretations of adaptive mechanisms on Earth, microgravity may finally become a necessary research tool in the hands of the experienced physiologist.

XII.

CONCLUSIONS AND SUMMARY

Readers of this review may feel that there is much more that we do not know about space endocrinology than what we know. Several reasons for this state of affairs have been given:

1. the complexity of the field of endocrinology with its still increasing number of known hormones, releasing factors and precursors, and of the interactions between them through various feedback mechanisms

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2. the difficulty in separating the microgravity effects from the effects of stress from launch, isolation and confinement during flight, reentry, and postflight re-adaptation 3. the experimental limitations during flight, such as limited number of subjects, limited number of samples, impossibility of collecting triple samples for pulsatile hormones like growth hormone 4. the disturbing effects of countermeasures used by astronauts 5 . the inadequacy of postflight samples for conclusions about inflight values 6. limitations of conclusions from animal experiments and space simulation studies The endocrinology field is divided in to nine systems or axes, which are successively reviewed:

1

2

3.

4.

5.

6.

7. 8.

Rapid bone demineralization in the early phase of spaceflight that, when unopposed, leads to catastrophic effects after three months but that slows down later. The endocrine mechanism, apart from the effect of exercise as a countermeasure, is not yet understood. The hypothalamic-pituitary-adrenal axis is involved in stress reactions, which complicate our understanding and makes postflight analysis dubious. In the hypothalamic-pituitary-gonadal axis, pulsatility poses a problem for obtaining representative values (e.g., for luteinizing hormone). Reproduction of rats in space is possible, but much more needs to be known about this aspect, particularly in women, before the advent of space colonies, but also in males because some evidence for reversible testicular dysfunction in space has been found. The hypothalamic-pituitary-somato-mammotrophic axis involves prolactin and growth hormone. The latter also acts as a stress hormone and its secretion is greatly decreased in spaceflown rats, but not in astronauts, which may be due to differences in the regulation of growth hormone secretion between rats and humans. The hypothalamic-pituitary-thyroid axis involves the thyroid hormones thyroxine and triiodothyronine, which are lowered in space, suggesting mild hypothyroidism. The renin-angiotensin-aldosteroneaxis, which regulates water and electrolytes, involves antidiuretic hormone and two natriuretic peptides and shows paradoxical behavior in space. Erythrocyte mass regulation involves erythropoietin, and space anemia is still not explained. The endocrine pancreas involves insulin and glucagon, with loss of insulin sensitivity in space due to lack of exercise, which phenomenon requires more study before the advent of space colonies.

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9. The sympathetic system acts through epinephrine, norepinephrine and dopamine and seems to have an increased activity in space in contrast to what had been widely believed.

From the foregoing conclusions, it is clear that much further study is needed in all fields of space endocrinology. On the other hand, future studies will allow us to understand what happens in a given endocrine subsystem in the absence of the "gravity factor", the perturbing factor to which the human race has become adapted through thousands of years of evolution. This should provide us with a fuller understanding of the internal homeostatic mechanisms. An important point is that some endocrine systems seem to undergo changes in space that resemble those observed during senescence, but after spaceflight, recovery always occurs within weeks or months after r e t ~ r n . l ~ ~This - l ~is' particularly true for the systems regulating bone and muscle metabolism and reproduct i ~ n , exactly ' ~ ~ as happens with the immune, neurosensory, and cardiovascular systems. Further space research may help us find new insights in the pathophysiology of aging and hopefully define novel preventive and therapeutic strategies for extending healthy life on Earth.'33-'40

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66. Vet-nikos, J., Dulln1;in M.E., Van Loon, G., Keil, L.C. Drug effects on orthostatic intolerance induced by bedrest. Journal ojClinical Pharmacology, 31 :974-984, I99 I. 67. Tietze, K.J., Putcha, L. Factors affecting drug bioavailability in space. Journul ofclinical Phurmacolog>~.34:67 1-676, 1994. 68. Noskov, V.B., Katkov, V.E.. Afonin, B.V., Chestukhin, V.V., Sukhanov, Y.V. Central venous pressure and hormonal regulation of the water balance when altered in antiorthostasis. Human Physiology, 12:341-346, 1986. 69. Shi, X., Squires, W.G., Williamson, J.W., Crandall, C.G., Chen, J.J., Krock, L.P. et al. Aerobic fitness: 1: Response of volume regulating hormones to head-down tilt. Medic,ine inzd Science in Sports r i d Ewrcise, 24:991-998, 1992. 70. Crundy, D., Reid, K., McArdle, F.J., Brown, B.H., Barber, D.C., Deacon. C.F. et al, Trans-thoracic fluid shifts and endocrine responses to 6 degrees head-down tilt. Avintion, Space, and Environmental Medicine, 62:923-929, 199 1 . 71. Haas, G., Hinghofer-Szalkay. H., Baisch, F., Maass, H., Lane, L. Blomqvist, C.G. Effect of head-down bed rest on blood/p!asma density after intravenous fluid load. Acta Physiologicu Scundinnvica, 604 (Suppl.): I 1 3-120, 1992. 72. HerbutC, S., Oliver, .I., Davet, J., Visa, M., Bballard, R,W., Gharib, C. et al, ANP binding sites are increased in choroid plexus of SLS- I rats after 9 days of spaceflight. Aviation, Space, and Environmenral Mrdic.int,,65:134-1 38, 1993. 73. Gharib, C., Maillet, A,, Gauquelin, G., Allevard, A.M., Guell, A,, Cartier, R. et al. Results of a 4-week head-down tilt with and without LBNP countermeasure: I. Volume-regulating hormones. Aviation, Syuce, and Environmentul Medicine, 63:3-8, 1992. 74. Maillet, A,, Fagettc, S.,Allevard, A,-M., Traon, A.P.-L., Guell, A,, Gharib, C. et al. Cardiovascular and hormonal response during a 4-week head-down tilt with and without exercise and LBNP countermeasures. Journal qf Gravitutiona! Pliysiology, 3 : 3 7 4 8 , 1996. 75. Anna(, G., Guell, A,. Guaquelin, G., Vincent, M. Plasma vasopressin, neurophysin, rcnin and aldosterone during a 4-day head-down bed rest with and without exercise European Journal of A p l ~ l i ~Physiology, d 5559-63, 1986. 76. Bestle, M.H., Jacobsen, H., Norsk, P., Bie, P. Ten days of head down tilt: Effects of isotonic and hypertonic saline load\ in normal man. Journal qf Gravitational Physiology, 4: 105-106, 1997. 77. Hammerum, M.S., Bie. P., Pump, B., Johansen, L.B., Christensen, N.J., Norsk, P. Vasopressin and renal water handling during water immersion in hydrated humans. In: lKth Annuul Internurionul Gruvitational Physiology Meeting (crhstrac~ts), April 20-25, 1997, Copenhagen, Denmark, p. 48. 78. Stadeager, C., Johansen, L.B., Warberg, J., Christensen, N.J., Foldager, N., Bie, P. et al. Circulation, kidney function, and volume-regulating hormones during prolonged water immersion in humans. Journal ofApplied Phy.siolog.y, 73:530-538, 1992. 79. Nakamitsu. S., Sagawa, D., Miki, K., Wada, F., Nagaya, K. Keil L.C. et al. Effect of water temperature on diuresis-natriuresis: AVP, ANP, and urodilatin during immersion in man. Journal qf Applird Physiology, 77: 1919-1925, 1994. 80. Norsk, P, Role of arginine vasopressin in the regulation of extracellular fluid volume, Medic,al Science in Sjmrts and Exercise. 28 (10, Suppl.):S36-S41, 1996. 8 I . Leach C.S., Altchuler, S.I., Cintron-Trevino, N.M. The endocrine and metabolic response to space flight. Medicine and Science in Sports und Exerci.ce, 15:432-440, 1983. 82. Leach, C.S., Johnson, P.C., Cintron, N.M. The regulation of fluid and electrolyte metabolism in weightlessness. In: Space Physiology (ESA SP-237) (J.J. Hunt, Ed.), pp. 3 1-36, ESA-ESTEC, Noordwijk, The Netherlands, 1986. 83. Smith, S.M., Krauhs, J.M., Leach, C.S. Regulation of body fluid volume and electrolyte concentrations in spaceflight. Advances in Spuce Biology undMedicine (S.L. Bonting, Ed.), 6: 123-165, 1997.

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84. Greenleaf., I.E. Problem: Thirst, drinking behavior, and involuntary dehydration. Medical Science in Sports and Exercise, 24:645-656, 1992. 85. Grigoriev, A.I., Noskov, V.B., Poliakov, V.V., Sukhanov, Iu.V., Gharib, C., Guaquelin, G. et al, Water-salt metabolism and its hormonal regulation studied in the 2nd joint Soviet-French space ?light.Aviukosmicheskaya Ekologiya i Meditsina, 26:36-39, 1992. 86. Kirsch, K.A., Baartz, F.J., Gunga, H.-C., Roecker, L. Fluid shifts into and out of' superficial tisiues under microgravity and terrestrial conditions. Clinical Investigator, 71:687-689, 1993. 87. Grigoriev, A.I., Popova, I.A., Ushakov, A.S., Metabolic and hormonal status of crew members in short-term spaceflights. Aviation, Space, and Environmental Medicine, 21:A121-A125, 1987. 88. Keil, L., Evans. J., Grindeland, R., Krasnov, I. Pituitary oxytocin and vasopressin content in rats flown on COSMOS 2044. Joiirntzl ofApplied Physiology, 73(2, Suppl.):S 166-S 168, 1992. 89. Rowe, J.W., Shelton, R.L., Helderman, J.H., Vestal, R.E., Robertson, G.L. Influence of the emetic reflex on vasopressin release in man. Kidney International, 16:729-735, 1979. 90. Stalla, G.K., Doerr, H.G., Bidlingmaier, f., Sippel, W.G., von Restorff, W. Serum levels of eleven steroid hormones following motion sickness. Aviation, Space, and Environmental Medicine, 56:995-999, 1985. 91. Fox, R.A., Keil, L.C., Daunton, M.A., Crampton, G.H., Lucot, J. Vasopressin, and motion sickness in cats. Aviation, Space, and Environmental Medicine, 28 (9, Suppl.):A143-A147, 1987. 92. Kohl, R.L. Hormonal responses or metoclopramide-treated subjects experiencing nausea or emesis during parabolic flight. Aviation, Space, and Environmental Medicine, 5 8 (9, Suppl.):A266-A269, 1987. 93. Drummer, C., Stromeyer, H., Riepl, R.L., Koenig, A., Strollo, F., Lang, R.E. et al. Hormonal changes after parabolic flight: implications on the development of motion sickness. Aviation, Space. and Environmental Medicine, 61:821-828, 1990. 94. Strollo, F., Strollo, G., Mangrossa N., Ferretti C., Riondino G., Enck, P. Endocrine effects of motion sickness experienced during parabolic flight. Proceedings of the VIth European Symposium on Lqe Sciences Research in Space (ESA SP-390), pp. I 11-1 14, Trondheim, Norway, June 16-20, 1996. 95. Kirsch, K.A., Roecker, L., Gauer, O.H., Krause, R. Venous pressure in man during weightlessness. Science, 225:218-219, 1984. 96. Strollo, F., Strollo, G., Mor& M., Riondino, G. Short-term antiorthostatic position endocrine adaptation test of microgravity [Decuhito antiortostatico di breve durdta quale test di adattamento endocrino precoce alla micrograviti]. Minerva Aerospaziale, 21: 13-18, 1989. 97. Strollo, F., Strollo, G., Mor& M., Riondino, G. Head-down tilt test and space-related endocrine physlology. Proceedings of the 4th European Symposium on Lijie Sciences Research in Spuce (ESA SP-3071, pp. 191-195, Trieste, Italy, May 28-June 1, 1990. 98. Hinghofer-Szalkay, H.G.,Noskov, V., Jezova, D., Sauseng-Fellegger, G., Fueger, G.F., Sukhanov, Y. et al. Hormonal changes with lower body negative pressure on the 6th day in microgravity in one cosmonaut. Aviation, Space, and Environmental Medicine, 64: 1000-1005, 1993. 99. Hinghofer-Szalkay, H., Noskov, V., Schmied, J., Roehrer, R., Viehboeck, F., Koenig, E.M. et al. Changes of blood plasma composition with LBNP on ground and in space in one subject. Aviation, Space. and Environmental Medicine, 65:214-219, 1994. 100. Roessler, A,, Hinghofer-Szalkay, H., Noskov, V., Laszlo, Z., Plyakov, V.V. Diminished plasma cGMP during weightlessness. Journal (if Gravitational Physiology, 4:101-102, 1997. 101. Drummer, C., Gerzer, R., Heer, M., Molz, P., Bie, P., Schlossberger, M et al. Effects of an acute saline infusion on fluid and electrolyte metabolism in man. American Journal of Physiology, 262:P744-P754, 1992. 102. Drummer, C., Heer, M., Dressendoerfer, R.A., Strasburger, C.J., Gerzer, R. Reduced natriuresis during weightlessness. Clinical Investigator, 71:378-386, 1993. 103. Heer, M., Baisch, F., Drummer, C., Gerzer, R. Longterm elevations of dietary sodium produce parallel increascs in the renal excretion of urodilatin and sodium. In: Scientific Results of the

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German Spacelah Mission 0 - 2 (P.R. Sahm, M.H. Keller, B. Schiewe, Eds.), pp. 708-798, WPF, Aachen, 1994. 104. Stadeager, C., Johansen, L.B., Norsk, P., Warbedrg, I., Bie, P., Christensen, N.3. et al. Influence of microgravity on endocrine and renal responses in humans to an isotonic saline infusion. In: Scientific Results of the German Spacelah Mission 0 - 2 (P.R.Sahm, M.H. Keller, B. Schiewe, Eds.), pp. 736-737, WPF, Aachen, 1994. 105. Norsk P., Drummer, C., Roecker, L., Strollo, F., Christensen, N.J., Warherg, J. et al. Renal and endocrine responses in humans to isotonic saline infusion during microgravity. Journal uf Applied Physiology, 78:2253-2259, 1995. 106. Heer, M., Kamps, N., Biener, C., Drummer, C. Is malnutrition a possible cuase for body fluid losses in microgravity? In: 18th Annual International Gravitational Physiology Meeting (abstract),April 20-25, 1997, Copenhagen, Denmark, p. 44. 107. Huges-Fulford, M. Review of the biological effects of weightlessness on the human endocrine system. Receptor, 3:145-154, 1993. 108. Udden, M.M., Driscoll, T.B., Gibson, L.A., Patton, C.S., Pickett, M.H., Jones, J.B. et al. Blood volume and erythropoiesis in the rat during spaceflight. Aviation, Space, and Environmental Medicine, 66:557-561, 1995. 109. Gunga, H.-C., Kirsch, K., Baartz, F., Maillet, A. Erythropoietin under real and simulated microgravity conditions in humans. Journal of Applied Physiology, 81:761-764, 1996. I 10. Hochachka, P.W., Gunga, H.-C., Kirsch, K. Our ancestral physiological phenotype: An adaptation for hypoxia tolerance and for endurance performance? Proceedings of the National Academy qfSciences (USA),95:1915-1920, 1998. 11 1. Van Helder, T., Symon, J.D., Radomski, W. Effects of sleep deprivation and exercise on glucose tolerance. Aviation, Space, and Environmental Medicine, 64:487492, 1993. 112. Greteheck, R.J., Schoeller, D.A., Gibson, E.K., Lane, H.W. Energy expenditure during antiorthostatic bed rest (simulated microgravity). Journal of Applied Physiologjj, 78:2207-2211, 1995. 113. Popova, LA, Vetrova, E.G., Zaitseva, L.B., Larina, O.N., Markin, A.A., Fedotova, N.Iu. Metabolism in cosmonauts: The results of biochemical research on the blood of the crew members of the 7 prime expeditions on the MIR orbital space complex Aviakosmicheskaya Ekologiya i Meditsina, 26:35-39, 1992. 114. Lane H.W., Smith, S.M., Rice, B.L., Bourland, C.T. Nutrition in space: Lessons from the past applied to the future. American Journal of Clinical Nutrition, 60:SEOl-S805, 1994. 115. Maass, H., Raabe, W., Wegrnann, H.M. Effects of microgravity on glucose tolerance. In: Scientzfic Results of the German Spacelab Mission 0 - 2 (P.R. Sahm, M.H. Keller, B. Schiewe, Eds.), pp. 732-735, WPF, Aachen, 1994. 116. Stem, T.P., Schluter, M.D., Boden, G. Development of insulin resistance by astronauts during spaceflight. Aviation, Space, and Environmental Medicine, 65: 1091-1096, 1994. 117. Acheson, K.J., Decomhaz, J., Piguet-Welsch, C., Montognon, F., Decarli, B., Bartholdi, 1. et al. Energy, protein, and substrate metabolism in simulated microgravity. American Journal qfPhysiology, 269 (2, Pt.2):R252-R260, 1995. 118. Macho, L., Fickova, M., Svabova E., Zorad, S., Serova, L., Popova, I. Changes of insulin in plasma and receptor for insulin in various tissues after the exposure of rats to space flights and hypokinesia. Journal o j Gravitational Physiology, 1:P23-P24, 1994. 119. Alekseev, E.I., Krasnov, I.B. Morphological studies of endocrine cells of the pancreas in rats after exposure to micro- and hypergravity. Aviakusnzicheskaya Ekologiya i Meditsina, 2913742, 1995. 120. Fagette, S., Lo, M., Gharih, C., Gauquelin, G. Sympathetic nervous system activity and cardiovascular variability after a 3-day tail suspension in rats. European Journal of Applied Phvsiology, 69:480-487, 1994.

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121. Fagette, S., Somody, L., Koubhi, H., Fareh, J., Visa, M., Gharib, C. et al. Central and peripheral noradrenergic responses to 14 days of spaceflight (SLS-2) or hindlimb suspension in rats. Avia-

tion, Space, and Environmental Medicine, 67:458-462, 1996. 172. Fareh, J., Cottet, Emard, J.M., Pequignot, J.M., Jahns, G., Meylor, J., Visa, M. et al. Norepinephrine content in discrete brain areas and neurohypophysial vasopressin in rats after a 9-day spaceflight. Aviation, Space, and Environmental Medicine, 64507-5 I I , 1993. 123. Krasnov, I.B. Hypoadrenergic syndrome of weightlessness: Its manifestations in mammals and possible mechanism. The Physiologist, 34:S23-S26, 1991. 124. Goldstein, D.S., Vernikos, J., Holmes, C., Convertino, V.A. Catecholaminergic effects of prolonged head-down bed rest. Journal of Applied Physiology, 78: 1023-1029, 1995. 125. Zhang, L.-F., Qin-Wen, M., Jin, M., Zhi-Bin, Y. Plasticity of arterial vascnlature during simulated weightlessness and its possible role in the genesis of postflight orthostatic intolerance. Journul of Gravitational Physiology, 4:97-100, 1997. 126. Newberg, A.B., Changes in the central nervous system and their clinical correlates during longterm spaceflight. Aviation, Space, and Environmental Medicine, 65562-572, 1994. 127. Youmans, J.R., Smith A.H. Gravitational fields and ageing. The Physiologist, 34:s 19-S22, 1991. 128. Kiebzak, G.M. Age-related bone changes. Experimental Gerontology, 1991, 26:171-187. 129. Grigoriev, A.I. Health in space and on Earth. World Health Forum, 13: 144-150, 1992. 130. Strollo, F., Semprini, A,, Strollo, G., Mor& M., Bollanti, L., Ciarmatori, A. et al. Even short-term hGH treatment induces endocrine changes in the elderly. In: Growth Hormone II-Basic and Clinical Aspects (B.B. Bercu, R.F. Walker, Eds.), pp. 338-346, Springer-Verlag, New York, 1994. 131. Timiras, P.S. Disuse and aging: Same problem, different outcomes. Journal of Gravitational Physiology, 15-7, 1994. 132. Strollo, F., Riondino, G. Recent progress in the chronobiology of pituitary, adrenal and reproductive hormones as a step towards integral space physiology. ELGRA News, 16:76-78, 1993. 133. Bigard, A.X., Lienhard, F., Merino, D., Serrurier, B., Guezennec, C.Y. Effects of growth hormone on rat skeletal muscle after hindlimb suspension. European Journal of Applied Physiology, 693377343, 1994. 134. Jiang, B., Roy, R.R., Navarro, C., Edgerton, V.R. Absence of a growth hormone effect on rat soleus atrophy during a 4-day space flight. Journal ofApplied Physiology, 74527-53 I , 1993. 135. Stump, C.S., Balon, T.W., Tipton, C.M. Effects of insulin and exercise 011 rat hindlimb muscles after simulated microhravity. Journal qfApplied Physiology, 73:2044-2053, 1992. 136. Stump, C.S., Woodman, C.R., Fregosi, R.F., Tipton, C.M. Muscle glucose uptake in the rat after suspension with single hindlimb weight bearing. Journal of Applied Physiology, 742072-2078, 1993. 137. Linderrman, J.K., Crosselink, K.L, Booth, F.W., Mukku, V.R.. Grindeland, R.E. Resistance exercise and growth hormone as countermeasures for skeletal muscle atrophy in hindlimb suspended rats. American Journul of Physiology, 267(2, Pt.2): R365-R37I, 1994. 138. Grindeland, R.E., Roy, R.R., Edgerton, V.R., Grossman, E.J., Mukku, V.R., Jiang, B. et al. Interactive effects of growth hormone and exercise on muscle mass in suspended rats. American Journal o f Physiology, 267 (1,Pt.2):R316-R322, 1994. 139. Turner, R.T. Effects of short-term spaceflight and recombinant human growth hormone (rHGH) on bone growth in young rats. Aviation, Space, and Environmental Medicine, 66:763-769, 1995. 140. Scano, A., Strollo, F. Life Sciences Experiments in Space Bring Benejits on Earth, ESA BR-119 (M. Perry, Ed.) European Space Agency, Noordwjik, The Netherlands, 1996.

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Chapter 5

CROWING CROPS FOR SPACE EXPLORERS ON THE MOON, MARS, OR IN SPACE

Frank B. Salisbury I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . A. Choice of Plants. . . . . . . . . . B. Economics of CELSS . . 11. Design of a CELSS . . . . . . . . A. Component . . . . . . . . . . . . . . . . . . . . . . . B. Problems to Be Overcome with Any CELSS . . . . . . . . . . . . . . . . . . . . . . . 111. CELSS for Different Locations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Mars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... B. The Moon . . . ... .......................... C. A Microgravity CELSS in a Space S h p . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cultivation of Plants in Space. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . EarlierExperiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Experiments with S .................................... A.

Experience with Biosphere-2, Arizona . .

Advances in Space Biology and Medicine, Volume 7, pages 131-162. Copyright 0 1999 by JAI Press Inc. A11 rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

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B. Experience with Bios-3, Siberia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,150 C. BIO-Plex under Construction at Johnson Space Center, Houston . . . . . . . ,156 ,157 VI. Conclusions: Lessons Learned . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Acknowledgments. . . . . . . . . . . . . . . . . . . . 160 References and Notes . . . . . .

1.

INTRODUCTION

The human exploration of space has required solving many challenging problems: ( I ) designing and constructing vehicles with enough propelling force to break their ties with Earth's gravity; ( 2 ) protecting the astronauts from the radiation and vacuum of space and providing them with reasonably comfortable living quarters; (3) providing an atmosphere containing sufficient oxygen and as little as possible of any toxic gas (humans can tolerate carbon dioxide concentrations of about 1%-compared to 0.036% in the Earth atmosphere-but a slightly higher concentration adversely affects their physiological responses, producing minor to very serious symptoms); (4) providing drinking water (about 2 liters per day), free of toxins and pathogens at harmful levels; (5) providing sufficient nutrients and chemical-bond energy to keep the astronauts alive and functional; Schwartzkopf, in a review published in 1997 in this series, estimates that an average human requires 408 kg.y-' of food, 304 kg.y-' of oxygen, and 2926 kg.y-' of water including 676 kg.y-' for drinking and the rest for food preparation and body hygiene, a total of nearly four metric tons of consumables per year!' On short-term missions, sufficient oxygen, water, food, and waste storage have been carried to last throughout the mission. CO, accumulating in the cabin atmosphere is removed by physicochemical means, for which supplies are carried. The Russian Space Station Mir, which has been in orbit for over a decade, has been regularly resupplied from the ground with these items, and waste has mostly been returned to Earth. An alternative to resupply and waste return is to recycle waste on board, which to a certain extent can be accomplished with physicochemical techniques (e.g., much effort has been expended on studies of physicochemical water I ecycling). Nevertheless, it has long been recognized that an alternative approach might be a biological technique based on green plants. In theory, this would convert C 0 2 into oxygen, recycle water, and provide food. Around the turn of the century, long before space travel was achieved, Konstatin Edwardovich Tsiolkovsky (considered to be the father of Russian space science), already suggested the use of green plants in future space exploration. Plants, through photosynthesis, remove CO, from the atmosphere and release oxygen and water vapor that can be condensed in nearly pure form. The photosynthetic process is driven by light energy with a maximum efficiency of 10 to 13% (chemical-bond energy/light energy).3 With a suitable choice of plants, part of this fixed chemical-bond energy can be used as

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food by space explorers. The edible portion of the total biomass, including roots, can vary from less than 30% (e.g.. for soybeans) to 80% or more (e.g., lettuce, potatoes); a typical value is 45% for wheat.4 This percentage is referred to as the hancst index. An additional distinct advantage is that green plants provide a pleasant environment for earthlings who are adapted to their presence on Earth. Green plants can also aid in waste disposal, e.g., they can utilize urine. Such a system using green plants is properly referred to as a bioregenerutive life-support system, for which I shall use the acronym CELSS, which stands for Controlled (or Closed) Environment (or Ecological) Life-Support System. A.

Choice of Plants

Which plants r i g h t be used in a CELSS? When the CELSS concept was developing during the 196Os, there was much discussion and considerable research ~-~ about the possibility of using green algae, especially Chlorella ~ u l g a r i s .Green algae are certainly efficient at supplying oxygen and removing carbon dioxide. Chlorella, however, is not easy to eat" and has caused nutrient deficiencies and illness in both test animals and in humans." The strains that have been studied mostly contain large quantities of proteins and nucleic acids but no carbohydrates, which is not a very suitable composition for human nutrition. Chlorella has been successfully incorporated into various baked products, but so far nobody has been able to develop a satisfactory process to make it suitable for human consumption in significant quantities. In contrast to most green algae, however, Chlorella 241 80 is a strain that produces maltose, which can be used in human nutrition. The strain has been employed in a prototype photo-bioreactor that can regenerate the air and provide some food in space, as has been described in this series by Luzian Wolf? He also reviewed earlier work on algal bioreactors and describes the technical problems of making such systems function in microgravity. All components of this bioreactor have been designed to function in microgravity, and some have been tested in space experiments. Bioengineering might further refine the nutrient contents of various algae, including Chlorella. Some cyanobacteria (blue-green algae, e.g., Spirolina and Nostoc) can be consumed directly and have been proposed as an organism in bioreactors for space exploration. Although these organisms are edible, astronauts would soon tire of a diet that included large quantities of Spirolina or its relatives. Nevertheless, an important feature of the cyanobacteria is that they can fix atmospheric nitrogen into forms that higher plants can use,12 and fixed nitrogen is likely to be lost in a CELSS as a result of waste processing and the activities of certain microorganisms. In any case, when air purification is the only goal for a bioregenerative system, algal systems are certainly viable alternatives that have been tested in laboratories all over the world.

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Higher plants are more difficult to grow, but a great advantage i s the edible food they can produce, as well as the psychological effect of having these familiar l i f e forms on board. A disadvantage i s that the harvest index is seldom, i f ever, 100%. Thus, part of the higher plants must be consigned to the recycling or waste dis-

Food production with waste processing (CELSS)

I

, 0 ‘

fl

0

0

0

0

/

0

-.-, _ . F .

atmosphere

and water

Mission Duration (e.g., years)

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Figure 1. An illustration of principles involved in determining the mission duration at which recycling is economically beneficial (after Myers,’ cited also by Schwartzkopf.’) The lines, which show the mass required for life support (ignoring the mass of the launch vehicle) as a function of mission duration, are based on the initial launch mass plus the mass that must be resupplied as the mission continues. The curve labeled resupply assumes that all water, atmosphere, and food is either taken in the initial launch or resupplied at intervals; hence, the longer the mission, the greater the amount of food and other supplies that must be launched. Since much of the required mass consists of water, the slope of the line is much less when water is recycled, but obviously the equipment required for water recycling adds to the launch mass. Recycling the atmosphere requires even more launch mass, and a bioregenerative life support system (CELSS) requires much more mass. Yet in each case, with additional recycling, less resupply is needed,so the curves are not as steep. When closure approaches loo%, the slope of the curve approaches zero, but 100% closure is unlikely ever to be achieved. The arrows represent break-even points where the duration of the mission justifies recycling of increasing complexity.

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posal system. The first question that comes to mind is whether a functional and reliable CELSS can be built for longterm use in space. Considerable research has hien devoted to this question, especially at the Institute of Biomedical Problems in Moscow and at the Institute of Biophysics in Krasnoyarsk, Siberian Russia. Active efforts to construct a functional ground-based CELSS, are currently under way in Japan and at the Johnson Space Center; the latter project is called B10-Plex (see section V.C). Building a CELSS is not simple, but most of us who are familiar with past and current projects think that the challenge can be met.

5. Economics of CELSS If a CELSS can be built, then the second question appears; namely, what is the cost of a CELSS versus resupply from earth? CELSS equipment will be rather massive, costing much to launch into space. Furthermore, it will require large amounts of energy to operate. The expense of launch, maintenance, and energy must be considered against the cost of resupply. It is also extremely costly to launch, preserve, and deliver food, oxygen, and other necessary items from earth to space explorers. The actual expenditure will depend upon whether these explorers are in near-earth orbit, on a base on the Moon or Mars, or in a spacecraft on a mission to prospect for asteroid material. Figure 1 illustrates the concept as proposed by Jack Meyers in 1963.13The graph plots launch mass of food and/or materials to produce water, air, and food for each person as a function of the mission duration. Obviously, the goal is to minimize the launch mass. If everything, including water, air, and food, is brought up and resupplied, and waste products are simply stored or jettisoned, the mass begins at zero for a mission of zero duration and increases with mission duration according to a steep curve. Because humans require large amounts of water, if water can be recycled, the curve will be much less steep. It will not begin at zero because the launch mass will have to include the mass of the water-recycling equipment (whatever it happens to be). Regeneration of the atmosphere requires still more equipment, but doing so leads to a curve that is still flatter. If equipment for food production and waste processing (recycling, when possible) is added, the initial launch mass is very high but the curve becomes almost flat. It is not completely horizontal because some materials, so-called deadlock substances, cannot be recycled in any practical way. Some resupply will be needed. The break-even times are when the curves for recycling of water, air, or food cross the steep line for completely expendable supplies. The break-even points have been calculated for various scenarios. Steven Schwartzkopf, for example, has calculated break-even for a lunar colony with a CELSS.'*14If the colony supports four crew members, Schwartzkopf calculates break even at about 2.6 years; if there are 100 crew members, break-even will come as soon as 1.7 years. Of course, many more data are needed before such a calculation can be carried out with real confidence. Nevertheless, it seems almost

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obvious that a CELSS would soon pay for itself as part of a Mars station where resupply would be very expensive.

II. DESIGN OF A CELSS A.

Components

Considering the functions required of a CELSS, the equipment will consist of four essential components:15

1.

biomass production unit-where the plants are grown; this unit must include equipment to control the environment, particularly light, temperature, atmosphere (controlled to a great extent by the plants themselves), pressure, humidity, and cultivation medium for growing the plants. 2. food preparation area-most crops need processing before they can go to the kitchen. Furthermore, special food technologies will have to be developed to make use of as many normally inedible plant parts as possible, thereby increasing the harvest index. 3. waste recycling system-this would be aimed at recycling as much waste as possible. Inevitably, some materials, the so-called deadlock substances, cannot be recycled without incorporating far more equipment and supplies (e.g., strong acids for liquid oxidation), so there must be storage facilities for such substances. Extra supplies are needed to compensate for the deadlock substances that are eliminated from the system or else they will have to be resupplied during the mission. 4. computerized monitoring and control system-such a system also requires spare parts, (e.g., lamps, sufficient for the planned life time of the CELSS), stored or resupplied. The vast resources of Earth allow for long durations and inefficiencies in production and recycling. Natural processes suffice, even when they are quite inefficient. Earth has such a huge buffering capacity that human activities have only in recent times begun to have an impact on such global phenomena as the climate. If farmland produced well below its potential, the farmer only needed to increase the growing area, but cultivatable land areas are now beginning to be limited. Slow microbial action or even geological processes are permissible means of recycling on Earth." However, since a CELSS facility is costly to build and to launch into space, it is essential to achieve optimal productivity and efficiency from the start. This will require the use of appropriately programmed computers. In a CELSS facility with its small buffering capacity, the processes are in a much more rapid state of flux than is the case with Earth's biosphere. For example, it may take eight years for the CO, in the Earth's atmosphere to be completely recycled through

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living organisms, but in a CELSS facility all CO, must be recycled in just a few days or less. The problems encountered in designing a functional CELSS facility strongly depend on where the CELSS is to be located. In the foreseeable future, three situations are apparent: the surface of Mars, the surface of the Moon, and a microgravity spacecraft in orbit or in free-fall transit to Mars, some other planet, or the asteroids. Some problems, however, are a function of the space environment and are, with very minor exceptions, common to all three locations once the Earth’s protective atmospheric mantle has been breeched. l 6 B.

Problems to Be Overcome with Any CELSS

Any CELSS facility must provide sufficient oxygen for the astronauts and sufficient carbon dioxide for the plants. In some cases, it will be necessary to supply all of the required CO, from Earth. The plants will also need water and minerals, including fixed forms of nitrogen. Nitrogen gas will probably make up about 80% of the CELSS atmosphere as it does in the Earth’s atmosphere. Since fixed nitrogen used by the plants may be converted to gaseous nitrogen, some means of nitrogen fixation will be required or enough fixed nitrogen for the entire voyage must be taken along. Cyanobacteria might serve this function or physicochemical methods might be used. Leaks from the space craft or planetary habitat to the vacuum of space or the low atmospheric pressure on Mars will be a serious hazard, as was demonstrated when the supply vehicle caused a small leak in the Spektr module of Mir on June 25, 1997. Enough food will have to be carried as backup in case of unexpected difficulties in growing the plants. Difficult decisions will have to be made about what to take and what to resupply. It will be necessary to take spare parts for everything that could wear out. Lights eventually bum out, lubricants evaporate or wear out, motors burn out. Virtually everything in the mechanical environment of a CELSS facility is subject to wear and tear and eventually complete failure. This is certainly a serious limitation, one that has now been encountered in the Mir after so many years in orbit. It is important to note that the plants are much more dependable than the machinery that creates and maintains a suitable environment for their growth. Energy to run a CELSS facility is a serious problem. It is especially serious if energy is required to irradiate the plants with visible light of suitable wavelengths. A logical energy source would be a nuclear power plant on Mars or the Moon or possibly even in a spacecraft. Solar cells have provided much of the energy for space exploration so far, but very large arrays would be required to produce enough power to irradiate the plants even with the most efficient lamps. The best solution would be to use direct sunlight for the plants, which would be possible on Mars. Formidable engineering problems would have to be overcome, however, to make this possible on the Moon or in a spacecraft.

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Many authors have suggested that, in the absence of a protecting atmosphere, hard cosmic radiation and occasional solar flares could be damaging or fatal to both plants and humans virtually anywhere in space. This point is somewhat controversial, however. Schwartzkopf, for example, defends an inflatable greenhouse on the surface of the Moon by saying that "dangers posed to plants from galactic cosmic radiation. solar flares, and meteorite strikes are statistically very low, even over an assumed 20-year lifetime of the facility".', l7

111.

CELSS FOR DIFFERENT LOCATIONS A.

Mars

Once we get there, Mars is the easiest location to build an extraterrestrial CELSS facility. Although the atmosphere is only about 0.1 % that of Earth, it is enough to make parachute landings possible and to provide slight protection from radiation. The atmosphere consists mostly of carbon dioxide, which is a big advantage because it would not be necessary to transport C 0 2 for the plants. There is actually 25 to 50 times as much CO, in the Martian atmosphere as in the atmosphere of Earth, depending on elevation on the Martian surface. Water is available in the form of ice, but it will be difficult to obtain. Nevertheless, this is another great advantage compared to a microgravity CELSS facility or probably one on the Moon, where water will be more difficult to obtain. The Martian regolith could perhaps be used as a plant substrate that is watered with a solution of minerals essential for plants, but it has still to be determined whether it is nontoxic. Silicate rocks can be used as a source of oxygen as well as mineral nutrients for the plants." The gravitational force at the surface of Mars is about one-third that of Earth, which is sufficient to facilitate convective cooling and circulation of nutrient solutions to plants. The fact that the Martian day has a length of 24.7 hours, which is close to a day on Earth, is a great advantage for plants that flower and that initiate and stop their developmental processes in response to the relative length of day and night (phot~period).~, 19, 2o Because Mars has a highly elliptical orbit around the sun, irradiance at its surface will vary from 37 to 52% of the light that reaches Earth's upper atmosphere. This is sufficient for excellent growth of plants, and is probably much more than many crops receive in cloudy regions on Earth. Indeed, it is seldom cloudy on Mars, although occasionally dust storms seem to obliterate all of the surface features on the planet as viewed from Earth. These storms might significantly decrease the light reaching the surface. Mars has seasons because it's equator is inclined 25" to the ecliptic, as compared with 23.5" for Earth and only 1.5" for the Moon. The Martian year is equal to 687 Earth days. However, Mars also presents some serious problems. The atmosphere is so thin that leaks are essentially as dangerous as on the Moon or in space. Explorers on

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Mars would need space suits when they move out of their artificial habitats. Mars also experiences extremely low temperatures. It appears that the surface (regolith) temperature at noon near the equator and in Martian summer may reach 20 "C, but at night, even in summer, heat radiates into space so much that the temperature typically drops to at least -75 "C. There is water on Mars, but as noted above, it will be difficult to obtain. Nitrogen could also be a problem since there is no evidence that the Mars atmosphere contains any nitrogen. Certainly the greatest problem with a Martian CELSS facility is to get there and to resupply it with whatever cannot be recycled or brought on the initial voyage.

B. TheMoon The Moon has a gravitational force of 0.165 G, which is about 1/6 of that encountered at the surface of the Earth, a sufficient amount to facilitate the circulation of nutrient solutions to plants and convective cooling. It should be possible to obtain oxygen and perhaps also hydrogen (although the latter remains to be determined) from silicate rocks. If both hydrogen and oxygen can be obtained, water could be produced and perhaps the energy of combustion of hydrogen and oxygen could be used in the system. The lunar regolith could also provide mineral nutrients. If lunar regolith is nontoxic and inert, it might be used as a solid substrate that is watered with a solution of minerals essential for plants, although hydroponic growth of plants is relatively simple and is probably more reliable. Several possibilities have been considered for energy production on the Moon. Solar cells could relatively easily be deployed on the lunar surface. However, such cells are expensive, relatively inefficient, and quite massive to transport to the Moon. Furthermore, it is dark for half of the lunar day, which is about 29.5 Earth days long. Another possibility would be to install a conventional nuclear fission reactor on the Moon. Finally, nuclear fusion, if ever developed to a practical level on Earth, might be an energy source, since it has been speculated that tritium exists on the lunar surface, adsorbed onto regolith particles. l 8 This tritium could serve as the energy source for a fusion reactor. This might also be a reason to go to the Moon in the first place-namely, to collect tritium for use in fusion reactors on Earth. In the absence of any atmosphere, the problem of leaks is very serious on the Moon. Furthermore, the Moon has no known source of carbon, which thus would have to be brought from Earth in order to be burned (with oxygen produced from silicates) to produce CO, for the plants. Solar flares are relatively common and, without a protective atmosphere, potentially lethal at the lunar surface. It has been recommended that any living being on the Moon should be protected by at least three meters of lunar regolith. If this is true, the lunar CELSS will have to be underground (but note the comment of Schwartzkopf' l 7 mentioned above). This means that no direct sunlight would be available for plants, although during the lunar day light could be 'piped' in through fibre optics or a similar system. The 9

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size of the collectors, however, would probably have to be twice as large as the area illuminated because of the inefficiency of moving light this way. The long lunar night would, however, remain a problem. Because the Moon turns once on its axis for each revolution around the Earth, the same side of the Moon is always facing the Earth. This means that if the lunar colony with its CELSS facility is located in the Sea of Serenity on the Moon, for example, the Earth will appear about 60” above the horizon, just west of south, and it will always be in that position. Because there is no atmosphere, the sky is always dark and the stars will be visible to the dark-adapted eye. Although the Apollo astronauts, who visited the Moon during lunar day, could not see them, the stars in the black sky along with the Earth should be visible for anyone looking out of a porthole in a dark or poorly illuminated room, even if the room would be beneath three meters of regolith. S ~ h w a r t z k o p fhas l ~ ~presented ~ details of a proposed lunar CELSS facility built on the surface of the Moon. Earth-watching from the Moon would certainly be a wonderful pastime. The diameter of the Earth as seen from the lunar surface would appear 3.7 times that of the Moon as seen from the Earth. The Sun, however, will appear essentially the same size as it does from Earth, and is almost exactly the same size as the Moon appears from the Earth, varying slightly with the Moon’s distance from Earth. The Earth will go through a similar series of phases as the Moon appears to do from Earth. When the Sun is nearly behind the Earth, only a small crescent of Earth will be illuminated (i.e., the “new Earth”). Once or twice a year, the Earth will come between the Sun and the Moon causing a solar eclipse. The Earth atmosphere will then refract red light all around the Earth-a circular, celestial sunset! When the Moon is located between the Sun and Earth, the Earth will be fully illuminated a (i.e., “full Earth”). This will be at lunar midnight, when the Moon is seen from the Earth as a “new Moon”. When the Moon is directly between the Sun and Earth, the Moon’s shadow with its umbra (the dark center) and penumbra (the lighter, graded, surrounding shadow) will be visible as it moves across the Earth’s surface, a phenomenon that has already been photographed from satellites. Seen from the Earth, this is called a solar eclipse, but seen from the Moon it is an eclipse of the Earth, even though only a small portion of the Earth will be darkened. Such an eclipse will occur once or twice a year. C.

A Microgravity CELSS in a Space Ship

Building a CELSS facility to orbit the Earth that would thus operate in a microgravity environment will surely present two challenges. Firstly, engineering the movement of liquids and gases in microgravity will be a technical challenge. Water or nutrient solutions will not drain through a porous substrate as they do on Earth, nor will heated gases rise. Liquids must be pumped and gases must be moved by fans or other means. Toilets in microgravity have had serious problems in the past; they only work when properly engineered with rapid air movement.

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The second problem is how microgravity might affect the crop plants that are to be used in a CELSS facility. Plants on Earth are highly sensitive to g r a ~ i t y .21-23 ~. When a young bean seedling is placed on its side, the tip of its stem will within hours have bent so that it will be growing upright. Even if such a stem is turned only a few degrees from the vertical, it will soon bend just enough to be vertical again. Even when a tree is tipped to its side, it will bend as compression wood forms on the bottom of the trunk and or tension wood forms on the top of the trunk. Gradually, the upper part of the tree will approach the vertical. Thus, it would certainly not be surprising if plants could not complete a life cycle in microgravity. Even if they appear to grow normally at some time in their life cycle, at some stage of their development an accelerating force would be required. So this stage cannot be completed in the absence of such an accelerating force, which on Earth is provided by gravity. Could this prevent the use of plants in a microgravity CELSS facility?

IV.

CULTIVATION OF PLANTS IN SPACE A.

Earlier Experiments

To determine whether plants can be grown for a full life cycle in microgravity, a few dozen experiments about plant growth have been carried out in orbiting space vehicle^.^^-^^ In some cases, the plants seemed to grow quite normally for the short duration of a shuttle flight,28but in other cases, even such short-duration flights led to serious a b n ~ r m a l i t i e s . ~Russian ~ . ~ ~ scientists have carried out several longterm plant-growth experiments in space, and in most of those instances, plant growth was quite abnormal. The paramount achievement was the growth of Arabidopsis thaliana through a complete life cycle, from seed to seed.27,32This experiment was carried out in the Phyton-3 device in Salyut-7 in 1982.The plants were grown under continuous light for 69 days from sowing until return to Earth. Five plants produced 22 fertile seed pods, while two plants produced 11 sterile pods. There were about 200 seeds, half of which were immature. Only 42% germinated to produce normal plants. Compared with plants grown in the same devices on Earth, growth in space was retarded and generally very poor; plants aged prematurely. This may have been due to the accumulation of ethylene in the cabin atmosphere (see discussion in section IV. C). It is premature to conclude that microgravity per se causes poor growth of plants in space. These findings clearly necessitate further and more extensive experiments in which all other factors have been optimized. This is discussed in section IV. B.

B.

Experiments with Super-Dwarf Wheat in Mir

In 1990, Russian and Bulgarian scientists sent a plant growth chamber called Svet (Russian for “light”) to the Russian Space Station Mir. The first plant exper-

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iment in Svet produced wilted plants that probably suffered from lack of water in the ~ u b s t r a t e . ” In ~ ~1992, ~ I was asked to form a United States team35 to work with the Russian and Bulgarian scientists, and this combined team has recently completed three experiments with a cultivar of super-dwarf wheat (Triticum aestivum L.) in Svet on Mir.36337Wheat is a good candidate for growth in a CELSS facility for the following four reasons: (1) it constitutes a significant portion of the diet of many peoples, (2) it can easily be stored, (3) it can simply be converted into edible and nutritious food by soaking or grinding and boiling to make a mush, or even by making bread, and (4) its yield in controlled environments can be surprisingly high. Bugbee and Salisbury4 were able to produce 60g m-2d-1,five times the world record yield in the field, thanks to continuous irradiation at the level of noan, summer sunlight (2000 ymol.m-2K1 PPF, which is photosynthetic photon flux in moles of photons between 400 and 700 nm in micromoles per square meter second), optimum temperature (23 “C) and humidity, enriched C 0 2 (1200 pmol.mol-’), and a nearly ideal hydroponic nutrient solution. At that rate, only about 15 m2 plant growth area would be required to provide adequate nutrition for a single crew member if that crew member were willing to eat nothing but wheat! With addition of other crops plus a safety factor, 50 m2 should suffice.15 Although super-dwarf wheat gives poor yields and would never be used in a CELSS facility, it is short enough (about 30 cm tall) to fit in Svet and otherwise provides a good model for higher-yielding wheat cultivars. Svet has about 0.1 m2 of growing area. The so-called root module has two compartments filled with a nutrient-enriched zeolite called balkanine. It is covered tightly with a metal lid that is perforated with small holes and with four open channels where plants can be grown. Our wheat seeds were attached to plastic strips in proper orientation, so shoots would go “up” into the air and roots “down” into the balkanine. The seed strips were placed between two layers of wick material that absorbed water from a so-called hydroaccumulator in the center of the balkanine in each compartment. Water diffused from the wicks into the balkanine. Originally, there were six pairs of fluorescent lamps, a system manufactured in the Soviet Union before 1990. The irradiation from the lamps was just high enough to permit growth of plants (-120 p n ~ l . m - ~ . sPPF), - ’ but not high enough for optimal growth. The lamps were cooled by a fan that pulled cabin air into Svet, past the plants, and over the lamps. When our Utah State University team began to cooperate with our colleagues in Moscow, it was agreed that our team would build additional equipment to attach to Svet. This equipment included four infrared gas analyzers for monitoring CO, and water vapor in air entering and leaving two plastic cuvettes, one attached above each compartment of the root module. This allowed measurements of photosynthesis, respiration, and transpiration of the plants. Sixteen moisture probes operating on a thermal principle for controlling moisture levels in the balkanine were provided and together with two probes already part of the original root module made a total of I8 probes. Two infrared sensors detected temperatures of the

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Figure 2. Schematic drawing of Svet as it is installed on the wall of the Krystal module of Mir. The hatch, to which the U.S. Shuttle attaches, is just to the right on the drawing. When the hatch door is open, it misses the Svet equipment by only 1 cm. The root module consists of two compartments, and two plastic cuvettes cover the plants above each compartment, although this is not illustrated in the drawing. There are two gas-exchange systems, one for each of the cuvettes. The cuvettes were used only in the second planting of the 1996197 experiment.

growing surface or plant canopy, two thermistors measured air temperature, and oxygen level and cabin pressure were also monitored. The instrumentation was controlled with a notebook computer. Figure 2 shows Svet mounted on a wall in

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Mir with the Utah equipment attached (sent to Mir with the Spektr module in June 1995). Our experimental plan was to grow the plants from seed to seed by allowing at least 90 days for the plants to mature and produce seed after planting. Plants were to be sampled into chemical fixative at five stages during their growth, and photographs and video recordings were to be made to document the course of the experiments. After the mature plants were harvested, a second crop was to be planted, partially to see if the balkanine would continue to supply nutrients. With proper timing, the second crop would be harvested after 30 to 40 days, coinciding with the arrival of the U S . Shuttle with a liquid nitrogen freezer. The frozen plants were to be tested on Earth for several plant hormones and other metabolites as well as mineral nutrients. In 1995, super-dwarf wheat was grown in Mir for 90 days (August 12 to November 9), but a series of equipment failures led to extremely poor growth and almost totally vegetative plants.37 Four of the six lamp pairs failed early in the experiment, leading to light levels (-80 pmol.m-2.s-') only slightly more than the photon flux needed to reach the compensation point at which photosynthetic CO, uptake just equals CO, loss in respiration. Although the plants remained alive for most of the 90 days, they were spindly and somewhat disoriented in the microgravity environment. However, much was learned about the equipment and about the difficulty of maintaining a suitable moisture level in the balkanine. Concerning the latter point, particle size proved to be extremely important. If the particles are too large, water will not move by capillary force; if they are too small, roots suffer from oxygen deficiency. This knowledge, which is critical for any microgravity CELSS to be built in future, was applied in our next experiment. In 1996, we were able to repeat the experiment with new equipment including a new lamp bank that utilized more modem fluorescent lamps (very low mercury content) that produced about 400 p m ~ l . m - ~ . sPPF, - ' about one fifth of the photonflux of full sunlight. The equipment functioned well (faulty equipment had been replaced), and plants were grown for 123 days from seed planting until final harvest (August 5 to December 6). Plant samples were taken at five stages and chemically fixed, and photographs and video recordings were made several times during the experiment. Unfortunately, new plastic cuvettes arrived too late to be installed (plants were already too large), so the gas-exchange measurements could not be carried out. On the day of harvest the second planting was completed, and these plants grew for 41 days until the arrival of the Shuttle Atlantis at Mir on January 14, 1997. During this period, the cuvettes were installed and successful measurements of photosynthesis, respiration, and transpiration were completed-the first time such measurements had been achieved in a space experiment with plants. These young plants, which were just forming heads, were harvested and frozen in the liquid nitrogen freezer. The samples and other returned materials are being analyzed, and the results will be reported elsewhere. The most encouraging observation was that the plants

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Figure 3. Two photographs, made from video recordings transmitted to Earth by photographing the screen, of Super-Dwarf wheat plants growing in Mir. The top image shows the dense canopy 34 days after planting, and the bottom image shows two large and healthy appearing wheat heads 86 days after planting.

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grew extremely well (Figure 3); the high irradiance and carefully controlled substrate-moisture levels produced much more biomass than had been produced in any other plant experiment in space. About 280 heads appeared on the plants growing in the 0.1 m2 area. The environment was monitored carefully throughout the experiment, including PPF, air and leaf temperatures, CO, and O2 concentrations, humidity, and moisture in the balkanine. Substrate moisture was measured at 18 locations at two different depths and two distances from the wicks. Thus we were able to show that healthy plants can be grown in microgravity at essentially the same rate as on Earth. This is encouraging for the construction of a microgravity CELSS facility. The frozen plants arrived back on Earth in excellent condition. They have been analyzed for several plant hormones and other factors, a measure that is providing insight into the stress levels encountered by plants growing in space. A surprising and, at the time, disappointing aspect of the experiment was that the harvested and sampled heads were all sterile. Not a single seed could be found in any of the heads! Ground studies (not yet published) have been and are being carried out by William Campbell at Utah State University, David Bubenheim at NASA Ames Research Center, and our Russian colleagues at the Institute of Biomedical Problems and the University of Moscow. Studies of the dry heads brought back from Mir invariably indicate the same three phenomena: development of the flowers stopped just at the stage where the stamen filaments began to elongate, pollen was not released, and the pollen found in the anthers often had only one or two nuclei instead of the normal three. Some anthers contained no pollen at all. It is clear that seeds failed to form because pollination did not occur, that is, the plants suffered from male sterility. The fact that this was the case for all heads, although they developed at different times during about two months, suggests that the arrested development did not occur in response to a single, temporary stress such as an interval of high temperature. Rather, development was stopped by some prevailing factor that was always present in Mir.

C . Discussion Is microgravity the direct cause of poor plant growth in previous experiments and the lack of pollination in the Mir studies? Not necessarily, because plant experiments in space have always been plagued with several conditions that are stressful for plants even on Earth: 1. Photon flux has been low in nearly all experiments, on the order of 150 pmol.m-2.s-1PPF. Although healthy plants have been grown at this low level on Earth in equipment closely similar to that used in space, this is a lower limit for acceptable plant growth and has surely contributed to poor growth in previous experiments.

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2. Carbon dioxide has always been high in previous experiments, and this factor has probably not always been duplicated for the ground controls. Optimum CO, levels for many plants are around 1200 pmol.mol-'. A cabin atmosphere of 5000 pmol.mol-'(O.S%) is common in spacecraft because maintaining the CO, below this level requires too frequent a change of the lithium hydroxide in the CO, scrubbers and this relatively high COz level is well tolerated by humans. 3. Temperature in spacecraft fluctuates much more than in ground situations, sometimes reaching levels as high as 37 "C, which can be harmful to plants. 4. Root environment is probably the most important factor. Poor drainage may lead to water logging around the roots, and attempts to compensate for this may overcompensate and lead to lack of water, as apparently happened in the first plant experiment in Mir in 1990.33,34Clearly we cannot draw conclusions about plant responses to microgravity when so many other stress factors may be present. However, we have reason to believe that an entirely different factor was responsible for the arrested development and sterility of the super-dwarf wheat. Samples of cabin atmosphere returned from Mir showed that ethylene was present in levels of 300 to 1200 pmol.mol-' air. Ethylene is a gaseous plant hormone that is responsible for many plant r e ~ p o n s e sIt. ~is produced by ripening fruit, for example, and through positive feedback is responsible for continued ripening. Low concentrations of ethylene can induce male sterility in wheat and other cereal^.^*-^' Most studies with cereals were carried out with the soluble compound ethaphon (Ethrel), which releases ethylene after application, so it is difficult to relate the concentration of applied ethaphon to the concentration of ethylene in the atmosphere. Nevertheless, in one study with oats (Avena sativa), only 150 nmol.mol-' of gaseous ethylene produced almost total ~terility.~' My colleagues at Utah State University (William Campbell and Bruce Bugbee) and at NASA Ames Research Center (David Bubenheim) have performed studies in which super-dwarf wheat was grown in atmospheres that contained various levels of ethylene.37 Their results show that the heads that formed were not only sterile but exhibited the exact symptoms of the Mir wheat: inhibited filament elongation, failure to release pollen, and defective pollen within the anthers. Other symptoms noted in the Mir wheat, namely, shortened internodes and excessive tillering (branching at the ground level), also appeared in wheat grown in an ethylene-containing atmosphere. The conclusion of these studies is that ethylene was almost certainly responsible for the sterility of the Mir wheat. Thus, in future experiments with plants in space, as well as in any future CELSS facility on the Moon, Mars, or in microgravity, it will be critical to monitor ethylene levels and to scrub it from the atmosphere used. The ethylene in Mir could have been generated in several ways, particularly by fungi that are known to grow in damp places in the relatively large Russian space

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Figure 4. Aerial photograph of Biosphere-2, located in the desert of Arizona, U.S.A. The photograph was kindly supplied by John Allen.

station!3 Cooled walls cau3e condensation, and fungi take up residence. It is virtually impossible to eliminate them. Indeed fungi were growing at the base of the plants in our experiment, apparently without any direct harm to the vegetative growth of the plants. It should be noted that the atmosphere in Mir has not been completely changed in the 1 1 years of its operation, and the activated charcoal in the air purification system does not remove ethylene. We feel confident that viable seeds of wheat as well as Arubidopsis can be produced in microgravity if the atmosphere and all other factors are properly controlled.

V. A.

G R O U N D EXPERIMENTS

Experience with Biosphere-2, Arizona

The $1 50-million Biosphere 2 facility in Oracle, Arizona, has often been likened to a future CELSS on the Moon or Mars. It is a large greenhouse (Figure 4) covering 1.2 hectares of Arizona d e ~ e r t . Seven ~ ~ . ~ so-called ~ biomes (ocean, fresh and salt marshes, tropical rain forest, savanna, desert, intensive agriculture, and human habitat) stocked with 3800 species of plants and animals attempt to duplicate similar biomes on Earth. In the first Biosphere-2 project, four men and

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four women were sealed i n the structure for two years from September 26, 1991 to September 26, 1993. Although the designers and operators were willing to apply extensive and necessary technological intervention like the provision of large amounts of energy in the form of electricity and natural gas in order to achieve the desired level of environmental control and stability, a primary goal was to see if such a complex assemblage of organisms might arrive at a natural balance of some kind. Ecologists were of a divided opinion: some suggested that complexity might inevitably lead to stability of an ecosystem since, if one component fails, others will take over its function; others suggested the opposite, namely, that complex systems have more points of vulnerability. Certainly, once a complex ecosystem (e.g., a rainforest) has been destroyed, it is very difficult to restore it. The Biosphere-2 project provided an opportunity to test these ideas in a controlled environment. In the publicity about Biosphere-2, it was often stated that it was a prototype for future structures on the Moon or Mars. Clearly, such a relatively flimsy, pressurized structure could not exist on the airless or nearly airless surfaces of the Moon or Mars, nor could a stronger structure of such complexity, including so many species, be built on Moon or Mars even in the distant future. The actual design of Biosphere-2 suggests that it was aimed at a better understanding of the biomes on Earth, and it is now being used for that purpose. Hence, many if not most CELSS scientists tended to ignore the project. Nevertheless, a number of the results of the two-year trial and of subsequent studies should be of interest to scientists who concern themselves with the design and operation of CELSS facilities. Indeed, Biosphere 2 is a bioregenerative life-support system, a controlled environment life-support facility; as such, it qualifies as a CELSS facility. John Allen, the prime mover in the Biosphere-2 project, has published a review that describes the results in much As expected, there was an inverse relationship between light and CO, removal: the more light, the more CO, was removed by photosynthesis. During the summer of 1992, CO, levels were between 800 and 2000 pmol.mol.', ideal for photosynthesis. Since both winters in the two-year trial were unusually cloudy and the superstructure of the facility blocked about half of the light, decay and respiration of organisms led to much higher CO, levels during the winters, reaching 4500 CO, pmol.mol-' during January, 1993. When levels exceeded 5000 pmol~mol~', was removed by a scrubber in order to protect the pH of the aqeous biomes. The high CO, level was accompanied by an unexpected, gradual drop in oxygen level, which reached 14.2% before fresh oxygen was added to the atmosphere, because the human inhabitants experienced difficulty in working. Clearly, much more oxygen was leaving the system than could be accounted for by the increase in CO,; the drop in oxygen was about 6% of the total atmosphere, while C 0 2 increased by less than 1%. The discrepancy between 0, loss and CO, gain was a mystery for some time. As it turned out, 0, was being used in respiration and decay of the large amount of organic matter taken in before the structure was

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sealed, but much of the C 0 2 that was produced by this respiration combined with the unsealed structural concrete. Because there was opportunity for biological waste management, Biosphere-2 was able to recycle all human and animal wastes for the first time in a closed system with human inhabitants. Fresh and salt water were also recycled. The inhabitants were able to produce 8 1 % of their diet in a sustainable agriculture system is an area comprising 0.2 hectare. Allen notes that 100% of caloric requirement could have been supplied with more light (10 mol.m-2.d-'). This capability was demonstrated when artificial lights were installed above the agriculture system after completion of the two-year Biosphere-2 e ~ p e r i m e n tNitrous .~~ oxide (N20) increased continuously during the experiment. This gas would probably have to be removed by physicochemical means in a functioning CELSS facility. Species diversity remained high during the two-year trial as more species survived than had been expected. The unbalanced C 0 2 and 0, levels and the increase in N 2 0 demonstrated that enclosing even a large volume with thousands of species is not necessarily sufficient for providing a balanced turnover of matter. This will therefore be even more difficult to achieve in a necessarily much smaller CELSS facility. Much of the computer and other regulatory technology at our disposal will be required to reach this goal. However, if we are willing to settle for only partial closure with resupply, the challenge becomes much simpler. We would almost certainly have to settle for an incomplete closure because of the impossibility of recycling true deadlock substances. However, it is estimated that it might be possible to achieve closures as high as 95%. B.

Experience with BIOS-3, Siberia

When the Russian space program began to develop around 1960, scientists in the Institute of Physics in Krasnoyarsk, central Siberia, became intrigued with the CELSS concept. Details are summarized in an article by Salisbury, Gitelson, and L i ~ o v s k y In . ~ 1965, ~ the Siberian scientists constructed a system, called Bios-1 , that could regenerate the atmosphere and produce sufficient oxygen to support one human. A sealed 12-m3 chamber was connected through air ducts with an 1 %liter algal cultivator containing Chlorella vulgaris. About 8 m2 of the algal culture were irradiated with three, 6-kW xenon lamps. In 1968, the structure was attached to a 2.5 x 2.0 x 1.7 m chamber for higher plants that the builders called a phytotron. (This term was first applied in the late 1940s by James Bonner and Samuel Wildman to the Earhart Plant Research Laboratory at the California Institute of Technology in Pasadena, in jest, to indicate that botanists could have something as imposing as the cyclotron being constructed then at the University of California, Berkeley. The term was soon being applied to controlled-environment facilities all over the world).

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In a later version of the Siberian facility called Bios-2, oxygen was regenerated not only by the algal culture but also by food plants, which additionnally provided a small amount of food for the individual living inside. The test subject could go through a sealable hatch from the chamber into the phytotron to tend the plants and to harvest the crop. The algae still provided about 75 % of the air purification. A third version, Bios-3, constructed in 1972, has since then been used almost continuously and in various ways, including three full-scale experiments with crew members sealed inside. The total time of closure for the three Bios facilities now exceeds two years. Bios-3 is completely underground, reached by a passageway from the main building of the institute.47 It is constructed of welded plates of stainless steel to provide a hermetical seal. The structure is divided into four compartments of nearly equal size (ca. 7 x 4.5 x 2.5 m). Each compartment has three doors that are sealed tightly with rubber gaskets, and one of these doors leads to the outside. Occupants of the system can escape through the outside door within 20 s in case of an emergency, but such an escape has never occurred. Each compartment can

Figure 5.

Interior of one of the three phytotrons of Bios-3. Two xenon lamps were installed in each water jacket. To make the lamps visible, the upper part of the black-and-white print (from a color slide) was given three times the exposure time of the bottom part; that is, the lamps are much bri hter relative to the plants than they appear in this print (from Salisbury et al).45

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be sealed independently in combination with any other compartment. There are large round windows in some doors and other large portholes in the living compartments. The crew area occupies one compartment. It is subdivided into three individual sleeping rooms, a kitchen, a lavatory, a control room, and equipment for processing wheat and inedible biomass, for making repairs and measurements, and also for purifying water and air. During the early years of Bios-3, one compartment included algal cultivators that provided enough air revitalization to support at least three crew members, although the remaining two compartments did not provide enough space to grow the vegetable requirements of three humans. Eventually, the algal cultivators were removed, and a common configuration was to grow wheat, chufa nut sedge, and a set of vegetable crops in each of the three compartments (Figure 5). The total growing area equals 63 m2, which provides ample air regeneration capacity. Each phytotron initially had 20 vertical, 6-kW xenon lamps, each surrounded by a vertical glass cylinder through which water circulated for cooling. The cylinders are inserted through a hole cut in the ceiling, allowing the lamps to be changed from the outside. By 1991, the number of lamps had been doubled in one phytotron by inserting two lamps into each water jacket. Light levels varied from 900 to 1600 p m ~ l . m - ~ . sunder -' single lamps (depending upon voltage applied) and from 1,300to 2,450 ymol.m-2.s-' under double lamps. The high irradiancesthe highest being well above summer sunlight--come at the expense of a relatively high air temperature (ca. 27 "C to 30 "C), which is too high for many crops including wheat. This required expanding the cooling system and providing additional energy above that needed for the lamps. In the experiments, so far, the lamps were operated continuously without a dark period, which excludes crops like tomato and potato that require a dark period. Maintaining inside pressures slightly above outside pressures to exclude entrance of pathogens causes calculated maximum leak rates of only 0.20-0.26 vol.% per day. Air is circulated to the crew quarters from the phytotrons and back. It is partially purified by the plants, but a thermocatalytic filter operating at 600-650 "C oxidizes all organic molecules to C 0 2 and H20 and completely eliminates ethylene from the atmosphere. Transpired water is condensed and reused for nutrient solution for the plants, for washing linen and dishes, and for general cleaning. Drinking water is additionally purified by ion exchange filters with the addition of small quantities of potassium iodide and fluoride for health and potassium chloride and some other salts to improve the taste. Samples are passed through small air locks to the outside for analysis, and the health of crew members is continually monitored, sometimes by attachment of various sensors to their bodies. Wires from these sensors are passed through the walls in sockets designed for the purpose. Occupants have privacy during their free time but are still monitored for medical parameters. There was no health deterioration after six months, although the microflora of skin, mucous membranes, and intestines changed significantly but without pathological consequences.

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Table 1.

153

Bios-3 Crops during the Third Experiment

Crop

Expected Crew Needs

(d4 I . Wheat. grain (dry mass) 2. Chufa, tubers (dry mass) 3. Pea, grain 4. Carrot, edible roots (fresh mass) 5. Radish, edible roots (fresh mass) 6. Beets, edible roots and leaves (fresh mass) 7. Kohlrabi, vteins and leaves (fresh mass) 8. Onion, leaves, bulbs (fresh mass) 9. Dill, greens (fresh mass) 10. Tomatoes (fresh mass) 11. Cucumbers (fresh mass)

12. Potatoes (fresh mass) Notes:

520 234 52 220 I10 130 180 120 30 150 100 250

Actual

Yield

Area

Area

(dm’di

(m21 40.0 9.0 4.0 1.4 0.9 0.9 1.1 0.7

fm’1

13 26 13 160 125 170 170 170 30 110

250 80

39.6 8.6 4.0 1.2 0.9 0.9 1.0 0.6

C

C

1.4 0.4 3.2

1.2 0.4 4.8

Harvest Harvest Indexh (dd) (%o) 496 34.7 120 48.1 26 25.4 236 54.9 266 59.8 132 67.5 164 37.1 110 90.1 16 93.0 88 33.1 276 54.6 22 5.9

‘Scientific names of crops are as follow^: 1 . Trificumurstivum; 2. Cyperus esculentus; 3 . Pisum sativunz; 4. Daucu,>carotu; 5 . Raphanus sativus; 6. Beta vulgaris; 7. Brassica olerucea gongylodes: 8. Allium sp.; 9. Arirthum graveolens: 10. Lycopersicon esculmtum; 1 I.Cucumis sativus; 12. Solanurn tuherosuni. ’Harvest index is calculated on a dry mass basis. ‘L. =crop was grown between other culture rows

All three of the closure experiments in Bios-3 were initiated during early winter to minimize invasion of pathogens from the outside. The first experiment lasted six months during the winter of 1972 to 1973 with three male crew members; some of them exchanged during the experiment. The second experiment during the winter of 1976 to 1977 lasted four months, again with three male crew members although one left during the experiment. The goal was to test the ability of the enclosure to supply food. In the third experiment, two male crew members were sealed in the facility for five months from November 1 1, 1983 to April 10, 1984.47 Table 1 lists the crops that were grown during this experiment. The plants were grown in artificial, solid substrates with hydroponic nutrient solutions. Each phytotron contained crops of three to seven different ages, allowing the continued availability of food during the experiment and contributing to stable oxygen levels. Chufa nut sedge (Cyperus esculentus), which has an oil-rich tuber, was an interesting addition to the crops grown in Bios-3. It was used as a delicacy by many peoples for millennia, but since its culture was never mechanized it has essentially dropped from modern use. However, it proved to be an excellent source of oil in the Bios-3 experiments. Chufa and some of its relatives are known in many parts of the world as the world’s worst weeds.48 The choice of plants4935”depends not only on taste, nutritional qualities, and caloric content, but also on their gas-exchange qualities. The average respiratory quotient for a human (RQ = C02/02) is about 0.89 to 0.90 rnol.mol-’, depending on the diet. Plants that store energy primarily as starch have an assimilation quo-

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FRANK B. SALISBURY

tient (AQ = O,/CO,) close to 1.0. Thus if only starchy plants such as wheat and potatoes are grown, oxygen will tend to decrease in the atmosphere as C 0 2 builds up. To achieve a better balance and also for the human diet, it is necessary to have oil crops, which have a lower AQ, the exact quotient depending on the crop and the growth conditions. Introducing chufa into Bios-3 lowered the AQ from about 1.0 to 0.95, and the gas concentrations remained relatively stable. The CO, varied from about 0.5% when crops were growing especially well to slightly over 2% shortly after the beginning of the second experiment. The Bios-3 crew members consumed about 20% of their calories in the form of meat stored at the beginning of the experiment or passed in through the air locks. This was generally lyophilized meat to which water was added for reconstitution. None of the crew members were vegetarians, and none of them were desirous of becoming such. This raises the question: Is production of meat in a CELSS facility needed? It is quite possible to be a completely healthy vegetarian, but many potential crew members find that idea unappealing. Nevertheless, growing the usual meat animals in a CELSS facility would require at least 10 times as much area to grow feed for the animals as that required for vegetarians. Actually, a few animals could be part of a CELSS facility by feeding them plant parts that are inedible for humans. Chickens and fish have been mentioned in this context. Nevertheless, construction and operation of a CELSS facility become much simpler as the crew moves in the direction of vegetarianism.49750Another important aspect is the recycling of waste material. In Bios-3, the urine was added to the nutrient solutions for wheat (contact only with roots). Other human wastes were dried and stored, so the Bios experiments made only a beginning at the recycling of waste material. A rather large team of researchers was involved in the Bios-3 experiments. There were chemists who studied mineral balances including trace metals released from the air purification and other systems. About six researchers studied the microflora (i.e., bacteria, fungi, actinomyces, and yeasts) of nutrient solutions, plant root and shoot surfaces, solid media, and human skin and intestines (fecal samples). It was found that, although stability was never achieved, populations of various micro flora never exceeded the normal limits encountered outside of Bios-3. On the other hand, it appeared that staphylococci on the skin had the potential to endanger the very existence of humans in the system. The microbial population varied extensively in the first experiment, so measures were taken in the second experiment to reduce this fluctuation. Linen was no longer washed, but clean linen enough for the duration of the experiment was stored at the beginning. The catalytic converter was added. Crew members wore gauze masks when they worked with the plants. This led to higher stability in the microbiological communities, although they never became completely stable. Several theoretical analyses were carried out based on the Bios-3 experience. One practical conclusion emphasizes the reliability of plants over machinery. When the correct environment is provided, plants are highly reliable; most prob-

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155

lems resulted from failure of the equipment providing that environment. While an algal reactor may contain 1013 cells, any one cell may regenerate the system because the ability to do so is encoded in its genome. The same is true for higher planta, where a single seed, or even a single plant cell if tissue-culture techniques are used, may regenerate the plant culture. Engineered components, on the other hand, have no such capability for self-regeneration. Chemical studies showed that it would be essentially impossible to recycle some substances-the so-called deadlock substances. The Bios-3 experience made it clear that, although it is a desirable goal to reduce unrecycled waste substances to the barest minimum, this might prove to be more costly than resupply. To convert the minerals in ash to plant nutrients, for example, might require sophisticated equipment that itself requires substances such as strong acids that would have to be resupplied. Would it be better to grow the plants in solid substrates rather than hydroponically, so that waste products could be composted and returned to the substrate? Unfortunately, such biological waste disposal is slow and has its own problems, like the potential for plant and even animal diseases. There are many possibilities that await future study. In any case, thinking about Bios-3 helps us to appreciate the balances that have existed for $0 long on Earth. Clearly, there are also balances in our industrialized society. Such a complex society, with its manufacturing capability, could hardly be compressed into the confines of a practical CELSS facility. Rather than trying to duplicate the balances in our biosphere and industrialized society, it will be necessary to learn to achieve balances and thus stability in the confined volume of a CELSS facility. Achieving stability proved to be a serious problem not only in the Biosphere-2 project, but also in the Bios-3 experiments. In the latter experiments the instabilities were mostly in microelements and microflora, and the recognition and evaluation of these instabilities was a clear achievement of the Bios-3 experiments. Microfloral instability poses a potential threat, both to plants and to crew members, and microbial communities may exhibit new processes not recognized in the design of the system. Viruses and plasmids remain to be studied in such systems. Stability was much higher in Bios-3 than it was in Biosphere-2. As noted, in spite of expensive environmental control and other human intervention, the goal in Biosphere-2 was to let the diversity of species lead to stability. There were a few instances in which this seemed to be taking Cockroaches, for example, multiplied exponentially until they were visible almost everywhere. Lizards (geckos) that fed on the cockroaches then multiplied until the cockroach population was brought back under control. Some crops, however, failed to produce as had been predicted, while others seemed to flourish. Apparently, too much carbon had been taken into the system at the start because of the concern that growing plants would soon exhaust the carbon dioxide in the atmosphere. This overcompensation led to a drop in oxygen levels. In any case, the high level of diversity in Biosphere-2 did not seem to be much of an advantage in achieving stability. In spite of some shortcomings, the Bios-3 experiments were more suc-

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FRANK B. SALISBURY

cessful in maintaining an acceptable level of stability through the application of advanced technology and human intervention in a relatively simple system, particularly in food production. It should perhaps not be surprising that it is easier to obtain stability in a simple system in which the parameters can be better understood and controlled. The CELSS experiments carried out so far, including Biosphere-2, strongly call our attention to the importance of size in a functioning ecosystem. The Earth, with its huge hydrosphere, atmosphere, lithosphere, and biosphere, has an immense capacity to buffer against change. Our industrialized society has been pumping CO, into the atmosphere at increasing rates for almost two centuries, and so far the changes have been relatively small, though significant. In a CELSS facility with its small buffering capacity, CO, levels fluctuate over hours instead of years. John Allen has called this phenomenon a time microscope.44 The clear conclusion is that a functioning CELSS facility must depend on technology that makes up for its tiny buffering capacity.

C.

BIO-Plex under Construction at Johnson Space Center, Houston

NASA is designing and building a facility called BIO-Plex at the Johnson Space Center (JSC) in Houston, The facility is essentially an updated version of Bios-3. It is hoped that lessons learned in the Bios experiments can be applied in B I O - P ~ ~The X.~ scientists ~ at JSC have consulted with their colleagues in Krasnoyarsk. Initially, the facility will consist of five cylindrical chambers, each 4.6 m in diameter and 11.3 m long, joined by an interconnecting transfer tunnel and accessed through an airlock, a configuration that has earlier been suggested for a lunar CELSS facility.I8 It will be possible to add two more chambers for a total of seven to meet future needs. Each chamber will have two decks and two hatches, one connecting with the tunnel and one for emergency entry or egress. The facility will be state-of-the-art with all the latest control systems, lighting systems for the plants, and so forth. Both physicochemical and bioregenerative life-support systems will be tested. Current plans are for a 120-day test in the year 2001 with three chambers providing 50% food production and 25% waste recycling (personal communication from Russ E. Fortson, Johnson Space Center, Houston, Texas). A 240-day test with five chambers is planned for 2003 when a laboratory chamber and a second biomass production chamber will be added. It is hoped to achieve 90% to 95% food production and SO% waste recycling. Present plans also include a 425-day test beginning in 2005 with 90% to 95% food production and 90% to 95% waste recycling. Of course, all these plans are subject to change. Actually, many preliminary tests have already been completed in smaller chambers at JSC, some including plants and others based on physicochemical systems. In one such test involving a single occupant, one crop of wheat was almost completely sterile. Although not proven, this may be the result of an ethylene concen-

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157

tration of about 200 nmol.mo1-' measured in the chamber. This observation is of interest in light of our experience with super-dwarf wheat in Mir. Additional CELSS facilities are being constructed in Japan53 and planned in Europe.

VI.

CONCLUSIONS: LESSONS LEARNED

Research in the field of bioregenerative systems, closed or nearly closed with respect to matter but open with respect to energy, has led to some important insights and generalizations, not only about the design and operation of a CELSS but also about earthly ecosystems. A number of the generalizations that can be concluded from this review are summarized here:

1. It is possible, at least over relatively short time intervals with the use of advanced technological control of the environment (temperature, light, air purity, etc.) and an outside energy source, to enclose in a relatively small volume a functioning ecosystem (i.e., CELSS), that accommodates humans who are dependent on green plants for recycling of the air (algae or higher plants) and for food production (mostly higher plants). 2. A CELSS will require a high input of energy to provide sufficient light for photosynthesis (if not obtainable from direct sunlight) and to maintain environmental control. 3. The challenge of creating and operating a CELSS facility is that its limited size leads to a highly limited buffering capacity against the changes in the environment that tend to occur as crops are grown and as humans interact with the system. The lack of buffering capacity must be compensated for by sophisticated control systems. 4. The time that such a CELSS facility can be maintained, even in semiclosed mode, is highly dependent upon the efficiency of waste management. Without resupply and removal of wastes, their accumulation will eventually limit the life of the facility. 5. Resupply of critical components will prolong the life of the CELSS facility. It seems clear that a practical facility will not achieve 100% closure with respect to matter but will depend on some resupply and waste removal. 6. The practicality of a CELSS facility for space exploration is determined by the break-even time when the extra mass required to operate the facility equals the mass of materials that would otherwise need to be resupplied (see Figure 1). Only if the break-even time is shorter than the duration of a proposed mission will a CELSS facility become practical (providing that costs also balance). 7. Biological recycling of organic wastes, as in Biosphere-2, is most efficient (i.e., produces products that can be used directly by plants), but is slow, requires relatively large mass and volume, and may harbor plant and ani-

FRANK B. SALISBURY

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ma1 pathogens. Physicochemical recycling, although limited in other ways, may be needed. 8. An important conclusion is that large size and complexity of an ecosystem are no guarantee for ecological balance and stability. If we are to build such a small system as a CELSS facility must be, we will have to learn many things about how best to intervene in the functions of the system in order to keep it relatively stable and under control. Much headway has been made, but much remains to be done. 9. The weakest link in a CELSS facility is not the plants, but rather breakdown of the mechanical equipment. Plants can regenerate (reproduce) themselves after a crop failure (probably caused by failure of mechanical equipment), but machinery has no such ability. Broken machinery must usually be repaired by living organisms-the crew members. 10. There is good reason to believe that the absence of gravity will not limit crop production in a microgravity CELSS facility. Plants will grow well in microgravity if other stress factors are maintained at a minimum. 1 1. Inclusion of animals in a CELSS facility will greatly increase its complexity and size. The more vegetarian the diet, the simpler and smaller can the facility be. It might be possible, however, even in a relatively simple system, to include some fish and fowl that can feed mostly on food that humans cannot consume. Meat might sometimes be provided through resupply. 12. The success of a CELSS facility operated in a gravity environment or in microgravity is to be found in the details, and often those details are not evident until experimentation is carried out. In the Mir experiments, for example, the importance of balkanine particle size and of ethylene present in the atmosphere only became apparent after failuies were experienced in space experiments. Another example is the importance of balancing the respiratory quotients of the crew members with the assimilation quotients of the various crops. Such details may be known, but they are often overlooked.

VII.

SUMMARY

An option in the long-duration exploration of space, whether on the Moon or Mars or in a spacecraft on its way to Mars or the asteroids, is to utilize a bioregenerative life-support system in addition to the physicochemical systems that will always be necessary. Green plants can use the energy of light to remove carbon dioxide from the atmosphere and add oxygen to it while at the same time synthesizing food for the space travelers. The water that crop plants transpire can be condensed in pure form, contributing to the water purification system. An added bonus is that green plants provide a familiar environment for humans far from

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their home planet. The down side is that such a bioregenerative life-support system-called a controlled environment life-support system (CELSS) in this paper-must be highly complex and relatively massive to maintain a proper compwition of the atmosphere while also providing food. Thus, launch costs will be high. Except for resupply and removal of nonrecycleable substances, such a system is nearly closed with respect to matter but open with respect to energy. Although a CELSS facility is small compared to the Earth’s biosphere, it must be large enough to feed humans and provide a suitable atmosphere for them. A functioning CELSS can only be created with the help of today’s advanced technology, especially computerized controls. Needed are energy for light, possibly from a nuclear power plant, and equipment to provide a suitable environment for plant growth, including a way to supply plants with the necessary mineral nutrients. All this constitutes the biomass production unit. There must also be food preparation facilities and a means to recycle or dispose of waste materials and there must be control equipment to keep the facility running. Humans are part of the system as well a5 plants and possibly animals. Human brain power will often be needed to keep the system functional in spite of the best computer-driven controls. The particulars of a CELSS facility depend strongly on where it is to be located. The presence of gravity on the Moon and Mars simplifies the design for a facility on those bodies, but a spacecraft in microgravity is a much more challenging environment. One problem is that plants, which are very sensitive to gravity, might not grow and produce food in the virtual absence of gravity. However, the experience with growing super-dwarf wheat in the Russian space station Mir, while not entirely successful because of the sterile wheat heads, was highly encouraging. The plants grew well for 123 days, producing more biomass than had been produced in space before. This was due to the high photon flux available to the plants and the careful control of substrate moisture. The sterile heads were probably due to the failure to remove the gaseous plant hormone, ethylene, from the Mir atmosphere. Since ethylene can easily be removed, it should be possible to grow wheat and other crops in microgravity with the production of viable seeds. On the ground Biosphere-2 taught us several lessons about the design and construction of a CELSS facility, but Bios-3 came much closer to achieving the goals of such a facility. Although stability was never completely reached, Bios-3 was much more stable than Biosphere-2 apparently because every effort was made to keep the system simple and to use the best technology available to maintain control. Wastes were not recycled in Bios-3 except for urine, and inedible plant materials were incinerated to restore CO, to the atmosphere. Since much meat (about 20% of calories) was imported, closure in the Bios-3 experiments was well below 100%. But then, a practical CELSS on the Moon might also depend on regular resupply from Earth. Several important lessons have been learned from the CELSS research described in this review.

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ACKNOWLEDGMENTS I wish to thank Mary Ann Clark for help with the manuscript. Preparation o f the paper was partially supported by the Utah Agricultural Experiment Station (paper # 6015) and by Grant NCC-2831 from NASA.

REFERENCES AND NOTES I. 2.

3. 4.

5.

6.

7. 8.

9.

10.

11. 12.

13.

14. 15.

16.

Schwartzkopf, S.H., Human life support for advanced space exploration. In: Advances in Space Biology and Medicine (S.L. Bonting, Ed.), pp. 231-253, JAI Press Inc., Greenwich, CT, 1997. Tsiolkovsky, K.E. Life in Interstellar Medium, Nauka Press, Moscow, 1964 (In Russian, reprinted. Tsiolkovsky died in the 1930s.) Salisbury, F.B. and Ross, C.W. Plant Physiology, 4th ed., Wadsworth, Belmout, CA, 1992. Bugbee B.G., Salisbury F.B. Exploring the limits of crop productivity. I. Photosynthetic efficiency of wheat in high irradiance environments. Plant Physiology, 88:869-878, 1988. Wolf, L., Bioregeneration with maltose excreting Chlorella: System concept, technological development, and experiments. In: Advances in Space Biology andMedicine (S.L. Bonting, Ed.), pp. 255-274, JAI Press Inc., Greenwich, CT, 1997. Shepelev, Ye. Ya. Biological life support systems. In: Foundations of Space Biology and Medicine (M. Calvin, 0. Gazenko, Eds.), vol. 3, pp. 274-308. Academy of Sciences USSR, Moscow, Russia, and NASA, Washington, DC., 1975. Krauss, R.W. Mass culture of algae for food and other organic compounds. American Journal of Botany, 29~425-435, 1962. Krauss, R.W., The physiology and biochemistry of algae with special reference to continuous-culture techniques for Chlorella. In: Bioregenerative Systems (Conf. Proc. Washington, D.C., 1966), NASA SP-165, pp. 97-109. NASA, Washington, D.C., 1968. Meleshko, G.I., Lebedeva, Y.K., Kurapova, O.A., Uliyanin, Y.N., Prolonged cultivation of Chlorella with recovery of the medium. Kosmologiia Biologiia Medicine 1(4):28-32, 1967. (Translation in: Space Biology and Medicine 1(4):41-47, 1967). Kamarei, A.R., Nakhost, Z., Karel M. Potential for utilization of algal biomass for components of the diet in CELSS. In: Controlled Ecological Life Support Systems: CELSS ‘85 Workshop, NASA Ames Research Center, Moffett Field, CA (R.D. MacElroy, N.V. Martello, D.T. Smernoff, Eds.), pp. 13-22, NASA TM 88215, 1986. Waslien, C.I. Unusual source of proteins for man. Critical Reviews in Food Science and Nutrition, 6:77-151, 1975. Packer, L., Fry, I., Belkin, S. Application of photosynthetic N2-fixing cyanobacteria to the CELSS program. In: Controlled Ecological Life Support Systems: CELSS ‘85 Workshop, NASA Ames Research Center, Moffett Field, CA (R.D. MacElroy, N.V. Martello, D.T. Smernoff, Eds.), pp. 339-352, NASA TM 88215, 1986. Meyers, J . Space biology: Ecological aspects: Introductory remarks. American Biology Teacher 25:40941 I , 1963. Schwartzkopf, S.H. Design of a controlled ecological life support system. BioScience, 42526535, 1992. Salisbury, F.B. Lunar farming: Achieving maximum yield for the exploration of space. HortScience,26:827-833, 1991. Calvin, M., Gazenko, O.G. (Eds.) Foundations of Space Biology and Medicine, Joint USA/ USSR, 3 vols., NASA, Washington, D.C., 1975.

Crowing Crops for Space Explorers 17

IX

19 20 21 22 23 24 25

26 27

28

29.

30. 31.

32.

33. 34. 35.

36.

37.

38.

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Schwartzkopf, S.H. Hazard and risk assessment for surface components of a lunar base controlled ecological life support system. Proceedings 22nd International Conference on Environmental Systems, SAE Technical Paper Series No. 921 285, July, 1992. Mendell, W.W. (Ed.) Lunar Bases and Space Activities of the 21st Century, Lunar and Planetary Institute, Houston, TX, 1985. Vince-Prue, D. Photoperiodism in Plants, McGraw-Hill, London, 1975. Salisbury, F.B. Photoperiodism. Horticultural Reviews, 4:66-105, 1982. Hart, J.W. Plant Tropisms and Other Growth Movements, Unwin Hyman, London, 1990. Sack, F.D. Plant gravity sensing. International Review qfCytology,127: 193-252, 1991. Salisbury, F.B. Gravitropism: Changing Ideas. Horticultural Reviews, 15:232-278, 1993. Dutcher, F.R., Hess, E., Halstead, T.W. Progress in plant research in space [experiments from 1987 to 19921,Advances in Space Research, 14(8):159-171, 1994. Halstead, T.W., Dutcher, F.R. Plants in space. Annual Review of Plant Physiology, 38:31-345, 1987. Mashinski, A.L., Nechitailo, G.S., Vaulina, E.N. Space biology. Biology, (Moscow), 10:64, 1988. Nechitailo, G.S., Mashinski, A.L. Space Biology: Studies at Orbital Stations. Mir Publishers, Moscow, 1993. Lewis, N. Plant metabolism and cell-wall ,formation in space (microRravity) and on Earth, 1992.1 993 NASA Space Biology Accomplishments, NASA Technical Memorandum, pp. 241244, NASA, Washington, D.C., 1995. Krikorian, A.D. Space stress and genome shock in developing plant cells. Physiologia Plantarnm, 98:901-908, 1996. Krikorian, A.D., Levine, H.G. Development and growth in space. In: Plant Physiology, A Treatise, Vol. X : Growth and Development. Academic Press, New York, 1991, pp. 491-555. Tripathy, B.C., Brown, C.S., Levine, H.G., Krikorian, A.D. Growth and photosynthetic responses of wheat plants grown in space. Plant Physiology, 110:801-806, 1996. Merkies, A.I., Laurinavichyus, R.S. Complete cycle of individual development of Arabidopsis thuliana Haynh plants at Salyut orhital station. Doklady Akademii Nauk SSSR 271(2):509-5 12, 1983. Ivanova, T.N., Dandolov, I.W., Moistening of the substrate in microgravity. Microgravity Science and Technology, 3:151-155, 1992. Ivanova, T.N., Dandolov, I.W. Dynamics of the controlled environment conditions in isvet? greenhouse in flight. Explorations Cosmiques 45(3):33-35, 1992. Members ofthe team included Frank B. Salisbury, William F. Campbell, John G. Carman, Linda Gillespie, Gail E. Bingham, Steven Brown, Pamella Hole, Liming Jiang, and Rubin Nan at Utah State University; David Bubenheim, Boris Yendler, Tad Savage, Gary Jahns, Kristina Lagel, David Pletcher, Sally Greenawalt, and Terry Schnepp at NASA Ames Research Center; Vladimir Sytchev, Margarita Levinskikh, lgor Podolsky, Lola Chernova, Irene Ivanova, Elena Nefedova at The Institute of Biomedical Problems and the Moscow University, Moscow, Russia; and Tanya Ivanova and her colleagues at the Space Research Institute, Sophia, Bulgaria. Others include Alexander Mashinsky, Galina Nechitailo, and Yuli Berkovitch, who worked with us during early stages of our project, and some students were involved for short periods of time. Salisbury, F.B., Campbell, W.F., Carman, J.G., Bingham, G.E., Bubenheim, D.L., Yendler, B., Sytchev, V., Levinskikh, M.A., Ivanova, I., Chernova, L., and Podolsky, I. Plant growth during the Greenhouse I1 experiment on the Mir orbital station. Advances in Space Research (In press.) Salisbury, F.B., Growing Super-Dwarf wheat in microgravity on space station Mir. Life Support and Biosphere S i e n c e , 4: 155-166, 1997. Foster, K.R., Reid, D.M., Taylor, J.S. Tillering and yield responses to ethephon in three barley cultivars. Crop Science, 31:130-134, 1991.

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39. Moes, J., Stobbe, E.H. Barley treated with ethephon: 1. Yield components and net grain yield. Agronomy Journal, 83:86-90, 199 I . 40. Rowell, P.L., Miller, D.G. Induction of male sterility in wheat with 2-chloroethylphosphonic acid (ethrel). Crop Science, 11:629-631, 1971. 41. Taylor, J.S., Foster, K.R., Caldwell, C.D. Ethephon effects on barley in central Alberta. Canadian Journal of Plant Science, 71:983-995, 1991. 42. Reid, D.M., Watson, K. Ethylene as an air pollutant. In: Ethylene and Plant Development. (J.S. Roberts, G.A. Tucker, Eds.), pp, 277-286. Butterworths, London, 1985. 43. Ables, F.B., Morgan, P.W., Saltveit, M.E. Jr. Ethylene in Plant Biology, 2nd ed., Academic Press, San Diego, 1992. 44. Allen, J. Biospheric theory and report on overall Biosphere-2 design and performance. Life Support and Biosphere Science, 4:95-108, 1997. 45. Nelson, M., Burgess, T.L., Alling, A,, Alvarez-Romo, N., Dempster, W.F., Walford, R.L., Allen, J.P. Using a closed ecological system to study Earth’s biosphere. BioScience, 43(4):225-236, 1993. 46. Eckart, P. Life Support & Biospherics: Fundamentals, Technologies, Applications. Herbert Utz Pub., Miinchen, Germany, 1994. 47. Salisbury, F.B., Gitelson, J.I., Lisovsky, G.M. Bias-3: Siberian experiments in bioregenerative life-support. BioScience, 47:575-585, 1997. 48. Holm, L.G., Pluckett, D.L., Pancho, J.V., Herberger, J.P. The World’s Worst Weed.s, Distrihution and Biology. University Press of Hawaii, Honolulu, 1977. 49. Hoff, J.E., Howe, J.M., Mitchell, C.A. Nutritional and cultural aspects of plant species selection for a regenerative life support system. In: NASA Contractor Report 166324, Purdue University, West Lafayette, IN, 1982. 50. Salisbury, F.B., Clark, M.A. Choosing plants to be grown in a controlled environment life support system (CELSS) based upon attractive vegetarian diets. Life Support & Biosphere Science, 2:169-179, 1996. 51. Henninger, D.L., Tri, T.O., Packham, N.J.C. NASA’s Advanced Life Support Systems Human-Rated Test Facility. Advances in Space Research, 18:223-232, 1996. 52. Tri, T.O., Edeen, M A . , Henninger, D.L. The advanced life support human-rated test facility: Testbed development and testing to understand evolution to regenerative life support. Proceedings 26th International Conference on Environmental Systems, SAE Technical Paper Series, No. 961592, July 1996. 53. Kibe, S., Suzuki, K., Ashida, A,, Otsubo, K., Nitta, K. Controlled ecological life support systemrelated activities in Japan. Life Support & Biosphere Science, 4:117-125, 1997

Chapter 6

ELECTROPHORESIS IN SPACE

Johann Bauer, Wesley C. Hymer, Dennis R. Morrison, Hidesaburo Kobayashi, Geoffry V.F. Seaman, and Cerhard Weber I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Early Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Column Electrophoresis ............................... B. Preparative Scale Continuous Flow Electrophoresis. . . . . . . . . . . . . . . . . . 111. Lessons Learned from Microgravity Experiments ............... A. General Conclusions . . . . . . . . . . . ............... B. Conclusions from Pitu C. Consideration of Future Improvements. . . . . . . . . . . . . IV. Proposed Developments and Experiments . . . . . . . . . . . . . . A. Calibration Standards ............................... B. Pituitary Cell System. . . . . . . . . . . ............... C. United States Commercial Electrop D. Octopus Continuous F Segmented Chamber Fluids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................... References Advances in Space Biology and Medicine, Volume 7, pages 163-212. Copyright 0 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

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164 164 164 170 183 183

193 194

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1.

INTRODUCTION

The term electrophoresis covers a wide variety of techniques including gel, capillary, static column, and continuous flow electrophoresis as well as isoelectric focusing and isotachophoresis. Most applications of electrophoresis are of analytical nature and carried out in gels that prevent diffusion, electroosmosis, and electrohydrodynamic effects. These are rather unsuitable for separating particles and for scaling up for preparative purposes. Electrophoresis without gels (i.e., free-fluid electrophoresis) is a powerful method for purifying biomaterials on a preparative scale and for separating particles such as cells and subcellular organelles. Yet the method has its limitations in separation efficiency and throughput of samples. Separation efficiency is limited by gravity-dependent phenomena, such as particle sedimentation, droplet sedimentation, and thermal convection. Particle sedimentation is a function of the density difference between the particles and the fluid buffer. Droplet (or zone) sedimentation occurs when distinct molecules accumulate in a narrow zone, an event that makes it denser than the surrounding solution, causing the zone to sediment as a droplet within the fluid.2,3 Thermal convection is produced by Joule heating of the buffer, which is proportional to the electric current flow. The gravity dependence of these phenomena has led to carrying out electrophoretic separations in space. Two types of space experiments have been conducted: (1) studies of fundamental electrophoretic phenomena, some of which become apparent only when gravity-dependent phenomena are eliminated (e.g., electrohydrodynamic effects) and (2) actual preparation, purification, or isolation of biological materials. For such experiments, suitable biological candidates for separation must be identified, which can be more effectively fractionated in space than can be done on the ground. Criteria for effective fractionation should include both product quality and the quantity 01' product required. Electrophoresis experiments were performed in microgravity during 20 spaceflight missions. In the beginning, mainly column electrophoresis devices were used, but since 1983 continuous flow electrophoresis (CFE) was preferred. Various microgravity cell electrophoretic studies were recently summarized by Morr i s ~These ~ ~ .are ~ treated briefly here, while others are described in more detail.

II. A.

EARLY EXPERIMENTS Column Electrophoresis

Experiments on Apollo- 14 and - 16

The column electrophoresis experiments carried out in space are summarized in Table 1. The first one was a free-zone electrophoresis during the Apollo-14 mis-

Table I .

Space Experiments in Various Modes of Static Column Electrophoresis

Mission Apollo-14

Year 197 1

Hardware Static Fluid

Mode Zone-Electr.

Macromolecules Particles/Cells DNA, hemoglobin

Apollo-I6

1972

Static Fluid

Zone-Electr.

latex particles

ASTP

1976

MA-01 1

ITP Zone-Electr

STS-3

1981

MA-01 1

ITP Zone-Electr.

Salyut-7

1982

Tavriya

IEF

erythrocytes kidney cells lymphocytes erythrocytes kidney cells bone marrow cells

STS-11

1984

MA-01 1

IEF

MIR STS-26

1987 1988

Svetlana MA-01 1

IEF

MIR

1988

Svetbloc

Gel-Electr.

Note:

genome serum albumin hemoglobin albumins vaccine hemoglobin albumins DNA

Electr. = electrophoresis; ITP = isotachophoresis: IEF = isoelectric focusing

Objective resolution fluid dynamics resolution fluid dynamics resolution cell function

resolution cell function resolution resolution fluid dynamics resolution resolution fluid dynamics standards

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sion using hemoglobin, deoxyribonucleic acid (DNA), and soluble dyes. The samples to be separated were inserted in cylindrical chambers with 0.64 cm inner diameter (i.d.) and 10 cm length.5 The voltage gradients were applied across the cylinders. Photographs taken of the columns after electrophoresis showed that the dyes separated as expected. However, the protein separation (resolution) was poor, which was attributed to a misaligned sample insertion assembly, causing the samples to be too near the column walls, and to bacterial degradation of the proteins. On Apollo- 16 standard particles, 0.23 mm and 0.80 mm diameter monodisperse latex particles, were separated.6 Three sample chambers were processed at a nominal voltage gradient of 26 V/cm; the first contained 0.23 mm particles, the second contained 0.80 mm particles, and the third contained a mixture of the two types of particles. All three latex samples migrated as elongated parabolic bands due to the strong electroosmotic flow (10 pm.cm.V-’s-’) of the fluid moving along the wall of the chamber in the opposite direction. Although electrolysis of the aqueous buffer produced bubbles in the electrophoresis chambers, that reduced the voltage gradient with time, particle velocities could be calculated from densitometry scans of the photographs. These experiments confirmed the potential advantages of electrophoresis in microgravity for the purification of certain subpopulations of living cells. The emphasis then shifted to the development of free zone and continuous flow electrophoresis systems for the separation of cells in microgravity.

Experiments during the ASTP Mission With the static column electrophoresis device MA-01 1 , four sets of different experiments were performed in order to demonstrate principles and advantages of both static free-zone isotachophoresis and electrophoresis of biological cells in mi~rogravity.~ Isotachophoresis (ITP) is based on the use of “leading and trailing” buffers, which tend to focus the migration of the cells into bands.* On ASTP, the isotachophoresis buffer system consisted of a leading buffer of 10 mM phosphate of pH 7.4 and a “trailing” buffer of 200 mM L-serine of pH 8.2 with Tris as the common counter-ion. Both buffers contained 4.2% dextrose as an osmotic contributor and 3 M glycerol as a cryoprotective. The columns were made of pyrex glass (0.64 cm i.d., and 15.24 cm long) coated with a neutral film to reduce the zeta potential of the column wall. The columns had a silver anode chamber at one end and a palladium cathode chamber at the other end. In one experiment a mixture of fresh frozen rabbit and human red blood cells (RBCs) was separated, while the second experiment involved separation of a mixture of formalin-fixed human and rabbit RBCs. During isotachophoresis of the fixed RBCs, the frontal boundary moved more than 1 mm/min and the fixed cell boundary was flat and sharp as predicted by the computer models. However, the unfixed red cells migrated only 60% of the predicted d i ~ t a n c e . ~

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167

In zone electrophoresis, similar electrophoretic columns were used as in isotachophoresis, but the anti-electroosmosis film was adjusted for the low conductivity buffer used for separation of living cells.739Frozen sample slides were loaded into the electrophoretic column during flight, the cell sample was allowed to thaw, and then the electric field (13 V k m ) was applied. The cells migrated toward the anode at rates proportional to their electrophoretic mobility (EPM) as proved by photographs. At the end of each electrophoretic run, the column was frozen, removed from the device, and stowed in a liquid N, freezer. Postflight harvesting was accomplished by extruding the frozen cylinder from the glass column and slicing it into some 28 fractions, which were then analyzed separately for cell type, viability, EPM, and physiological functions. Results of red blood cell separations indicated that the electroosmotic flow along the column walls was almost eliminated by the special coating. Fixed horse RBCs separated from the mixture of human, rabbit, and horse RBCs, but the human and rabbit cells did not separate as well as predicted and cell recovery was 70 to 90% of the theoretical yield. Densitometry scans of the photographs and EPMs of cells from the frozen sample slices showed that the horse RBCs moved faster than predicted when compared to the human RBCs. Two samples of human embryonic kidney cells were successfully separated and recovered with good viability. Kidney cell bands could not be detected in the photographs. However, postflight cultures of the 28 different sample slices showed that secretory functions were retained and about one-forth of the fractions produced significant quantities of an enzyme called urokinase (used clinically to dissolve blood clots) and one of cell fractions produced six to seven times more urokinase than could be produced by cells separated by any other ground-based methods.’ This was the first successful demonstration of electrophoretic separation of subpopulations of secretory cells from a mixed population cultured from human organs in microgravity. However, insufficient cells were recovered in the fractions to measure accurately the surface charge character of those kidney cells which produced the highest levels of urokinase’ An experiment designed to separate 1.5 x lo6 human lymphocytes did not work properly because of a blocked buffer recirculation conduit to the electrode chambers. The lack of buffer circulation allowed electrolytic gas bubbles to stick to the electrode surface. The accumulating gas bubbles caused a high resistance at the electrode, that greatly reduced the voltage gradient. As accumulating hydrogen dissolved, the buffering capacity was exceeded and the increasing acidity killed most of the cells. Only 6% of the recovered lymphocytes were viable compared to 90% after normal recovery.9

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Experiments during the STS-3 Mission

The MA-01 1 static column hardware was used again on shuttle mission STS-3 to conduct two separations of fixed RBCs and six separations of human embryonic kidney cells in order to do the following: I. 2. 3. 4.

verify the Apollo-Soyuz results test new anti-electroosmosis coatings test a new buffer that could improve cell viability after two freezings separate urokinase producing kidney cells at high concentrations.

Photographs of RBC migration were made during the flight and analyzed by micro-densitometry.10The separation of human and rabbit RBCs was not as clear as predicted; the second band was somewhat diffuse, possibly due to interaction and aggregation of the cell^.^' The kidney cell bands could not be seen clearly enough on the photographs to measure the effective EPM, and malfunctioning of the flight sample freezer caused the separated kidney cells to be thawed before the column could be sliced into fractions for postflight culture and urokinase secretion assays. The need to freeze live cells before and after space electrophoresis proved to be a major disadvantage of the early microgravity experiments. This prompted the development of methods to harvest and transport live cells to space (without freezing), i n high concentrations, and then resuspend them in the electrophoresis buffers just prior to CFE experiments. Experiments during the STS-7 I and STS-26 Missions

During the STS-I 1 mission, isoelectric focusing (IEF) of proteins was performed in order to determine whether fluid convection is predominantly caused by electroosmotic or by electrohydrodynamic effects under microgravity. IEF, which requires buffers capable of forming stable pH gradients, is usually performed in gel electrophoresis. In gel-free electrophoresis, especially in microgravity, larger quantities of specimens can be processed, but local variations of field strength and pH cause convection and diminish resolution. The electrophoretic device was equipped with 8 cylindrical glass tubes (4.5 cm long, 0.625 cm i.d.), a camera, and amperemeters. The tubes were either coated with anti-electrostatic substances or noncoated, and segmented by screens or not segmented. The behavior during IEF in space of three human proteins, carbon monoxide complexes of hemoglobin-A and -C and albumin stained with bromophenol blue, was investigated. These proteins with isoelectric points of pH 7.0, 7.35, and 4.8, respectively, were added to a buffer solution composed of 54.0 ml water containing 40% of three kinds of ampholine solutions (LKB Bronia, Sweden, pH range 3.5-10, 3.5-5, and 5-7) and 1 ml antibiotic solution (penicillin, streptomycin,

'*

Electrophoresis in Space

169

fungisol). The solution had an electrical resistance of 1,900 to 2,000 ohms and the pH was adjusted to 7.3. After starting the experiment by applying a 75 V potential, the columns were photographed at 3-minute intervals during the first 30 minutes, followed by 30 photographs at 2-minute intervals. The actual current strengths were recorded at 3-minute intervals. The analysis of current densities suggested that the best focusing was achieved in segmented tubes, while unpartitioned columns showed an initial current decrease followed by a sudden reversal to the original levels of current. The current reversal indicated the onset of convection, leading to remixing of the components already partially focused to their isoelectric points. Less convection was observed in uncoated tubes than in coated ones. Photographs of columns segmented by screens showed reasonably good focusing of red and blue colored proteins, but all other columns exhibited only brown mixtures of unfocused red and blue proteins. This could not be explained by effects of gravity or electroosmosis but appeared to be due to electrohydrodynamic effects arising when proteins are concentrated and neutralized while approaching their IEP. At this point, differences occur between the conductivities in the sample bands and the surrounding buffer, which cause distortions of the electric field and generate local convective patterns that tend to spread the sample stream. 13 Further IEF experiments were performed with the same device during the STS-26 mission using hemoglobin-A and -C and albumin. In order to distinguish between an electroosmotic effect (wall effect) and an electrohydrodynamic effect (bulk effect), the surface-to-volume ratios of the electrophoretic columns were varied: electroosmotic effects would be most visible in columns with high surface-to-volume ratio whereas electrohydrodynamic effects would be more pronounced with a reversed ratio. So during STS-26, the device was equipped with 4 cylindrical tubes, each with an i.d. of either 0.635 cm or 0.317 cm and 4 rectangular tubes with a width of 0.635 cm and depths of 0.101, 0.152, 0.304, and 0.609 cm. Arnpholyte buffers consisted of mixtures of arginine, cycloserine, and p-aminobenzoic acid instead of carrier amopholytes. After 70 V had been applied, the electrophoretic columns were photographed and the actual strengths of currents were recorded at 3-minute intervals. Analyses of electric currents and evaluation of photographs revealed low fluid turbulence in the cylindrical tubes with 0.317 cm i.d. segmented by nylon screen and in any one of the rectangular tubes. More turbulence and poorer focusing was seen in the thicker rectangular tubes as compared to the thinner ones. This suggests that convection during carrier-free IEF in space is mainly due to electrohydrodynamic effects, because thin rectangular tubes with high shearing forces are the most effective means for flow stabilization. Yet, electroosmotic effects must also play a role, since at the start of focusing no major conductivity or density gradient could have arisen.

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170

Experiments during Soviet Missions

The Soviet free-fluid electrophoresis system “Tavriya” was used on Salyut-7 to separate bone marrow cells. This system allowed up to five fractions to be harvested simultaneously via lateral outlets along one side of the separation chamber, while buffer was injected from opposite ends.14 Tavriya was also operated on Mir as an isoelectric focusing system to separate five different variants of human serum albumin in an artificial pH gradient of borate-poly01.’~The Soviet gel electrophoresis system “Genom” was used on Salyut-7 to separate DNA molecules according to molecular weight. The separated fractions were immobilized in a very dilute gel from which they could be harvested by means of a syringe needle. ] 5 ,

*

B.

Preparative Scale Continous Flow Electrophoresis

Experiments during the ASTP Mission

Several continuous flow electrophoresis (CFE) experiments were performed during space missions that are summarized in Table 2. The first CFE experiment was performed during the ASTP mission in 1976. The purpose was to determine how temperature gradients and different carrier buffer flow rates affect resolution and throughput. The MA-014 CFE system had a flow chamber 3.8 mm thick, 2.8 cm wide, and 18 cm long and was operable at various voltage gradients of up to 60 V/cm. Sample fractions could not be harvested, but the separated sample streams were monitored by ultraviolet light transmission recorded by a photodiode array positioned at the end of the rectangular chamber.17 The cell samples, consisting of mixed rabbit and human RBCs and of rat bonemarrow, spleen cells, and lymphocytes, were injected at 1 x 10’ cells/ml; voltage and temperature were recorded. Sample flow rates were stepped down from 5 ml/hr to 3 ml/hr; carrier flow rates were 16.5 and 11.1 ml/min at voltage gradients of 60 V/cm and 40 V/ cm, respectively. Each electrophoretic run continued for almost 6 minutes. The relative position of the separated fractions and performance data were compared to those in ground separations, where the sample injection concentration had to be limited to 1 x lo7 celldml. Rabbit and human RBCs appeared to separate. Unfortunately, the ultraviolet illumination became so intense (halogen lamps bum brighter in microgravity due to lack of thermal convection) that it saturated the photodetector, so recordings were only obtained during periodical drops of the spacecraft power levels. Postflight analysis showed that the bone marrow cells had a bandspread almost twice as wide as in the ground controls while RBCs, spleen, and lymph cells separated into streams that were comparable in bandspread and resolution to the ground controls.’

’

Table 2.

Space Experiments with Preparative Continuous Flow Electrophoresis

Mission ASTP

Year 1976

Hardware MA-014

zone-electr.

STS-4

1982

CFES

zone-electr.

STS-6

1983

CFES

zone-electr.

STS-7

1983

CFES

zone-electr.

albumins erythropoietin hemoglobin erythropoietin polysaccharides erythropoietin

STS-8

1983

CFES

zone-electr.

erythropoietin

STS-41D STS-5 ID STS-61B Texus rocket Texus rocket Spacelab-J

1984 1985 1985 1988 1989 1992

CFES CFES CFES TEM06-I 3 TEM06-13 FEU

zone-electr. zone-electr. zone-electr. zone-electr. zone-electr. zone-electr.

erythropoietin erythropoietin erythropoietin

Mode

Macromokcu~es

erythrocytes bone marrow cells spleen cells

1994

RAMSES

zone-electr.

IML-2

1994

FFEU

zone-electr. pH-gradient

Objective resolution & basic aspects

resolution & throughput

latex particles

resolution & throughput resolution & throughput resolution & cell function

pituitary cells kidney cells pancreas cells

erythrocytes erythrocytes cytochrome c albumins trypsin inhibitor

zone-electr. IML-2

Particles/cells

clinical trials purity tests clinical trials resolution resolution resolution

Salmonella sp.

resolution throughput & fluid dynamics

pituitary cells spleen cells hyhridoma cells

resolution & cell function resolution resolution

hernoglobin albumins bacterial extracts DNA

J O H A N N BAUER et al.

172

Experiments during the STS-4, -6, and -7 Missions

On these missions, the McDonnell Douglas Astronautics Company continuous flow electrophoresis system (CFES) was used. The flight unit had a rectangular chamber 3 mm thick, 16 cm wide, and 120 cm long with 197 fraction outlets. Cells could be injected at concentrations of lo7 to 10' celldm1 at 4 ml/h. The carrier flow rate was 20 ml/min; a voltage gradient of up to 40 V/cm could be applied. During the first test on STS-4 in 1982, proteins were separated in a barbital carrier buffer of pH 8.3 with a conductivity of 250 pnhokm, while the conductivity of the sample stream was 970 pmhokm, mostly due to the 25% protein solution. A mixture of 12.5% rat serum albumin and 12.5% ovalbumin (25% protein w/v) was separated with a four-tube peak separation similar to the ground controls in which only a 0.2% protein solution was used. A 400x greater sample throughput was achieved in microgravity with essentially the same resolution as the ground controls. On STS-6 the sample throughput was increased to 556x with a 4-fold increase in resolution. On STS-7, three different sizes of polystyrene latex spheres colored red, white, and blue were separated on the basis of charge and size to test for resolution and the effects of conductance discontinuities between the particles and the carrier buffer (2.25 mM sodium propionate, pH 5.0, 155 pmho/cm). The latex spheres had electrophoretic mobilities of 3.5 & 0.2 (red), 2.4 k 0.2 SD (white), and 1.6 + 0.1 (blue) ym.cm.V-'.sec-'. Analysis of the 197 samples showed that significant band broadening occurred when the sample conductivity was increased 3-fold to 455 pmhokm, confirming that field distortions can result from conductivity mismatches. 19320

Experiments during the STS-8 Mission

For the STS-8 mission, the McDonnell Douglas machine was adapted in order to enhance throughput so as to demonstrate commercial feasibility of space electrophoresk2' The fraction collection system was reduced from 197 to 99 outlet tubes in order to permit collection of 12 ml volumes of suspended cells. Duplicate separations of dog pancreatic islet cells, human kidney cells, and rat pituitary cells were carried out at sample concentrations of 10' cells and a voltage gradient of 40 V/cm. The separated cell fractions were collected and stored at 4 "C for 5 days until return to the ground. The pancreatic cell experiments were designed to determine whether in this way diabetes could be cured by transplantation of islet cells, a method that would require about lo9 cells per patient. The cell distribution from the STS-8 experiments and the insulin and glucagon contents of the fractions indicated that beta cells predominated in fractions 17 to 23 and alpha cells in fraction 27 (Figure 1).

Electrophoresis in Space

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Figure 7. Separation of pancreatic cells on STS-8 and hormone secretions of the cell fractions. The flight sample concentration was 1O8 cells; hormone levels are expressed in ng/106 cells after four days of culture. (With permission from Hymer et

This confirmed the ground control separations and the combined advantages of higher resolution and much higher throughput under microgravity conditions. Human embryonic kidney cells, which produce the clot-dissolving enzymes urokinase and tissue plasminogen activator (TPA), were subjected to electrophoresis at cell concentrations of 2.5 x lo6 cells/ml and 8 x lo7 cells/ml (Figure 2).22 In each run, 45 fractions of kidney cells were collected of which 36 fractions could be subcultured to determine the amount of enzymes secreted and for how long it occurred. The electrophoretic mobility of the flight cells was approximately 30% greater than that of the ground controls. The bandspread of the mobility distribution was significantly increased in microgravity (Figure 2).23,24The

JOHANN BAUER et al.

174

CFES SEPARATION OF HEK CELLS O N S T S 8

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Figure 2.

Comparison of CFE separations of human kidney (HEK) cells on Earth and on STS-8 (up er panel). Sample input concentrations were as follow: ground = 7 x 10 cells/ml; STS-8 Run 3 = 2.5 x l o 6 cells/mI; STS-8

f?

Run 4 = 8 x 107 cells/ml. Urokinase (lower left panel) and tPA (lower right panel) levels were secreted by cell fractions recovered from Run 4 on STS-8 mission. (Adapted from Morrison et and Lewis et

flight mobility distribution was essentially the same at 2.5 x lo6 and 8 x lo7 cells/ ml, whereas the mobility distribution of the ground separation was significantly reduced at an enhanced cell concentration of 7.0 x lo6 cells/ml. Microscopic classification of cells cultured from the flight fractions showed that the separated cells differentiated into four morphological types,25 while fibrinolytic assays on culture medium from each cell fraction showed that all cells produced significant

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t

0

S

m

Q X

2

S

%

W

0

S

Figure 3. Results of two rat pituitary cell CFE experiments performed on the ground (Expt. 1 ) and on STS-8 (Expt. 2). (Reproduced with permission from Hymer et

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levels of urokinase and TPA.23,26Three to five cell fractions from the microgravity experiments were found to produce two to four times more urokinase than any of the other fractions, while five to six cell fractions also produced high levels of TPA.27 The elevated production of urokinase in each of the cell fractions continued for at least twelve days in continuous culture after return from STS-8.,* These results confirmed the earlier separation of urokinase-producing kidney cells obtained on the ASTP and STS-3 missions by means of free zone electrophoresis. Somatotrophic cells from rat anterior pituitary were also separated during the STS-8 mission. A pool of 5 x lo7 freshly trypsinized cells, prepared from the anterior pituitaries of 100 adult male rats about 18 hours before launch, were washed, suspended in low ionic strength triethanolamine buffer, and stored in a syringe for 72 h at 4 "C prior to injection into the CFE device on flight day 3. The electrophoresis buffer consisted of 0.65 mM triethanolamine, 30 mM glycine, 0.2 mM K-acetate, 0.3 mM MgCl,, 0.027 mM CaC12, 220 mM glycerol, and 44 mM sucrose, 296 mOsmol, brought to pH 7.25 by adding glacial acetic acid. Cells were collected into 50 individual bags preloaded with serum-containing medium. The fractionated cells could thus be kept viable for 5 days until cell recovery and analysis after landing. The separation profile of cells producing growth hormone (GH) was remarkably similar to that routinely seen during ground-based separations (Figure 3) except for band spreading, which was increased as was also seen in human kidney cells in the same flight experiment (Figure 2). The most anodal fractions contained 63% GH cells. Radioimmunoassays of alkaline extracts of these cells indicated that they contained more than 600 ng GH/I .5 x lo5 cells. The corresponding fractions obtained in ground separations had 60% GH cells, containing 600 ng/ GH/I .5 x los cells. The cells producing prolactin, the only other cell class monitored in this flight experiment, showed some enrichment in the lower mobility fractions, another aobservation consistent with ground trials. Experiments during the S T S - 4 1 0 , - 5 1 0, and -6 ID Missions

On these missions, the protein hormone erythropoietin was purified. This was done in order to obtain pure hormone for clinical trials and improvements in process control. Experiments during Sounding Rocket Flights

The electrophoresis system TEM 06-13 was built by MBB-ERN0 for the Texus sounding rocket and Biotex Spacelab programs. It had a separation chamber 0.5 mm thick, 7 cm wide, amd 20 cm long, a sample volume of 10 ml, a video camera, an ultraviolet detector at the outlet, and 7 fraction collectors and operated at electric fields up to 143 V / ~ r n . ,The ~ unit successfully separated rat, guinea pig,

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(l--T@?) b

u

f

f

e

r reservoir tank

a,

-0

0

5 (d u

separation chamber ion exchange mernbi‘ane

detector (254 nm) 60 channels plunger pump I 1 : I / , , , I . , , . , I / I . , I

60 channels fraction collector

&

subunit

buffer tank

separation chamber cover

sample cassette sample feeder separation tube

waste bag

front panel

Figure 4. Diagram of the Japanese Free Flow Electrophoresis Unit (FFEU) Upper Panel: separation chamber. Lower Panel: external view of the device.

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and rabbit erythrocytes and demonstrated possibilities for enhancing resolution.30,31 Experiments during the Spacelab-J Mission

During the Spacelab-J mission, a smaller free-flow electrophoresis unit (FFEU; Figure 4) constructed by the Japanese space agency NASDA and Mitsubishi Heavy Industries, was used to separate both proteins and bacterial cells. The unit has a separation chamber 4 mm thick, 6 cm wide, and 10 cm long that can be operated at field strengths up to 100 V/cm and a carrier flow rate of 25 ml/min (Figure 4). Sixty 0.6 ml fractions can be collected with a resolution of 0.1 cm, while the outlet is monitored by an optical detector system (280 nm or 600 nm). Experiment L-3 tested the effects of protein concentrations, sample flow rate, and carrier flow rates on the ability to resolve a mixture of cytochrome-C, conalbumin, bovine serum albumin, and trypsin inhibitor at concentrations of 25 and SO mg/ml. The flight samples were compared with similar ground-based experiments using a chamber only 0.8 mm thick.32333Experiment L-8 separated three strains (SL1027, SL3749, and SL1102) of Salmonella typhimurium with mobilities of 0, 3.1, and 4.2 pm.cm.s-'.V-', r e ~ p e c t i v e l y .The ~ ~ ,three ~ ~ strains were grown separately on 0.5 % polypeptone medium at pH 7.0 and 37 "C for 16 h, harvested, washed and suspended in 10-mM triethanolamine-acetate buffer (pH 7 3 , and then injected into the separation chamber. Electrophoresis was carried out at 33.3 V/cm and SO V/cm. No real-time data of monitoring optical densities could be obtained because of a malfunction of the optical equipment. So, bacteria were collected in various fractions and then counted. The evaluation showed that the SL1027 and SL3749 strains were separated into two peaks, while the SLI 102 strain overlapped with the elution peak of SL1027. The migration distances of the SL1027 and SL3749 strains were as predicted from the results of ground-based studies. This means that, in this case, electrophoretic separation in microgravity did not give significantly better results than obtained on the ground. Experiments during the IML-2 Mission

A French electrophoresis device, called RAMSES, was flown to qualify the design and to improve our understanding of the phenomena involved in continuous flow electrophoresis in microgravity. It has interchangeable chambers and an illuminated detector system. The chamber is 3 mm thick, 6 cm wide, and 30 cm long. It operates at a field strength of up to 50 V/cm, and can also be used for isoelectric focusing p ~ r i f i c a t i o nFor . ~ ~the IML-2 mission, three zone electrophoresis experiments were aimed at separating hemoglobin and bovine serum albumin, or bovine serum albumin and a-lactalbumin, either at low concentrations (2-3 mg/ ml) or at high concentrations (20-30 mg/ml). Two further experiments were performed to purify gamma interferon from a crude bacterial extract with protein

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contents of 4.9 mg/ml and 19.3 mg/ml, respectively. The results demonstrated that, with the use of more concentrated samples, the throughput at higher protein concentrations can be increased by a factor of 5 in m i c r o g r a ~ i t y . ~ ~ During the same mission, the Japanese free flow electrophoresis unit (Figure 4) was used to separate two genes in a DNA sample, hybridoma cells from antibodies, and subcellular granules of rat anterior pituitary cells. The walls of the sepa-

10

(sec)

a 2,n

L'".

1.5

1 0

20

30

40

unc-6 probe

3

gm

1.0

n,

1I

figure 5. Ultraviolet absorption profiles monitored every 10 seconds during free flow electrophoresis of C. elegans DNA mixed with the markers adenosine (left peaks) and NADP (right peaks) (upper panel). After electrophoresis the sod-4 and unc-6 genes were found in different fractions at different concentrations as determined with the polymerase chain reaction method (lower panel).

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ration chamber were cooled from both sides by thermomodules, controlling the temperature at 4OC. An iron electrode used as anode was rinsed by a 0.5 M Tris-acetate (pH 7.5) solution containing 10 mM NaC1, while a silver chloride cathode was rinsed by a 0.5 M HEPES-tetraethylammonium hydroxide (pH 7.4) buffer. Ion exchange membranes separated the electrodes from the chamber buffer, which had a flow rate of 3 cm/min. This flow rate was maintained by a 60-channel plunger pump at the bottom of the separation chamber and by the pressure generated by a coil spring behind the diaphragm of the buffer reservoir tank, The sample was loaded at a speed of 2.5 c d m i n at the top of the separation chamber through a nozzle with a diameter of 0.5 mm (Figure 4). The detector system, measuring absorbance at 254 nm, was a 512-channel linear PCD sensor attached to a detection window of the separation chamber. The signals of the detector were transferred at 10-s intervals to the Payload Operations Control Center at Marshall Space Flight Center, Alabama, by the down-link system. They were converted in real-time to three-dimensional electropherograms, which served to follow the separation process and to determine the fractions to be colI e ~ t e d . ~Bacterial ~ - ~ ' contamination of the specimens, a serious problem in previous experiments, was avoided by disassembling the apparatus before launch and disinfecting the parts by a suitable combination of mild disinfectant^.^^ This cleaning operation, carried out at the Kennedy Space Center after extensive verification tests and personnel training, achieved sterility of all electrophoresis buffer solutions throughout the mission. The DNA sample contained the sod-4 and unc-6 genes of the worm Cuenorhabditis elegans. The buffer solution for electrophoresis of this sample consisted of 0.01% hydroxypropylmethyl cellulose and 0.3% ampholyte (Pharmalite 2.5-5) dissolved in water (pH 3.87, conductivity 87 pSiemens/cm). A 1.5-L volume of the degassed and filtered solution was introduced into the buffer tank. To the 3-ml sample containing 300 mg DNA was added 6 ml of a marker solution containing 5 mM of adenosine and 5 mM of NADP (pH 6.05, conductivity 440 pS/cm). In preliminary experiments on the ground as well as in space, adenosine showed little mobility and stayed near the injection point, while NADP migrated towards the anode (Figure 5). The migration of these two markers indicated the position of the genes since these always migrated between the two markers; the position of the markers could simply be monitored by the 254-nm detector system.The fractions were collected and stored in a freezer at -20°C until landing. Afterwards they were analyzed for the gene contents by application of the polymerase chain reaction (PCR) method. Figure 5 shows that the genes were found in fractions 32 to 38, and that the ratio of sod-4 to unc-6 was 1 to 1 in fraction 32, and it was 7 to 1 in fractions 35 and 36, indicating that a partial separation had taken place. STKl hybridoma cells, which secrete an immunoglobulin-G antibody, were also subjected to electrophoresis in the Japanese FFEU apparatus. The hybridoma cells were cultured in space for 7 days in an incubator containing 5% CO, (at 37 "C and 60% of humidity) by means of the CCK cell culture kit.39 They produced

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Figure 6. Ultraviolet absorption profile of a free-flow electrophoresis experiment on separation of hybridoma cells in microgravity.

twice as much antibody (50 m g ~ m - as ~ )they did on the ground. The culture medium, containing cells and antibody were introduced above fraction 30 in the separation chamber. Electrophoresis was carried out stepwise at 0 V, 1.50 V, and 300 V. The left hand three-dimensional electropherogram in Figure 6 shows that cells behaved as expected at 0 V, but at higher voltages, anomalous profiles were recorded, which was thought to be due to a large air bubble in the electrophoresis chamber. Despite this air bubble, space electrophoresis appeared to give much stabler performance than that on the ground. The separation of subcellular granules of rat anterior pituitary cells was accomplished s u c c e s s f ~ l l y It. ~was ~ assumed that different types of growth hormonecontaining granules have different electrophoretic mobilities. Rat pituitary cells were cultured in space by means of the CCK cell culture unit. The cells were lysed to provide a granule suspension for electrophoresis. Although air bubbles in the chamber precluded satisfactory separation in space, advantages of processing this type of sample in microgravity were noted: about 6x as much sample could be processed in a given time, and more variant forms of GH molecules could be resolved.

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Mobility from stari

Mobilily from start

Figure 7. Electrophoretic mobility profiles of rat pituitary cells. After trypsinization cells were cultivated in cell culture kits (CCKs). Mobilities of flight cells in panels A and E, of ground cells in panels C and C . Medium changed 4x in CCK # I ; no medium change in CCK #3. GH released from cells after electrophoresis and 6-day postflight culture shown for flight cells in panel B, for ground cells in panel D. lntracellular GH in different electrophoretic fractions prepared from cells originally cultured in CCK #3; flight cells panel F, ground cells panel H. (Reproduced with permission from Hymer, et aL40)

Electrophoresis of 14-day flight-exposed pituitary cells at Kennedy Space Center in Florida within 8 h after landing showed that changing cell culture media in space affected the electrophoretic mobility of the GH cells on Earth (Figure 7). Cells undergoing four changes of medium (CCK #1) had mobilities ranging from 3.9 x cm2.V-'.s-' (Figure 7A), while those for cells from to 7.2 x unchanged media averaged 1.7 x loe4cm2 V' s-' (Figure 7E). The former cells

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from 19 of 40 electrophoretic fractions, when plated in culture wells, released detectable GH into the culture medium (Figure 78). Most of the hormone cm2.V-'.s-', appeared to come from pituitary cells with mobilities above 6 x which is consistent with other data showing a high mobility of the GH cell type.42 Surprisingly. these high-mobility GH cells were absent in all of the other treatment groups (unchanged flight cells; changed ground cells; unchanged ground cells).

111.

LESSONS LEARNED FROM MICROGRAVITY EXPE RI MENTS A.

General Conclusions

Static Column Electrophoresis

The operational limitations of free-zone electrophoresis, gel electrophoresis, isotachophoresis, and isoelectric focusing have been explored with different equipment on nine flight missions (Table 1). In the absence of flowing carrier buffers, the free-zone electrophoresis and isotachophoresis experiments demonstrated the fundamental advantages of higher electric field strength and reduced zone sedimentation possible in microgravity. Scparations of standard particles with well-characterized electrophoretic mobility distribution have demonstrated the effects of electroosmosis, conductivity mismatches, and Poiseulle flow in closed free-fluid electrophoresis systems. The separations of particles and protein mixtures have enabled investigators to improve the computer models of the electrohydrodynamic effects in both free zone and continuous flow electrophoresis. Although the advantages of purification of dissolved macromolecular biologicals in space were demonstrated, the major emphasis was to demonstrate that fixed and living cell samples could be separated at concentrations 2 to 3 orders of magnitude greater than could be achieved on the ground. This is illustrated by the ASTP and STS-3 experiments with the MA-01 1 system, which showed that the electrophoretic mobility of human and rabbit RBCs were the same at lo9 cells/ml in microgravity as they were at lo6 cells/ml in I - G . l o ~ lThe ' effect of electroosmosis has been eliminated by the use of special wall coatings to reduce the chamber wall zeta potential. This enhances resolution by eliminating parabolic flow and simplifies the design of collection ports.43 Continuous Flow Electrophoresis

Continuous flow electrophoresis in space has the advantage of much greater sample throughput and higher resolution than on the ground. Six different flight systems have been constructed, five of which have been tested in microgravity.

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Table 3. Comparison of Continuous Flow Electrophoresis Spaceflight Systems

Device

MA-014a CFESb TEM06- 13' FFEU~ RAMSES~ USCEPS' Notes:

thick (mm)

Chamber Wide (cm)

3.8 3.0 0.5 4.0 3.0 1.5-4.5

2.8 16 7 6 6 12.7

Optimal Field long (cm) (V/cm)

18 120 20 10 30 102

60 40 I43 100 50 40

Sample Collection Tubes

Described in Reference

no 197 7 60 40 6x99

17 22 29 32 36

'MA-014 was equipped with a 128-array ultraviolet-detector. bTEM06-13, RAMSES, FFEU, USCEPS have fraction collectors and optical detectors 'USCEPS has not yet flown in space; described in U S . patent # 5,562,812

Table 3 shows a comparison of the dimensions, operational field strengths, and fraction collection capabilities of these systems. It should be noted that the CFES and USCEPS systems have chambers at least four times longer (120 cm and 102 cm, respectively) than any of the other systems. In separations of macromolecular substances, differences in buffer ion concentration between sample band and carrier buffer can produce electrohydrodynamic distortions of the sample bands that may cancel the advantages of operation in microgravi ty .43 In cell separations, bandspread and resolution are significantly reduced when the sample concentration exceeds a certain threshold. For example, a 50% reduction in electrophoretic mobility distribution of human kidney cells occurs at sample concentrations above 2.7 x lo6 celldm1 in 1-G, whereas in microgravity this .~~ cells show the same effect is absent up to at least 8 x lo7 c e l l ~ / m l Pituitary effect at concentrations exceeding 2 x lo7 cells/ml on the ground.22 During cell separation in microgravity, ranges of operating conditions can be extended in the absence of sedimentation and thermal convection. This helps to explain sample band spreading and occasional retrograde (cathodal) migration at high cell concentration~.~~-~~

B.

Conclusions from Pituitary Cell Separation Experiments

Pituitary Gland Complexity and Cell Separation

In order to understand the purpose of the two attempts to achieve separation of pituitary cells by electrophoresis in microgravity, some background information about this important endocrine gland is required. The anterior pituitary gland of the rat, which has served as the study model for the human pituitary for more than 50 years, contains six classes of hormone-producing cells: cells producing growth

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hormone (GH), prolactin (PRL), follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), and adrenocorticotropic hormone (ACTH). A single gland of a male rat weighs about 10 mg and contains about 2 x lo6 hormone-producing cells, roughly 40% GH, 20% PRL,, and 10% of each of the other four classes. This distribution may change with age, sex, and physiological status of the animal. The first application of cell separation techniques, over 25 years ago, showed that subpopulations of cells existed within the hormone-producing classes.47 More recent experiments show that the position of the cell within the gland also affects its function, probably due to paracrine control of hormone production exerted by neighboring cells. In addition to this cellular heterogeneity in the pituitary gland, there is also heterogeneity in the hormone molecules produced by them. For example, at least 12 molecular variants of both GH and PRL are k n ~ w n . ~ They ' , ~ ~ result from transcriptional, translational and posttranslational events that deviate from the traditional processing pathways described in textbooks. It appears that the biological activity of the secreted hormone depends on its molecular configuration (i.e., variant form) as well as its origin from a defined cell type that is strategically located within the tissue mass. This conclusion results from the replacement of biological hormone assays by the more sensitive and specific radioimmunoassay techniques. Yet, there are good reasons to view data obtained from antibody-based detection techniques with some skepticism. The dichotomy between immunoreactive (iGH) and bioactive GH (bGH) provides a case in point. The classic way to measure the biological activity of a GH preparation is to inject it into a young adult rat from which the pituitary gland has been surgically removed. After four days of injections, the widths of the tibia1 epiphyseal plates are measured and any increases are known . ~ ~ and Grindeland have shown that to reflect the GH activity of a p r e ~ a r a t i o nEllis the GH activity of a preparation measured in this way often does not correlate with In fact, these investigators estithe activity measured by radioimm~noassay.~~ mated that the ratio of bGH to iGH in human plasma often exceeded 100! Table 4. Purumeter density frequency ultrastructure GH molecules released in vitro biological activity o f GH molecules released in vitro response to 1 pM hvdrocortisone

Differences Between Growth Hormone Cell Subpopulations in Rat Pituitary Type 1

Type 2

< 1.070g/cm3

> 1.070 g/cmj 50%

50% few secretion granules only monomer (22 kDa) modest

increased release of bGH

many secretion granules 22 kDa plus disulfide linked oligomers potent in bone growth assays modest effect

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The earlier techniques used to separate pituitary cells were based on differences in cell size and density. About 80% of protein in the human pituitary gland is GH protein,52 a figure that probably reflects the importance of this hormone for controlling the musculoskeletal, immune, vascular, and endocrine systems of the body. This fact, coupled with the knowledge about the different hormone-producing cells in the pituitary, made the GH cell a natural target for early pituitary cell separation studies. By 1975, the application of two techniques, velocity sedimentation at unit gravity and density centrifugation through linear gradients of bovine serum albumin, had shown that two populations of GH cells were contained within the rat pituitary gland. They differed in their morphology and density, in the quantities and activities of GH released by them In vitro, and finally in the molecular forms of the secreted hormone (Table 4). Later separation techniques were based on cell surface charge density. In 1983, the first report describing results of the electrophoretic separation of rat GH cells appeared.42 Two preparative techniques were used, density gradient and continuous flow electrophoresis, and two analytical methods, microscopic and laser tracking electrophoresis. Continuous flow electrophoresis was carried out with a device designed and built by McDonnell Douglas in St. Louis, M i s s o ~ r i . ~ ~ , ~ ~ When cells in individual fractions were cultured for 14 days in order to evaluate their GH secretory potential, the most anodal fractions produced about 6 times as much immunoreactive hormone as the least mobile cells.42 Positive evidence for an electrophoretic mobility difference of the GH cell type was obtained in similar ground-based experiments using the McDonell Douglas device. Hymer and colleagues.22 reported that higher mobility GH cells released 5 times as much bioactive GH in culture (as measured by the tibia1 line assay) as lower mobility GH cells did. Also, when the two types of GH cells (Table 4) isolated by density gradient centrifugation were subjected to density gradient electrophoresis in a cylindrical water-jacketed glass column (2.5 x 10.4 cm) of the type described by Boltz and Todds3 at 4"C, the somatotroph-enriched cells (d > 1.070 g/cm3) showed significantly slower migration profiles than their less dense counterparts. However, no difference in electrophoretic mobility of dense GH cells and less dense GH cells or other hormone cell classes were found using either a Zeiss Cytopherometer or a Pen Kem automated light-scattering electrophoresis device42 The electrophoretic mobility of GH cells that is predicted by analytical electrophoresis data suggested the possibility that the GH cell enrichments obtained by preparative electrophoresis methods might actually be artifactual. For example, it was postulated that gravity-driven vertical sedimentation of dense GH cell subpopulations during their upward flow in the flow electrophoresis device might be sufficient to explain the differences in the actually observed GH elution profiles. Of course, gravity-induced sedimentation can be eliminated by performing electrophoresis in microgravity. This was the primary rationale for the electrophoresis trials in space between 1983 and 1994.

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After the two space experiments on electrophoresis of pituitary cells, it appears that sedimentation of dense GH cells during separation in preparative free flow electrophoresis units is not the explanation for their electrophoretic behavior on the ground. In other words, dense GH cells have greater net negative surface charge density than their less dense counterparts and some of the other hormoneproducing cell classes. Why the analytical particle electrophoresis data indicated no mobility differences between the cell classes remains unexplained, although results from the 1994 free flow electrophoresis experiment in space indicated that other factors such as changing of cell culture media must be considered.

Microgra vity Operations A cell separation experiment in low gravity is demanding. It requires the following: 1. proper cell handling during all phases of processing, from loading into suitable culture vessels before launch to postflight recovery of fractionated cells 2. capability for changing and storage of spent culture media 3. capacity for preparing and storing fresh cell culture media and several liters of electrophoresis buffer 4. suitable cell culture hardware operating at controlled temperature and in a suitable gas environment 5. a largely automated continuous flow electrophoresis device 6. trained personnel to operate and monitor all phases of the separation experiment In standard operation in a laboratory on Earth, the transferring of fluids from one vessel to another is probably the most common manual feature in experiments. Bubbles rarely interfere with this procedure on Earth, but they do in a microgravity environment! In ground laboratories, associated techniques are common: cell harvesting + centrifugation + counting + cultivation + media change + cell washing + continuous flow electrophoresis + analysis of fractionated cells. Thus far, these are rarely executed in microgravity. However, as explained below, some progress has been made toward achieving the “coupled technology” goal that will be absolutely required for processing of biological samples on a commercial scale when the International Space Station becomes operational after the year 2000. The main purpose of the second continuous flow electrophoresis experiment with pituitary cells was to achieve this “coupled technology”. Significant advances in spaceflight hardware design during the last I 1 years permitted the design of a more “user friendly” experiment. Thus, pituitary cell culture at 37 “C in MEM cell culture medium supplemented with 5% horse serum and antibiotics

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under a controlled gas environment (9.5% air; 5% C02) prior to actual electrophoresis in flight was r e a l i ~ e d . ~In” ?addition, ~~ effects of changing culture media in flight on several parameters (including hormone release) were studied. The data in Figure 7 could not have been obtained without the cell culture hardware that permitted pituitary cell maintenance under more “user friendly” conditions than was possible in the 1983 experiment. So in future experiments “state-of-theart” cell biology methodology must be coupled with relatively primitive free flow electrophoresis space technology in order to demonstrate the practicality of doing biotechnology operations on a future space station platform. C.

Considerations of Future Improvements

Considering the experiments described above, two central questions remain:

1. Is the process in space more effective compared with the best procedures conducted on Earth? 2. Which biological separation problems will be significantly decreased by operating in space? In order to answer these questions we must reconsider the physical parameters important for electrophoresis in microgravity and to select future experiments that optimally utilize the uniqueness of the space environment. Physical Parameters Important for Electrophoresis in Space

The major attribute of the space environment relevant to biological systems is microgravity. Gravitational effects on biological systems include sedimentation of cells and organelles, flotation of some lipid materials, buoyant convection, and segregation of components by density and perhaps by flows originating from the interplay of density gradients and interfacial tension. Indirect effects of rnicrogravity on the behavior of biological systems include decreased removal of metabolically derived heat due to the absence of thermal and fluid convection and modified long-range transport and concentration oscillations.” Terrestrial life has evolved in an environment of unit gravity, which means that cells and cell organelles have not had to contend with the near absence of inertial acceleration and the resulting reductions in hydrostatic pressure, buoyant flow, and sedimentation. While it is self-evident that gravity acts on large systems, its effects on cells and macromolecular cell components is less obvious.55 The continuous prolonged freefall condition inherent in spaceflight is termed weightlessness, near zero gravity, or microgravity. In reality, a background acceleration of to 1 0-7G is imposed on the spacecraft by atmospheric drag and by accelerations associated with experiments located some distance from the center of mass of the spacecraft. It should also be noted that practical experimental con-

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ditions are able to generate flows in fluids of a few cm.sec-2 with potentially devastating consequences for the unwary experimentalist. Additional accelerations are produced by normal operations in the spacecraft, that are usually random in nature (G-jitter) and range from to I 0-4G according to accelerometer measurements. Such G forces will generate significant buoyancy-driven flows in particle and cell suspensions. Some of gravity’s effects on Earth can be compensated for in various ways such as, by rotation of a device or vessel around a horizontal axis as in a clinostat or rotating reactor, but inertial forces (Coriolis forces) are produced by this rotation. Sedimentation and flotation of components with concomitant convective effects are produced by gravity in heterogeneous multiphasic systems in which the components differ in density, such as cell suspensions or blood. In normal gravity, a particle, droplet, or bubble immersed in a fluid will be subjected to buoyancy forces which will produce a rising or settling of the disperse component. Stokes sedimentation describes the constant equilibrium velocity of a particle that is falling through a fluid in which gravitational, buoyant, and viscous drag forces are balanced. For a sphere of radius a and density p, in a suspending medium of density po, the sphere has an equilibrium velocity v, given by Stokes law as

where q is the viscosity of the suspending medium and g is the acceleration due to gravity.56 Equation 1 shows that the sedimentation rate of a particle depends on its radius squared and the density difference between particle and suspending medium. A stable particle suspension can usually only be maintained for particles below 1 pm diameter, when the gravitational potential energy approaches the thermal energies of the molecules in the suspending medium and Brownian motion maintains suspension stability. Although in coarser suspensions, such as blood, sedimentation on Earth can be minimized by stirring, slow rotation of the container, or matching the densities of medium and particles, each of these approaches can introduce problems. Stirring may produce aggregation or coalescence of the particles, promote breakage of large molecules such as DNA, or create complex fluid motions. Rotation of the container may induce fluid motions and will produce a centrifugal force, thereby distributing particles according to their density. Density matching will introduce molecules that may affect the properties of the particles (particularly living cells) and increase viscosity and osmolarity of the suspending medium. On Earth, sedimentation is a problem in studies where a concentrated suspension of particles or cells has to be added to or inserted into a quiescent liquid. Particle concentrations above certain well defined limits will act as an assembly

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because of close contact. The sedimentation rate then becomes that of a large droplet rather than of the individual particles. Mason57 has pointed out that partcles will sediment as cohesive assemblies when the number of particles per unit volume exceeds

where a is the particle radius, D is the diffusion coefficient, q (x) is the viscosity, which varies with distance in the gradient, and d p (x) / dx is the variation of fluid density through the gradient. It should be noted that droplet sedimentation is a special case of the more general phenomenon of buoyancy-driven convection, which arises in two situations: (1) when a density gradient exists that is perpendicular to the gravitational force vector and (2) when a configuration exists with a more dense fluid located above a less dense fluid. In the first case, stable convection or flow ensues immediately upon even the slightest difference in temperature or density between two adjacent fluid elements whereas in the second case, thermal, unstable convective flow develops only when the driving force exceeds some critical value. Stable convective flow is characterized by the Grashof number, G,, which is a dimensionless parameter indicating the ratio of buoyant to viscous forces.

where g is the acceleration due to gravity, 1 is a characteristic length, h is the viscosity, and Ap is the characteristic density difference. A reduction in the length over which the density difference in a fluid occurs will significantly reduce natural or stable convection. Unstable convective flow is described by the Raleigh number Ra, which has the same form as the Grashof number. The critical value depends strongly on the vertical dimension and less on fluid properties. The influence of Joule heating, as arises in electrophoresis, on various types of convective flows has been studied by O ~ t r a c h While . ~ ~ this analysis is directed toward an examination of degradation of resolution in continuous flow electrophoresis, it has a general applicability to density-driven convective flows. Electrokinetic effects may also occur, as biological systems are complex mixtures of proteins, lipids, carbohydrates, nucleic acids, and other macromolecular complexes. Separation and purification of such systems often involves the use of electrophoresis in water-based gels, which are generally recognized as providing the highest resolution for analytical separation of proteins and analysis of nucleic acid sequences. Gels are used on Earth because most gravitational and electrohydrodynamic effects are nearly completely suppressed in them. Separation of subpopulations of cells, particles or organelles can also be accomplished by free fluid electrophoresis. For small particles, or macromolecules whose radii of curvature

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are similar to that of a dissolved ion, the electrophoretic mobility p is, according to Smoluchowski,

where is the zeta (electrokinetic) potential and E is the dielectric constant. For large particles such as cells or organelles, equation 4 changes to

While g does not formally appear in these electrophoretic equations, the applied electric field produces Joule heating with resultant fluid density differences and heat-induced convective flows. The results of electrophoretic experiments with well characterized model molecules and particles have identified some of the advantages and limitations of space-based purification processes. 19,11 A significant finding is the role of electrohydrodynamics in electrokinetic separations. l 3 Significant hydrodynamic distortion encountered in space has led to the use of wide gap chambers. On Earth, narrow gap chambers are required to minimize gravity-induced thermal convection, but they also restrict electrohydrodynamic effects. Thus, what is a secondary disturbance on the ground becomes a primary disturbance in space. Another problem in space is the slower removal of heat and catabolites produced by cell metabolism through thermal or solutal convection. The heat generated by a typical nucleated cell is about one picowatt (10-l2 J.sec-') in rest and about a hundred picowatts during activity. Under these circumstances, diffusion may be insufficient for removing the generated heat leading to a sustained rise in cell temperature. Electrophoresis involves a tangential motion of one phase with respect to the other phase when an electric field is applied, but only when the two phases carry free charges of opposite sign. Charges on molecules or at the surface of a material in an electrolytic medium arise from ionization of functional groups (amino, carboxyl, phosphate, etc.) and/or ion redistribution (adsorption, desorption, or exclusion). In some instances, charge is either gained or lost as a result of chemical interactions with a component of the suspending medium. Upon application of an electric field, a charged particle or molecule accelerates rapidly until the electric force is balanced by the frictional forces in the medium, whereupon it moves at constant velocity. This velocity at an applied electric field strength of 1 V/cm is known as the electrophoretic mobility p, and its dimensions are cm2.sec-'.V-'. The theoretical interpretation of electrophoretic mobilities has been discussed by a number of author^,^^,^' who conclude the following:

1. When a particle of arbitrary shape is large compared to the thickness of the electric double layer surrounding it, the electrophoretic mobility is indepen-

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dent of its size, shape, and orientation. The relationship between electrophoretic mobility p and zeta potential ( is given by the Helmholtz-von Smoluchowski equation (equation 5). For low surface potentials (< 25 mV), the zeta potential is approximately proportional to the surface charge density at the hydrodynamic surface of the particle, a condition generally satisfied by biological cells. 2. When the concentration of ions or particles is sufficiently low, their electrophoretic motions are independent of one another. 3. It is fair to assume that the ions or particles are exposed to a uniform electrical field, all nonlinear terms may be neglected, the particles have a negligible electrical conductivity (nonconductors), and the particle can be treated as a rigid sphere. Evaluation Criteria for Future Experiments

The NASA electrophoresis program has been in progress for about 25 years and it is believed that the major disturbances that can arise during the electrophoretic process have been identified. It has been established in the continuous flow electrophoresis experiments reviewed here that larger quantities of material can be separated in space than on the ground and that fluid disturbances, which arise from convection and sedimentation, are minimal. At this stage, it is important to continue to investigate the fundamental fluid phenomena involved in the electrophoretic process from both a theoretical and experimental point of view. In this context, it is necessary to develop a set of evaluation criteria that most probably will be a function of the particular biological separation problem addressed. The criteria should be based on measurable variables such as quantity, resolution, viability, retention of biological function, and rate of separation. It is advisable first to develop standard materials with well defined properties that can be used in evaluating separation equipment on the ground as well as in space, and in developing minimum performance specifications for this equipment. A major objective should be the development of standardized equipment for the routine separation of biological materials in space with the best possible resolution and recovery of the separated fractions in adequate amount for the purpose intended. On Earth, the separation of cells and macromolecules requires the elimination of convection and sedimentation problems by density stabilization and use of high viscosity suspending media such as gels. However, in many media, cells undergo changes in volume and shape as well as pinocytosis (ingestion of a substance by the cell by pinching off of a vacuole from the cell membrane). Moreover, the polymeric additives used in density gradients may adversely affect the quality of the separation. In addition, the concentration of cells that can be used is limited on Earth by droplet sedimentation. The cell separation procedure should provide a sufficient number of cells for cloning, injection, culturing, or histological examination (requirement lo4 to lo7 cells) or for isolation of a cellular component (e.g.,

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an enzyme or hormone) that is present in the cells in small amounts (requirement lo7 to lo9 cells). In the latter case, the isolation procedure should be able to process lo9 to 10'' cells within four to 10 hours in order to supply a sufficient number of viable cells. A microgravity environment is likely to be advantageous in the study of phenomena that present difficulties on Earth. Large biological cells, like megakaryocytes, fertilized egg cells, and nerve cells, are difficult to separate because of their very high sedimentation rates. Additional difficulties are presented by endothelial cells, which have a very active pinocytotic process and develop ultrastructural changes indicative of damage in the media usually employed in preparing density gradients. Even greater difficulties are posed by studies on the kinetics of cell aggregation, the behavior of cellular aggregates, cell adhesion, cell-sorting phenomena, and cell contact relationships, especially in regard to embryological development and formation of neuronal networks. Another benefit of space experiments is that they increase our understanding of fundamental processes in electrophoresis. Much of what has been accomplished in experiments to date is subject to the criticism that it could have been accomplished by carefully designed ground-based experiments. Over against this, it can be said that the space experiments have enabled us to recognize fundamental processes such as electrohydrodynamics, which limit the electrophoretic resolution but which are obscured on Earth by the effects of gravity. Future experiments on electrophoresis in space should therefore give proper attention to such fundamental processes, including the possible utilization of the almost unlimited capillary rise or development of interfacial tension gradients in weightlessness. This will require model experiments designed to increase our understanding of fluid dynamics in a near-zero-gravity environment. In addition, the use of standard calibration particles is needed to assess instrument performance on the ground and in space. It should, however, be recognized that spaceflight does not provide a true zero-gravity environment, but that appreciable flows and gravitational accelerations still occur.

IV.

PROPOSED DEVELOPMENTS A N D EXPERIMENTS A.

Calibration Standards

The use of well defined model particles, such as polystyrene microspheres with various functional surface groups, hydrophobicity, and other surface properties, is likely to provide a firmer rationale for electrophoresis in space. Acquisition of valid electrophoretic mobility data typically requires the elimination of errors that come from two principal sources: (1) the imprecision and inaccuracy of the measuring instrument, which is often caused by the effect of electroosmosis on the measured particle velocity; (2) the alteration of particle surface properties and

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hence their electrophoretic mobility by undefined materials in the suspending medium due to impurities in chemicals and water, by contamination from unclean containers, by leaching of components from containers (e.g., silica from glass)61 and filter membranes, and by agents used in processing (e.g., by wetting agents). A two-step standardization process for elucidating these two sources of errors may be undertaken by the use of hydrophilic and hydrophobic latex particle standards. Polystyrene microspheres modified by hydrophilic carboxylate may be used for instrument calibration checks since they are relatively insensitive to the presence of trace contaminants in the suspending medium or in processing buffers. Latex particles modified by hydrophobic sulfate possess, on their surfaces, large hydrophobic areas which serve as “docking” areas for the adsorption of both large and small molecular species. These latex particles may be used to detect the effect of contaminants. This dual use of two types of standard particles will significantly improve the quality of the collected electrophoretic data and provide more reliable comparisons between electrophoretic data collected in space and those collected on the ground.62 B.

Pituitary Cell System

Pituitary Cell Biology in Microgravity The ground-based continuous flow electrophoresis experiments on pituitary cells returned from a spaceflight raised an interesting question: How might microgravity change the net surface charge density of a GH cell?40 Many possibe answers to this question exist. In order to find the right answer(s), it seems useful to review what is known about pituitary cell biology in microgravity. In the period

Table 5. Payload rats rats rats pituitary cells pituitary cells pituitary cells Notes:

Spaceflights Involving Rat Pituitary Tissue

Operations in microgravity none none

Mission

Year

Duration in days

Refs. microgravity

1985 1987

I 13

63 63

none CFE

SL-3 Cosmos microgravity Cosmos 2044 STS-8

1989 1983

14 8

64 22

P cell culture

STS-46

1992

8

65,66

A cell culture

STS-65

1994

14

40

-

During SL-3, animals were not treated in microgravity; pituitary cells were isolated after flight by trypsinization. P cells cultured in sealed vials; no media exchange. A: cells cultured in chambers permitting exchange of media.

700

Cells in space

Rats in space I

I

Z

0

100

0 media changes 4 media changes

I

I I

Cells in mace

Rats in soace

0 media changes 4 media changes

fY lmmunoreactive Prl

Figure 8. Growth hormone and prolactin released by cultured pituitary cells. Left Panel: Comparison of bioactive and immunoreactive GH released from mixed pituitary cell cultures in microgravity with and without medium change and from pituitary cells prepared from rats postflight and subsequently cultured on Earth. Right Panel: Same for Prl release. Data from rats in microgravity are averaged from three flight experiments: SL-3, Cosmos 1887 and Cosmos 2044 (see Table 5). Those without medium change in microgravity are from STS 46 and STS 65, while those with four medium changes are from STS 65 (see Table 5).

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of 1983 to 1994, six spaceflight experiments were done to begin building a pituitary cell biology database (Table 5). The experimental designs have been detailed in several publications22,40,54,63-66 and thus need not be repeated in detail here. As already explained, particular attention was paid to the growth hormone (GH) system because of the significant role of this hormone in the regulation of the adult musculoskeletal system. The total amount and activity of GH released from cells in the culture medium is a relatively easy and straightforward measure of cell function. This is done by either the standard immunoassay (iGH) with a highly specific antibody to GH or by the standard bioassay (bGH) by injection of the preparation into the hypophysectomized rat and measurement of the bone growth 4 days later. Exposure of rats to microgravity results in a significantly decreased release (50% relative to that from cells of ground-based controls) of both bGH and iGH from postflight pituitary cell cultures. However, when isolated pituitary cells are flown in space, this postflight decreased release (specifically for bGH) only occurs if the culture medium has been changed during flight (Figure 8). Without medium change, the release of bGH and iGH is actually increased. Apparently, physiological “fidelity” requires inflight change of tissue culture medium. Prolactin (Prl) cells have been studied alongside GH cells during spaceflight for two reasons. First, because these two pituitary hormone systems often counteract each other so that when release of one hormone is activated, release of the other hormone is repressed. Secondly, both hormones control the immune system. Prolactin may, like GH, be determined by immunoassay (iPrl) as well as by bioassay (effect on division of rat lymphoma cells; bPrl). When Prl cells are removed from the body and studied in vitro, they are removed from the inhibitory influence of brain catecholamines on prolactin release, which may explain at least in part why Prl cells, during 9 days of cultivation, release 20 times the amount of hormone initially present in the cells.67 Exposure of rats to microgravity results in a modest, but statistically significant decrease in the release of both bPrl and iPrl from cells during postflight culture. In spaceflown cells, a corresponding decrease in Prl release occurs but only when culture media are not changed during flight. When media are changed, there is an increased release of bPrl and iPr1 (Figure 8). This is the opposite effect to that shown for GH in spaceflown cells in Figure 8. The stimulus-secretion coupling mechanism of hormone release from the pituitary cell is thought to involve secretory granules, which are physically anchored to a microtubular network. In turn, the microtubular network is connected to cell membrane components and to the nucleus. This suggests that cell size (derived from forward laser light scatter in flow cytometer), area of the cytoplasm occupied by GH-binding granules (derived from 90” laser light scatter),68 amount of GH bound to granules (determined from GH-specific cytoplasmic immunofluorescence), and electrophoretic mobility may serve as important biological parameters of the state of the spaceflown GH cell.

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.* *

--

IGH release (?22%) bGH release ($4S%) * size (tI 5%) * G I I cytoplasmic area (.122%) GH immunofluorescence(t4%) GH cell 90° light scatter (&SO%)

iPRL (fS7%)

bPRL(?165%)

-

GH cell EPM (t3-10X)

-+

-. -* * *

IGH release (7314%) bGH release (?13S%) size (-11 1%) GH cytoplasmic area (-141%) GH immunofluorescencc(?Sl%) GH cell 90" light scatter (-130%) GH molecular size (no change) GH cell EPM (no change) f--

Figure 9. Integration of data from cell culture experiments in microgravity with those from free flow electrophoresis experiments in microgravity. Changes in parameters, relative to synchronous ground controls, are shown (*). Most changes were statistically s i g n i f i ~ a n t . Key ~ ~ findings ~ ~ ~ ~ are ~ ~as- ~ ~ follows: (1) major increase in Prl release by spaceflown cells after four medium changes (top left), which affects (2) release of GH, (3) biophysical parameters of GH cells, and finally (4) net negative surface charge density of GH cells. No change in molecular size of secreted GH. Preliminary data from a passive cell culture e ~ p e r i m e n t(not ~ ~ shown in figure) suggest that spaceflown G H cells lose receptor sensitivity to the natural hypothalamic releasing hormone (GHRH). i = immunoreactive; b = bioactive

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Linkage between Pituitary Cell Biology and Continuous Flow Electrophoresis

Although each of the six space electrophoresis experiments listed in Table 5 had a different design, they are related because of their common focus on the rat pituitary gland. In ground-based investigations, one has the luxury of combining several disciplines in trying to comprehend the function of this important gland: biochemistry, biophysics, physiology, and histology. In space experimentation, it cult to follow such a multidisciplinary approach due to ( I ) limited opportunities for the necessary laboratory work, (2) limited space in which to do it, and (3) limited access to flight hardware (which sometimes does not even exist). The database concerning both pituitary cell electrophoresis and pituitary cell biology “coevolved” between 1983 and 1994 and originated from a single team of investigators. Obviously this commonality affected experimental design, methodology, and rationale. The data thus obtained are combined in the model presented in Figure 9. This model combines the findings for the Prl aiid GH systems, which were studied together because of their interrelationship. It attempts to offer an explanation for the large change in GH cell EPM in microgravity when culture media are changed with a frequency that is often used in pituitary cell cultivation on Earth. Bearing in mind that the data relating to GH-specific fluorescence staining intensity and laser light scatter by GH cells result from one pass of 10,000 cells through a flow cytometer, it appears that the simple procedure of changing culture media in microgravity can affect the biological state of the GH cell, as indicated in Figure 9. We suggest that, in microgravity, GH cells after medium change are exposed to more Prl than the ground control cells, which could negatively affect total bGH release in space. On the other hand, failure to change culture media over 14 days in microgravity seems to have the opposite effect. Since more than 80% of intracellular GH is associated with secretory granules, it seems likely that massive (on a subcellular scale) granule movements occur in microgravity, as are noticed in other cells after the stress of heat and shear. These changes in GH cells have been seen in more than one spaceflight experiment. One question remaining is whether they reflect changes in the GH cells in rats during spaceflight. Another question is whether these changes can be related to alterations of the net surface charge density of the isolated GH cells. Living cells are active metabolic units that constantly transport nutrients, ions, and metabolites into and out of the cell. Albre~ht-Buehler~~ suggested that this continuous transport can affect the density of the immediately surrounding medium. At normal gravity, microconvective currents remove metabolites and carry fresh nutrients to cells in a slow process that must operate for hours and days to be effective. This process stops in microgravity, resulting in cells “getting stuck in their own dirty bath water”. Early changes to be expected, according to Albre-

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cht-Buehler, concern the cell membrane potential and the composition of the glycocalyx, a glycoprotein layer on the outside of the cell membrane. The net result or various intracellular changes, which he assumes and which are in many ways directly related to what has been found experimentally in GH cells, would result in a “change in choice of the membrane state”.h9 In future experiments, it would seem logical to test the effect of the number of cells in the culture vessels on cell-to-cell contacts, and hormone secretion, and the effect of the frequency of medium changes on the electrophoretic mobility of the cells. Such experiments might profitably involve cell dissociation of intact tissue prepared from the animal in microgravity followed by immediate electrophoretic separation. In addition, it would be desirable to study the dynamics of fluid flow around and between pituitary cells in situ in the living animal in microgravity. The resulting information may then serve as a baseline for additional in vitro studies in microgravity. These studies would be useful for the space countermeasures program as well as for the space bioprocessing program on the international space station. C.

United States Commercial Electrophoresis Program in Space

The U. S. has played a leading role in space electrophoresis for a long time, as pointed out by M o r r i ~ o n However, .~ after the McDonnell Douglas Corporation terminated its program (including flight experiments) in 1988, a hiatus occurred and lasted until the IML-2 flight in 1994. Since the commercial biotechnology efforts at Penn State University were focused on electrophoresis in microgravity and were supported by NASA, the entire McDonnell Douglas effort was transferred in 1991 to the University’s Center for Cell Research, which is dedicated to stimulating the commercialization of space biotechnology. At the Center for Cell Research, several goals were set for the United States Commercial Electrophoresis Program in Space (USCEPS). First came the design, fabrication, ground testing and eventual spaceflight of the device for continuous flow electrophoresis, code-named USCEPS. This new unit, shown in Figure 10, incorporates some of the features of the older McDonnell Douglas hardware (CFES) but also some significant improvements. Next, Penn State scientists set out to develop a unique product application for USCEPS and additional applications in the bioprocessinghioseparation field for ground-based USCEPS units. Design of USCEPS

Specifications of the USCEPS device are shown in Table 6; a complete description of the device is found in U S . patent 5,562,812. Some of its characteristics are as follows:

1. it is rugged and designed to fit in a SpaceHab single rack

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JOHANN BAUER et al.

USCEPS electrophoretic device. A. entire unit; B. output collection canister with total collection capability of 594 chambers; C . crosssectional view of canister. Figure 10.

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2. it can operate in sterile fashion 3. it can operate in semiautomatic or manual mode 4. it can accommodate a variety of test samples (macromolecules, subcellular particles, and cells) 5 . it can be readily modified to accommodate a variety of configurations and space platforms. A ground device is located permanently at Penn State with separation chambers identical to those in the flight unit, which so far has not yet been tested in microgravity. As contamination of samples and buffer media has been a frequent problem in earlier space electrophoresis devices, USCEPS was designed to permit easy, partial disassembly and sterilization at autoclave temperatures (120 "C). Since polycarbonate material is subject to surface cracking at this temperature, the USCEPS separation chamber is made of polysulfone, which can withstand such temperatures. Furthermore, the chamber in the ground unit can easily be converted by insertion of a spacer to a flight chamber with three discreet chamber depths (1.5, 3.0, and 4.5 mm). This means that the performance of the unit can be tested on the ground with various chamber depths before sending the same chamber on a spaceflight. In this way, the benefits of space processing can be unequivocally established. Another unique feature of the USCEPS unit is its hexagonal fraction collection system composed of 6 collection canisters, 1 for each of the 6 samples to be processed in a single run (Figures 10B and IOC). One collection canister consists of 99 chambers, making a total of 594 chambers. Each chamber can hold up to 7 ml Table 6. Physical Dimensions Total unit: Height: 190 cm Width: 44.45 cm Depth: 84.85 cm Weight: 400 lbs (est.) Cooling chamber: Height: 88.9 cm Width: 12.7 cm 1.1 cm Depth: Separation chamber: Height: 101.6cm Width: 12.7 cm 1.54.5 mm Depth: Notes:

USCEPS Specifications Power Requirements

Flow rate

Peak: 600 W (DC) Total Power = 3.6 kW/hr

Recycled coolant flow cooling to 20 "C with 300 W air + 300 W water

1200 ml/min coolant flow

25-50 V/cm

100 ml/min

Operational temperature: 15-32 "C; Operational tluid volume: <25 liters Samples: 6 (individual) x 25 ml (each), processing rate of about 1 ml/min

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of sampIe. The canister contains 33 ‘pie slice’ chambers (seen in cross section in Figure 1 0 0 . Delivery of processed samples from each of the 99 outlets into these ‘pie slices’ is accomplished by a motorized unit, which drives each outlet needle through a plenum, into the chamber. During a parabolic flight test this collection system was found to perform flawlessly. Application of USCEPS

In U.S. Patent Number 5,562,812, the isolation of a 4-kD peptide from the human pituitary gland is described with electrophoresis in USCEPS as a part of the separation strategy. This peptide promotes bone growth in the hypophysectomized rat. Its high anodal electrophoretic mobility was the key feature that enabled its initial discovery. Since this material appears to be present in minute quantities, the well-documented throughput advantage offered by preparative free flow electrophoresis in microgravity may be very useful for its isolation. A number of mammalian cell types and polystyrene latex beads as test particles are being studied in USCEPS on the ground. D. Octopus Continuous Flow Electrophoresis Device with Segmented Chamber Fluids Ion Permeable Membranes and Segmented Media

The microgravity electrophoresis experiments to date have confirmed that gravity-related problems can be eliminated in space and that instead electrohydrodynamic and electroosmotic effects become dominant factors in distorting the sample streams. In recent years, a free flow electrophoresis chamber, called Octopus, has been constructed in which the latter two effects can be considerably reduced.71 Moreover, it can be adjusted within minutes to many different separation conditions with regard to buffer systems, electric field strength, residence times, and operational modes such as zone electrophoresis, isotachophoresis (ITP), and isoelectric focusing (IEF). In order to avoid disturbance of laminar flow by accumulation of buffer components within the chamber near the membranes, ion-impermeable or semipermeable membranes have been replaced by cellulose acetate membranes, which exhibit a low degree of ion selectivity in either direction. The novel membranes allow electrolytic products inside the electrode compartments (usually H+ and OH-) to be transported across these membranes by the electric field. This may deteriorate the operational conditions by changing pH and conductivity, and ion depletion zones may arise near the cathodic membranes. In order to eliminate these detrimental effects, a chamber front plate was designed with several fluid inlets at the bottom through which different buffers can be pumped into the chamber. The medium flow in the chamber is thus divided into various segmental

counterflow I 96 outlets for fractions

anodal membranm

cathodal 'membrane

-

-

countemow 96 outlets for fractions I

anodal membrar

cathodal

I 'membrane __

'C

-

I,

electrode

electrode I

margin buffer

\ -

sample separation medium

margin buffer

4 444! I 1 marain buffer

1 1 "

separation medium

sample + spacer spacer 2

Figure 1 7 . Octopus electrophoretic device: Principle of electrophoresis chamber with a segmented medium system. Left: arrangement for zone electrophoresis and isoelectric focusing. Right: arrangement for isotachophoresis.

Table 7. Examples of Segmented Buffer Systems Used in Continuous Flow Electrophoresis CenfralBuffer 1. zone electrophoresis a. cclls 10 mM TEA, acetic acid 2 mM Na-acetate, pH=7.2 50 mM NaC1, 180 mM sucr. 1. zone electrophoresis b. proteins 50 mM MOPS, SO mM GABA 0.2% HPMC, pH=5.6

N

0 P

2. IEF 0.5% Servalyt carrier ampholytes 0.2% HPMC pH 3-5 3. ITP Leading: 10 mM imidazole, 10 mM HCI, 1 mM KCl, 2 mM NaCI, 280 mM sucrose

Margin Bufferrs)

Anodic Buffer

50 mM TEA, acetic acid 20 mM Na-acetate, pH 7.2 250 mM NaCl, 126 mM sucr.

50 mM TEA, acetic acid 20 mM Na-acetate, pH 7.2 250 mM Na,S03

anodal: 50 mM acetic acid 50 mM GABA, 0.2% HPMC cathodal: SO mM Tris, 50 mM MOPS, 0.2% HPMC

50 mM acetic acid 50 mM GABA

anodal:

0.1 mM H3P0, 0.2% HPMC 0.1 mM NaOH 0.2% HPMC

0.1 mM H3P0,

100 mM HCI, 100 mM imidarole, 20 mM NaCI, 10 mM KCI 230 mM sucrose

100 mM HCI, 100 mM imidazole 20 mM NaC1, 10 mM KCI

cathodal:

anodal:

Carhodic Buffer

50 mM TEA, acetic acid, 20 mM Na-acetate, 250 mM NaC1, pH 1.2

50 mM Tris 50 mM MOPS

0.1 mM NaOH

I : 10 mM imiduole, 10 mM glutamic acid, 1 mM KCI, 2 mM NaC1.280 mM sucrose Spacer 2 : 10 mM imidazole, 10 mM MES, 1 mM KC1, 2 mM NaC1,280 mM sucrose Spacer 3: 10 mM imidazole, 10 mM MOPS, 1 mM KC1, 2 mM NaCI, 280 mM sucrose Terminating: cathodal 100 mM HEPES, 100 mM glycine 100 mM HEPES, 100 mM imidazole, 100 mM imidazole 100 mM imidarole 220 mM sucrose 220 mM sucrose Notes: HEPES, N-(2-hydroxyethyl)-piperazine-2'-(2-ethanesu1fonicacid); MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; TEA = triethanolamine: HPMC = hydroxypropylmethylcellulose

Spa&

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205

flow^.^' If only one pump is used to drive the buffers through the inlets, all buffers move at equal speed and are forced to flow adjacent to each other between the glas? plates to the top of the chamber. When isoelectric focusing or zone electrophoresis is performed, a central buffer containing the specimen flows between two margin buffers, which contact the membranes and protect the sample against electrode effects (Figure 1 1, left side). When isotachophoresis is performed, the leading buffer, several spacers, and the terminating buffer flow between the margin buffers (Figure1 l, right side). The central media between the margin buffers, which usually contain the specimen to be separated, are composed in such a way as to allow optimal separation as well as optimal conservation of the specimen. The two margin buffers. which flow past the membranes, are adjusted to the electrode fluid and also to the central buffer by an increased ion content. When possible, the pH of the medium is stabilized by chemical compounds, as described by Bier and colleague^.^^ This provides a high buffering capacity with low levels of charged compounds, giving low conductivity, and in turn high voltage at low electrical current and thus low heat development. The use of membranes with high ion exchange capacity requires that each medium is supplemented by suitable electrode buffers. Cathodic electrode buffers usually contain equal quantities and types of ions, but no uncharged molecules as contained by the margin buffers (Table 7). Anodic electrode buffers contain similar quantities of cations to those of the margin buffers. If high voltages are to be applied for zone electrophoresis, C1- has to be replaced by S20;', because chloride ions are a source of aggresive, corrosive, and poisonous electrolyte products, mainly chlorine. SzO,-' ions inactivate C10- ions, which may be generated when C1- ions from the electrophoresis compartment enter the anodic compartment. Test of Proper Medium Flow Table 7 shows examples of buffer systems for the various modes of electrophoresis. Often margin buffers contain the ionic components of the respective central media at elevated concentrations while the nonionic components are partially or totally removed. The increased concentration of the ionic component and partial or total removal of nonionic components (1) permit adjusting density and viscosity of the margin buffers to those of the central cell suspension buffer, ( 2 ) generate a sufficient ion reservoir near the membranes, and (3) prevent loss of the biocompatibility of the central sample-containing buffer upon application of electrical current. During an electrophoresis experiment, the temperature of the medium in the fluid curtain is influenced in opposite directions by the electric current and by the cooling capacity of the rear cooling plate. The rear cooling plate has an equal capacity at each point of the electrophoresis chamber but electrical power affects the margin buffers and central cell suspension buffer differently. Consequently, the temperature is not the same in the central and margin buffers, and their preadjusted viscosities and densities may change differently.

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IOHANN BAUER et al.

The stability of the contact zones of the various buffers during electric current flow needs to be checked on the ground as well as in microgravity. For this purpose, bromophenol blue and maxilon blue are injected into the central sample buffer. When the dyes enter the respective margin buffers, their migration is retarded because of the high conductivity of the margin buffers resulting in dye accumulation at the contact zones and transport of the dyes toward the sample collection device by buffer flow. Only in the case of laminar fluid flow do the concentrated dyes form a sharp line. Thus they are sensitive indicators of any disturbances within the contact area.73 This test also appears to be suitable for testing the medium flow in microgravity, where density effects are negligible but other factors are dominant. Examples of Application on the Ground

Octopus allows electrophoresis of cells suspended in a medium containing at least 50 mM NaC1.7' Although this is still far below physiologic levels, uncontrolled Ca2+ transmembrane fluxes are significantly lower in the presence of 50 mM NaCl than in normal low ionic-strength buffer^.^' The device is also very suitable for protein electrophoresis in zone mode, because the modifications not only increase its versatility but also open ways to reduce electroosmotic and electrohydrodynamic effects. Electroosmosis is significantly reduced by addition to the medium of hydroxypropylmethylcellulose(HPMC). Electrohydrodynamic effects can be eliminated in zone electrophoresis by changing from a continuous to a discontinuous operation. In the continuous operation, the samples pass through the separation chamber once and the separated streams of analytes are led continuously via outlets to collecting vials. In the discontinuous operation, the samples are injected until the tip of the sample stream reaches the top of the chamber, then the buffer flow is stopped and the electric current is applied. After sufficient separation, the current is switched off and the samples are washed out followed by the next injection. In ground experiments, proteins could be preparatively fractionated in this way,74but discontinuous electrophoresis of cells was not possible due to sedimentation. In microgravity, however, sedimentation does not occur. Thus it would be of great interest to study discontinuous electrophoretic cell fractionation in space and to test whether a similar improvement can be achieved as in protein fractionation. In isoelectric focusing, the analytes are concentrated and focused at their isoelectric point, which creates zones where the separated species have reduced ionization and solubility. Hence, under gravity the throughput is limited because aggregation and precipitation usually occur after proteins are concentrated at high levels, but resolution can be excellent as demonstrated by fractionation of a commercially available amyloglucosidase sample, which shows microheterogeneity with very small differences in PI values. When the protein was dissolved in the central ampholyte medium (Table 7) at a concentration of 1 mg/ml and the sample

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was directly injected at a rate of 1 ml/h into the Octopus separation chamber, all band? seen in an analytical gel could be isolated p r e p a r a t i ~ e l y at ~ ~a) voltage of 1460 V, resulting in 15 mA current at a temperature of 10 "C and a residence time of sample and media in the separation chamber of 23 min. In microgravity, where proteins at concentrations of 25% (w/v) can be subjected to electrophoresis,22isoelectric focusing performed in this way should be even more successful, because throughput will be increased at equal resolution. In isotachophoresis, sample species often accumulate between a leading and a terminating electrolyte with little chance to obtain individual species in separate fractions. This problem was solved by using spacers and creating blanks between the zones of the neighboring analytes. The Good's buffers75 offered a wide choice of suitable spacers. After this modification, two subpopulations of organelles from rat liver cells could be separated.7@The organelles were diluted in a MOPS (3-[N-morpholino]propanesulfonicacid) spacer solution and directly injected with a peristaltic pump at a rate of 5.1 ml/h in the area between the spacers glutamic and MES (2-[N-morpholino]ethanesulfonicacid). The total flow of the media (including sheath liquids) was 220 ml/h and the voltage 762 V, resulting in a 61 -mA current at a temperature of 7 "C. The residence time of sample and media in the separation chamber was 6 min. Adjustment to Microgravity Conditions

The Octopus device seems to meet important requirements for electrophoresis in space since it appears to solve many space relevant biological or biochemical fractionation problems in a single device. Being able to do so in a single device is very useful in view of the lack of space in shuttles and space stations. The Octopus device allows to choose the optimal operation mode and to reduce electroosmotic and electrohydrodynamic effects, and the use of front plates with multiple inlets and segmented buffer systems seems to be advantageous for electrophoresis in space. The system still has to be tested in space. A simple bromophenol blue/maxilon blue contact zone test can reveal whether or how the liquid-liquid multiphase system of the adjacently flowing media can be transferred from gravity to microgravity. This test could be performed even in a sounding rocket, as was done with TEM 06- 13 in the Texus rocket.29 The dye test can establish whether a discontinuous cell electrophoresis experiment in space is feasible. If free flow electrophoresis devices are operated in space with segmented chamber buffers, the combination of the improved resolution, which has been demonstrated in various ground laboratory experiments, with the enhanced throughput possible in microgravity should permit the separation of proteins either by zone electrophoresis, isotachophoresis, or isoelectric focusing. It should also permit the fractionation of particles by zone electrophoresis or isotachophoresis. The high throughput possible in space would then render electrophoresis in microgravity quite profitable.

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V.

CONCLUSIONS AND SUMMARY

Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of “sensing” the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities-quipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface.

REFERENCES Vanderhoff, J.W., Van Oss, C.M. Electrophoretic separation of biological cells in microgravity. In: Electrophoretic Separation Methods, (P.G. Righetti, C.M. van Oss, J.W. Vanderhoff, Eds.), pp. 257-274, Elsevier, Amsterdam, North Holland, 1977. 2. Todd, P. Microgravity cell electropboresis experiments on the space shuttle: A 1984 overview. In: Cell Electrophoresis, (W. Schutt, H. Klinkmann, Eds.), pp.3-19. Walter de Gruyter & Co., Berlin, 1985. 3. Todd, P. Separation physics. In: Progress in Astronautics and Aeronautics: Low Gravity Fluid D-ynarnics and Transport Phenomena, Vol. 130. (J.N. Koster, R.L. Sani, Eds.), pp. 539-672, American Institute of Aeronautics and Astronautics, Washington, DC, 1990. 1.

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Morrison, D.R. Cell Electrophoresis in Microgravity: Past and Future. In: Cell Electrophoresis, (J. Bauer, Ed.), pp 283-313, CRC Press, Inc., Boca Raton, 1994. McKannan, E.C., Krupnick, A.C., Griffin, R.N., McCreight, L.R. Electrophoresis Separation In Space-Apollo 14, National Technical Information Service, Washington, DC, NASA TMX64611, 1971. Snyder, R.S. Electrophoresis Demonstration on Apollo -16, National Technical Information Service, Washington, DC, NASA TMX-64724, 1972. Allen, R.E. et al. Electrophoresis Technology Experiment MA-01 1. In: Apollo-Soyuz Tesr Project Summary Science Report, I , pp. 307-334, National Technical Information Service, Washington, DC, NASA SP-412, 1977. Bier, M., Hinckley, J.O.N., Smolka, A.J.K. Potential use of isotachophoresis in space. In: Protides of the Biological Fluids: 22nd Colloqiuna. (H. Peeters, Ed.), pp.673-678, Pergamon Press, New York, 1975. Allen, R.E. et al. Column electrophoresis on the Apollo-Soyuz Test Project. Separation and Purification Methods, 6: 1-59, 1977. Morrison, D.R., Lewis, M.L. Electrophoresis tests on STS-3 and ground control experiments: A basis for future biological sample selections. In: Proceedings of 33rd Znternational Astronautics Federation, pp. 82-152, Paris, 1982. Snyder, R.S. et. al. Analysis of free-zone electrophoresis of fixed erythrocytes performed in microgravity. Electrophoresis, 6:3-9, 1985. Bier, M. Isoelectric focusing in space and spinoff. In: Proceedings of the AIAMIKI Microgravity Science Symposium, May 13-17, Moscow, USSR, 1991. Rhodes, P.H., Snyder, R.S., Roberts, (3.0.Electrohydrodynamic distortion of sample streams in continuous flow electrophoresis. Journal of Colloid and Interface Science, 129:78-90, 1989 Mitichkin, O.V. et al. Biotechnological experiment Tavriya carried out aboard Salyut-7. In: Scientific Readings on Cosmonautics and Aviation. (U. Gagarin, Ed.), Nauka Press, Moscow, 1984. Avduyevsky, V.S. (Ed.) Manufacturing in Space: Processing Problems and Advances, MIR Publishers, Moscow, 1985. Babsky, V.G., Zhukov, M.Y., Yudovich, V.I. Electrophoresis of biopolymers under low gravity. In: Fluid Mechanics and Heat and Mass Transfer in Low Gravity, Nauka Press, Moscow, 1982. Hannig, K., Wirth, H., Schoen, E. Electrophoresis experiment MA-014. In: Apollo-Soyuz Test Project Summary Science Report, 1, pp.335-352, National Technical Information Service, Washington, DC, NASA SP-412, 1977. Hannig, K., Wirth, H. Free-flow electrophoresis in space. Progress in Astronautics and Aeronautics, 52:411-418, 1977. Snyder, R.S., Rhodes, P.H., Miller, T.Y., Micale, F.J., Mann, R.V., Seaman, G.V.F. Polystyrene latex separations by continuous flow electrophoresis on the Space Shuttle. Separation Science and Technology, 21: 157-185, 1986. Todd, P. The loading and unloading of cells in electrophoretic separation. In: Cell Electrophoresis (J. Bauer, Ed.), pp. 75-101, CRC Press, Boca Rdton, FL., 1994. Clifford, D.W. Commercial prospects for bioprocessing in space, Commercial opportunities. In: Space, Progress in Astronautics & Aeronautics, 110 (F. Shahrokhi, C.C. Chao, K.E. Harwell, Eds.), pp. 177-184, American Institute of Aeronautics & Astronautics, Washington, DC, 1988. Hymer, W.C. et al. Continuous flow electrophoretic separation of proteins and cells from mammalian tissues. Cell Biophysics, 10:61-85, 1987. Morrison, D.R. et al. Electrophoresis separation of kidney and pituitary cells on STS-8, Advances in Space Research, 4:67-76, 1984. Morrison, D.R. et al. Properties of electrophoretic fractions of human embryonic kidney cells separated on Space Shuttle flight STS-8. Advances in Space Research, 4:77-79, 1984.

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25. Todd, P., Kunze, M.E., Williams, K., Morrison, D.R., Lewis, M.L., Barlow, G.H. Morphology of human embryonic kidney cells in culture after spaceflight. The Physiologist, 28(Suppl.): 182184, 1985. 26. Lewis, M.L., Barlow, G.H., Morrison, D.R., Nachtwey, D.S., Fessler, D.L. Plasminogen activator production by human kidney cells separated by continuous flow electrophoresis. Progress in Fibrinulysis, 6: 143-1 46, 1983. 27. Todd, P., Plank, L.D., Kunze, M.E., Lewis, M.L., Morrison, D.R., Barlow, G.H. Electrophoretic separation and analysis of living cells from solid tissues by several methods. Journal of Chromatography, 364:ll-24, 1986. 28. Stewart, R.M., Todd, P., Cole, K.D., Morrison, D.R. Further analyses of human kidney cell populations separated on the Space Shuttle. Advances in Space Research, 12:223-229, 1992. 29. Mang, V. Technical description of the electrophoresis facility for sounding rockets in the Texus program. Applied Microgravity Technology, 252-55, 1989. 30. Hannig, K., Bauer, J. Free flow electrophoresis in space shuttle program (Biotex). Advances in Space Research, 9:91-97, 1989. 31. Hannig, K., Kowalski, M., Klock, G., Zimmermann, U., Mang, V. Free flow electrophoresis under microgravity, evidence for enhanced resolution of cell separation. Electrophoresis, 11:600-604, 1990. 32. Kuroda, M., Tagawa, K., Tanaka, T., Uozumi, M. Separation of biogenic materials by electrophoresis under zero gravity. In: Proceedings of the 7th SL-J Post Flight IWG Six Month Science Report, April 26, NASDA Tsukuba Space Center, 1993. 33. Yamaguchi, T., Nakajima, H., Akiba, T. Separation of animal cellular organella by means of free-flow electrophoresis. In: Proceedings of the 7th SL-J Post Flight IWG Six Month Science Report, April 26, NASDA Tsukuba Space Center, 1993. 34. Akiba, T. Electophoretic Separation of Cellular Materials Under Microgravity Gravity. In: Summary report on the Science Results of IFuwatto’92 Space Experiment, December 6 7 , pp. 134136, NASDA Tsukuba Space Center, 1993. 35. Akiba, T., Nishi, A,, Takaoki, M., Nagaoka, S., Tomita, F. Electophoretic free mobility and viability of microbial cells: A preliminary study in preparation for space experiments. Applied and Theoretical Electrophoresis, 4:65-69, 1994. 36. Bozouklian, H., Sanchez, V., Clifton, M., Marsal, O., Esterle, F. Electrokinetic bioprocessing under microgravity in France as illustrated by space bioseparation: A programme initiated in France and in cooperation with Belgium and Spain. Advances in Space Research, 9:105-114, 1989. 37. Clifton, M.J., De Balmann, H., Sanchez, V., Bleuzen-Mariotte, V., Schoot, B.V. Purification of biological molecules by continuous flow electrophoresis in the second international microgravity laboratory. Journal of Biotechnology, 47:341-352, 1996. 38. Kobayashi. H., Ishii. N., Nagaoka, S. Bioprocessing in microgravity: Free flow electrophoresis of C. elegans DNA. Journal of Biotechnology, 47:367-376, 1996. 39. Okusawa, T., Tsubouchi, K., Haga, R., Ishikawa, K., Hamano, N., Baba, K. Experiments of separating animal cells from culturing solution. In: High Concentration in Microgravity. Summary report on the Science Results of IML-2 Space Experiment, November I , pp. 63-66, NASDA Tsukuba Space Center, 1995. 40. Hymer,W.C. et al. Bioprocessing in microgravity: Applications of continuous flow electrophoresis to rat anterior pituitary particles. Journal qfBiotechnology, 47:353-365, 1996 41. Takaoki, M., Gyotoku, J . Biological cleaning of FFEU. Chromatography, 17:231-242, 1996. (In Japanese.) 42. Plank, L.D. et al. A study of cell electrophoresis as a means of purifying growth hormone secreting cells. Journal of Biochemical Biophysical Methods, 8:275-289, 1983. 43. Snyder, R.S., Rhodes, P.H. Electrophoresis experiments in space. In: Frontiers in Bioprocessing (S.K. Sikdar, M. Bier, P. Todd, Eds.), pp. 245-286, CRC Press, Boca Raton, FL, 1989.

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65. Hymer, W.C., Grindeland, R.E., Salada, T., Nye, P., Grossman, E.J., Lane, P. Experimental modification of rat pituitary growth hormone cell function during and after spaceflight. Journal QfApplied Physiology, 80:955-970, 1996. 66. Hymer, W.C., Salada, T., Avery, L., Grindeland, R.E. Experimental modification of rat pituitary prolactin cell function during and after spaceflight. Journal of Applied Physiology, 80:971-980, 1996. 67. Wilfinger, W.W., Davis, J.A., Augustine, E.C., Hymer, W.C. The effects of culture conditions on prolactin and growth hormone production by rat anterior pituitary cells. Endocrinology, 105:530-536, 1979. 68. Hatfield, J.M.,Hymer, W.C. Flow cytometric analysis and sorting of live male rat anterior pituitary cell types by forward angle and perpendicular light scatter. Endocrinology, 119:26702682, 1986. 69. Albrecht-Buehler, G. Possible mechanisms of indirect gravity sensing by cells. ASGSB Bulletin, 4:25-34, 1991. 70. Weber, G., Bocek, P. Optimized continuous flow electrophoresis. Electrophoresis, 17:19061910, 1996. 71. Bondy, B., Bauer, J., Seuffert, I., Weber, G. Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis. Electrophoresis, 16:92-97, 1995. 12. Bier, M., Ostrem, J., Marquez, R.B. A new buffering system and its use in electrophoresis and isoelectric focusing. Electrophoresis, 14:1011-1018, 1993. 13. Bauer, J., Weber, G. Sodium chloride in preparative free-flow cell electrophoresis: Recent developments. Electrophoresis, 17526-528, 1996. 74. Bauer, J., Weber, G. Interval carrier free electrophoresis for high resolution protein purification. Journal ofDispersion Science and Technology, 19:937-950, 1998. 75. Good, N.E., Izawa, S. Hydrogen ion buffers. Methods in Enzymology, 24:53-68, 1972.

Chapter 7

TEACHING OF SPACE LIFE SCIENCES

Didier A. Schmitt, Pierre Francon, and Peter H.U. Lee I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 11. SpaceBiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 218 A. Cellular and Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 B. Developmental Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 C. . . . . . . . . . . 219 D. Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 E. . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 F, . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 G. 111. Space Physiology. A. Cardiovascular and Circulatory Function . . . . . . . . . . . . . . . . B. Respiratory Function. . . . . . . . . . . . . . . . . . . . . . . . . 221 C. Musculoskeletal System . . . ........................... 221 D. Neuroscience . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 E. Endocrinolog tory Physiology. . . . . . . . . . . . . . 222 F. Immunology, Hematology, and Microbiology. . . . . . . . . . . . . . . . . . . . . . . 222 G. Animal Physiology and Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 IV. Problems Encountered in Space Life Sciences Research . . . . . . . . A. Access to Spaceflight and to Microgravity . . . . . . . . . . . . . . . B. Control Experiments . . . . . . . . . . . . . . . . Advances in Space Biology and Medicine, Volume 7, pages 213-245. Copyright 0 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0393-X

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C. Spaceflight Simulation . . . . . . . . . . . . . . . . . . . . . . D. Medical Care. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 E. Psychology and Performance . . . . . . . . . . . . . . . . . . . . . . . 230 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 . . . . . . . . . . . . . .233 A. International Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 B. Intercultural Issues . . . . . . . . . . . . . . . . . . . . . . . . . C. Interdisciplinary Issue . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 V1. Tele-Education as a Teaching Tool. . . . . . . . . . . . . . . . . . . . . . . V. International, Intercultural, and Interdisciplinary Issues

B. Tele-Education Systems . . . . . . . . . . . . . . . . . . . . . C. Tele-Education Applied to Space Life Sciences. . . . . . . . . . . . . . . . . . . . . ,241 VII. Existing Teaching Programs . . . . . . . . . . . . . . . . . . . . . . . . . 241 A. NASA-Sponsored Programs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,241 B. Universities and Institutions . . . . . . . . . . . . . . . . . . . . . . 242 VIII. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,243

I.

INTRODUCTION

The first airplane flight by Clement Ader in 1897 marked the beginning of modern aeronautics. As plane flights increased, both in duration and altitude, some medical concerns appeared. Ultimately, a new discipline emerged-aeronautic medicine and physiology. The teaching of this discipline to physicians and investigators is now well established. The next frontier, outer space, was reached by humans in 1961. However, the first conference on space medicine was held as early as in 1948, and in the same year the first animal flew on a rocket. Space medical research was first focused on the health of the astronauts and cosmonauts, as was the early research in space biology. The driving force was actually the challenge given by President J.F. Kennedy to “bring a man on the Moon and return him safely back to Earth”. As the duration of spaceflights increased, more research could be performed. In the last two decades, a fair amount of data have been gathered in fundamental space life sciences. In the near future the International Space Station may be expected to accelerate the pace at which knowledge in this field is accumulated. Consequently, this area will no longer be restricted to a few specialists educated in their respective research fields and thus the already existing need for a specific education in space life sciences will increase. Space life sciences teaching is probably more challenging than it was for any other newly emerging discipline. Firstly, this is due to the fact that many different subjects such as cell biology, human, animal, and plant physiology, and medicine must be addressed. Secondly, space not only implies microgravity, but also radiation and vacuum, which have their distinct effects on cells and organisms.

Teaching of Space Life Sciences

Table 1. Biological Materials Science Molecular and Cellular Biology

Developmental and Reproductive Biology Plant Biology

Cardiopulmonary Physiology

Musculoskeletal Physiology Neurosciences

Regulatory Physiology

Behavior, Performance, and Human Factors Medical Support Systems Life Support Systems

Environmental Health

Radiation Health

Exobiology Biospherics Research

21 5

Research Areas as defined by the International Space Life Sciences Working Group Concerns space research related to both macro-molecular crystal growth and separation processes related to biological materials. Examines how gravity and other space flight factors influences biological function at the molecular and cellular level, including the identification of how single cells “sense” gravity, how this information is translated into biological responses, and how cells respond to both acute and longterm variations in gravity and other flight factors. Focuses on influence of gravity and other flight factors on reproduction, genetic integrity, differentiation, growth, development life span, senescence, and subsequent generations of animals. Concerns effects of gravity and other flight factors on growth, development, reproduction, movement, and orientation of plants and on the underlying mechanisms responsible for these effects. Examines acute and long-term cardiovascular and pulmonary adaptation to space flight and subsequent readaptation to the normal environment of Earth, including all associated underlying mechanisms of action. Focuses on responses and consequences of muscle and skeletal adaptation to space flight and subsequent readaptation to normal Earth environment, including associated underlying mechanisms of action. Concerns the acute and long-term adaptation of the central and peripheral nervous system to space flight and the subsequent readaptation to the normal environment of Earth, including all associated underlying mechanisms of action. Examines following areas of acute and long-term adaptation of animals to space flight and subsequent readaptation to normal Earth environment: circadian rhythms, endocrinology, fluid/electrolyte regulation, hematology, immunology, metabolism and nutrition, and temperature regulation. Examines basic mechanisms underlying behavioral adaptation to space flight, measurement and interpretation of performance during space flight, and factors influencing capabilities and limitations of crewmembers on space missions of varying duration. Focuses on providing preventive, diagnostic, and therapeutic capabilities for space flight operations and includes both countermeasure development and clinical studies. Concerns integration of biological, physical, and chemical processes to promote self-sufficiency in life support by improving regeneration of air, water, and food, by managing and recycling metabolic and other wastes to achieve optimum resource recovery and use. Examines effects of spacecraft environment on humans and other organisms in order to manage those environments properly to minimize risks associated with living and working in space: incl. barophysiology, microbiology, toxicology, and environmental monitoring. Focuses on developing scientific basis for radiation protection of humans engaged in space exploration, with particular emphasis on establishing firm knowledge base to support risk assessment for future long-term missions. Concerns research related to processes involved in origin, evolution, and distribution of life in the universe. Examines ways to measure and predict biological changes on Earth on regional and global scale due to changes in atmosphere and biosphere.

9 LZ

Figure 7.

The Various Fields Covered by Space Life Sciences

Thirdly, flight constraints and the need for developing specific hardware make it difficult to conduct space experiments. Finally, knowledge of space life sciences is not only required for life scientists involved in spaceflight, but also for the engineers who will build and operate the International Space Station and future space colonies so that these facilities will be as efficient and safe as possible. The three primary thrusts of space life sciences have been defined as “to develop an extended understanding of living systems and ecology in the universe, to provide the scientific foundation and technological support systems to enable safe and productive human space activity, and to use the knowledge acquired in the pursuit of space life sciences to improve the quality of life on Earth.” In fact, this definition does not encompass fundamental research in biology or physiology. A simpler definition would be this : space life sciences is the area of life sci-

Teaching of Space Life Sciences

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ence research and technology concerned with the interaction of living systems and bioprocesses with the space environment. Therefore, space life sciences include the adaptation of living organisms to spaceflight and extraterrestrial environments, the origin, evolution, and distribution of potential life outside of the Earth, the medical support for manned mission, and the scientific and technical aspects of human presence beyond Earth orbit (Table 1, Figure 1). A historical survey of the development of the field of space life sciences must, of course, include the history of human spaceflight, considering the three major phases in manned space exploration: adventure (1960-1970), learning (19701985), and exploitation (1985-). However, when considering the first human spaceflights, we should not forget the animal flights in sounding rockets starting in 1948 with monkeys and rats on modified V-2 rockets leading to the first animal in orbit in 1957. It should also be mentioned that, contrary to aeronautic medicine, space medicine was created in 1948 well before the first human flights and that the predictions for the survival of humans in space was very pessimistic. However, the important point to discuss might be the question “Why humans in space?” In recent times such questions have been raised with regard to the development of rocketry and propulsion in the 1920s and 1930s, war in the 1940s, and the Cold War and nationalism in the 1950s and 1960s. However, this is probably only part of the picture, the other part being the fundamental attraction of humans for discovery and escape from the planet. Evidence for this is already noticeable in the Babylonian and Chinese civilizations 2000 years ago, where beliefs, dreams, mythology, and even religion were in many ways space-related. Fiction with all kinds of ways to escape the earth written between 1600 B.C. and 1600 A.D. and current science fiction can also be considered a part of the history of human space exploration. This paper will review all aspects of space life sciences that should be addressed by those teaching in this field as well as provide a structure for doing this. In addition, we review the interdisciplinary, intercultural, and international aspects of space life sciences and consider the use of tele-education.

II. SPACE BIOLOGY Space biology must first address the question of whether and how cells, plants, and animals are affected by changes in the gravity vector, which we may call gravitational biology that is, in turn, subdivided into cellular and molecular biology, developmental biology, plant biology, multicellular organisms, and the application in space biotechnology. However, the space environment is also characterized by high levels of radiation and some other factors, which will be discussed in the last two subsections of this section.

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A.

Cellular and Molecular Biology

The field of space cell biology addresses mainly the question of how cells respond to the absence of gravity with respect to their proliferation, differentiation, and function. Gravitational biology must explain the adaptation to hypergravity as well as to microgravity. Hypergravity is easily understood, since every cell biologist has used centrifugation for cell washings or fractionations. However, the concept of microgravity, as in freefall, is more difficult to understand, which is also true for the concept of “quality” of microgravity (it can actually range from to10-6 G). It is therefore very important that all physical consequences of microgravity be described, not only the absence of a counterforce but also the absence of concentration and thermally driven convection, hydrostatic pressure, and the presence of Marangoni convection. In fact, gravitational biologists must have an understanding of the fundamentals of fluid physics in microgravity. At the cellular level, changes in signal transduction from the cell membrane to the nucleus, cell-cell and cell-surface contacts must be particularly emphasized. Hypergravity and microgravity affect isolated cells, but whether microgravity affects cell mechanisms directly or indirectly is not known. To avoid confusion about microgravity effects, it must be made clear that, in freefall, the liquid environment of a cell is in microgravity, but the interior of a small cell (10-20 pm diam.), due to cytoskeletal movements and active metabolism, is not a true microgravity environment. The different cell types that have been flown in space are bacteria, protozoa, isolated animal cells and plant protoplasts. Cell biology should also encompass the function and organization of tissues, both of which have been studied in microgravity. Cells are known to adapt to changes in pH, temperature, osmolarity, and in some cases stretch forces, vibrations, hydrostatic pressure, fluid sheer stress, and light intensity. The mechanisms of these adaptations should be understood to some extent before looking into gravitational biology. The main difference between gravity and the factors mentioned above is that gravity has never changed during the history of life on Earth. A special case in cell biology is presented by “multinucleated” organisms such as the acellular slime molds. Changes in cytoplasmic streaming as an indirect consequence of microgravity due to their size (several cm up to one meter) should be mentioned. The biochemical mechanisms of gravitaxis in specialized unicellular organisms like protozoa and algae, are of special interest because microgravity research has shown that gravitaxis is different from geotaxis. Gravitaxis has also been studied during space flight in small organisms such as jellyfish. This field, although usually addressed in space biology, can better be discussed under animal physiology and biology (section 1II.G.).

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Teaching of Space Life Sciences

B.

Developmental Biology

Developmental biology encompasses areas ranging from fertilization to postnatal development. Spaceflight data are essentially focused on fertilization and embryogenesis. Two situations must be considered: (1) fertilization and embryogenesis of invertebrates and amphibians, which is the simplest model studied, deal directly with the effects of gravity on single cells, cell proliferation and differentiation, tissue development, self-organization, and organ formation; (2) mammalian development, which is different in the sense that fertilization and embryogenesis occur in vivo, and physiological changes in the mother due to spaceflight will affect this process. Mammalian development, including postnatal development, should therefore be taught after space physiology. C.

Plant Physiology

Two important aspects in plant biology in space are gravitropism, and the plant growth and reproduction cycle. In a I -G environment, the positive gravitropism of the roots must be paralleled by the negative gravitropism of the shoots. In this context, other environmental factors influencing plants can be described, namely, phototropism, plagiotropism, and the factors that induce circadian rhythms in plants. Plant reproduction and formation of new seeds during long-duration spaceflights have been described. Abnormalities in various plant organs observed in these experiments must be discussed in the context of the controversy about artifactual effects, especially environmental factors (see section 1I.H.). D.

Biotechnology

Biotechnology in space was an important topic in the early 1980s. However, production of pharmaceuticals in a space station no longer appears to be a useful research topic, due to the difficulties of space experimentation as well as to the rapid technological advances on the ground, for example, in protein production (by cell cloning) and in protein separation by electrophoresis. (However, see chapter 6 on space electrophoresis by Bauer and colleagues). Protein crystal growth, another topic on the borderline between physical sciences and life sciences, is still a promising area. This field should be explained in the context of the fluid and material sciences under microgravity. New topics of interest, such as tissue engineering, will probably emerge in the near future. E.

Radiation Biology

The diversity in the electromagnetic spectrum (high energy-ionized particles, X-rays, gamma rays) and in the origin of space radiation (primary cosmic radiation, geomagnetically trapped particles, and solar flare events) in low Earth orbit

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should first be explained. The various units (rem, rads, Sieverts) and terms (radiation dose, dose equivalent, etc.) and the ways to quantify radiation doses (dosimetry) should also be described. Radiodosimetry will determine radiation both qualitatively and quantitatively by active and passive methods. Radiation biology should be taught in parallel with radiodosimetry to show the kind of damage caused by different types of radiation. Radiation-induced alterations can be divided into molecular, cellular, and tissue effects, and both short- and long-duration effects must be addressed. The major interest in radiation biology is the effect on DNA stability. This field must be emphasized on the cellular level as well as on the organismal level (gametogenesis and embryonic development). However, one must keep in mind that low doses of radiation might also affect other cellular mechanisms, as has been shown in recent ground-based research. It must be noted that the effect of radiation on biological material can be studied inside a spacecraft but also by exposing biological samples (spores, seeds) outside the spacecraft. In any case, the effects of microgravity must be evaluated and also the possible synergism between radiation and microgravity, despite earlier negative findings. The section on radiobiology can also be extended to radiation health (see section IV.D., “Medical Care”). F.

Exobiology

This is the study of the appearance, evolution, and distribution of life in the universe. It therefore involves, among other subjects, the detection of organic molecules in meteorites or in collected cosmic dust, and more recently detection of potential primitive life forms in meteorites coming from other planets. Exobiology can also be extended to the study of the atmosphere of other planets (e.g.,Titan), to remote detection of extraterrestrial organic molecules (particularly aminoacids), and to the study of the effects of ultraviolet radiation, high vacuum, and temperature extremes on organic molecules and living organisms. The findings of exobiology can have a bearing on prebiotic evolution on Earth. C.

Other Spaceflight Factors

In the space environment, microgravity and radiation are the most obvious factors that differ from the Earth environment. However, one should not forget to mention electromagnetic field changes in low Earth orbit. Not much is known yet about the effects of weak electromagnetic fields on biological processes, especially in the absence of gravity. However, it is known that strong electrical fields can induce root bending against gravity. In addition, there are the effects of extreme vacuum, ultraviolet radiation, and low temperature in outer space, which are not studied in space biology, except in exobiology.

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111.

SPACE PHYSIOLOGY

Before dealing with space-related changes of the different physiological functions it is obviously necessary first to explain the normal function and structures and their adaptation to the Earth environment. The microgravity effects on these systems must, when possible, be distinguished from other effects of spaceflight like stress, lighting, and sleep deprivation. The effect of pressure changes during extravehicular activity (EVA) can also be addressed by reviewing the hypobaric effects on relevant physiological systems. Ethical aspects of human research should also be discussed. A listing of the physiological functions with a summary of the main spaceflight effects follows. In teaching, the many links between these physiological functions should be indicated.

A.

. .. .. . 0

0

Cardiovascular and Circulatory Function

Fluid shifts: early changes in central venous pressure; changes in heart load condition Cardiac muscle deconditioning and changes in cardiac mass Arterial and cardiopulmonary baroreflex deconditioning Changes in venous tone and compliance, and general loss of control of vasomotoric function Capillary blood flow changes Changes in neurohormonal regulatory systems related to the cardiovascular system Interstitial fluid dynamic changes Postflight loading condition and the various factors involved in orthostatic intolerancelhypertension

B. Respiratory Function 0

. . 0

0

Changes in ventilation-perfusion ratio Thoracic wall movements Changes in pericardial and intrathoracic pressure Endocrine changes involved in blood volume regulation Blood redistribution in the lungs C.

. 0

0

0

Musculoskeletal System

Changes in muscle fiber structure and contraction properties Changes in neuromotor control Changes in tendons, muscle insertion, and cartilage structure Bone mineral loss

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Changes in the equilibrium between skeletal build-up and breakdown (bone remodeling) Disturbance of Ca2+ homeostasis Hormonal changes in relation to bone metabolism D. Neuroscience

Visual-vestibular and vestibulo-ocular reflex changes Visual and motion illusions Changes in cognitive processes Space motion sickness: origin and treatment Changes in posture and movement (proprioceptive modifications) Changes in taste and olfaction E.

Endocrinology, Metabolism, and Regulatory Physiology

Changes in energy expenditure Modification of hormone levels, particularly stress hormone Nutritional problems Changes in sleep regulation and biorhythms (can be included under this section, but can also be discussed in the neuroscience section.) Endocrine changes in bone metabolism and cardiovascular-fluid regulation should be included in the discussion of these topics.

F.

immunology, Hematology, and Microbiology

Topics to be discussed include humoral responses, cellular responses (cell proliferatim, cytotoxicity), space anemia (origins and implications), wound healing, and microbial contamination. The immune system has numerous interactions with other systems such as the bone/mineral regulatory system, neuroendocrine/stress hormones, general metabolism, and even the cardiovascular system. As a consequence, some of the changes found after spaceflight may be due to landing effects, like changes in the levels of stress hormone and other hormones. The immune system offers a good example of the interaction of physiological systems and should be emphasized as such to draw the attention to the impact that one system may have on another. The immune system should also be mentioned under “radiation health” in view of the increased risk of cancer from radiation, especially on missions to the Moon and Mars. Since hematology is linked to immunology through the white cells, the hematology of white cells should be treated in the immunology section. The effects of an acute exposure to solar flares, especially on bone marrow, should also be discussed in this section. Concerning red blood cells, the most important points to be

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mentioned are space anemia, its origin, and its consequences. Effects of spaceflight on other hematological parameters, such as blood clotting factors, have not bccn well studied. Microbiology can also be taught in conjunction with immunology because of the obvious interactions, but it is also related to hygiene and can therefore fit under “Prevention of Medical Risks” (in section 1V.D. “Medical Care”). However, the microbiological risks can also be mentioned under “Environmental Control and Life Support Systems” (section 1V.G.) or under “radiation biology” (section 1I.E.) in relation to the potential effects of microgravity and radiation on microorganisms. The relevant changes in these various body systems as a consequence of microgravity,\ or of spaceflight in general should also be discussed in the context of the various simulations of weightlessness (“Spaceflight Simulation,” section 1V.C.). The implementation of countermeasures should be explained and their relevance discussed. Potential mission impacts of the physiological changes and the relationship between these changes and the medical aspects of spaceflight can also be highlighted. The field of integrative physiology covers a variety of interconnected research fields such as the influence of nutrition on physiological parameters and even pharmacokinetics. These interesting interdisciplinary aspects should be emphasized because spaceflight and the corresponding ground simulations are well suited for this kind of research. C.

Animal Physiology and Biology

Animal physiology in relation to space research has connections with several subjects, namely, human physiology, reproduction and developmental biology, some aspects of cell biology, and also the history of human space exploration. It is therefore often addressed in these various topics, but it can also be a section by itself. First of all, animals were flown in space before humans were in order to explore the then still unknown and potentially high risks for humans during spaceflight. The earliest example is probably the flight organized by the Montgolfier brothers, who flew animals (sheep, roosters, and ducks) prior to their own maiden balloon flight in 1783. In preparation for human spaceflight, monkeys and rats were flown in sounding rockets in 1948 and a dog on an orbital flight in 1957. Since then, animals have been used for the same reasons as in ground research, (reasons that are even more important in space research) namely, that a larger number of individuals and specimens is available, that biopsies can be obtained more freely (e.g., bone and muscle biopsies in mammals), and that one can work with identical strain, age, conditioning, and housing. Another advantage is that animals, in contrast to crewmembers, are not subjected to countermeasures. A variety of animals have been flown in space for various scientific reasons. One should mention them for historical as well as scientific reasons: monkeys, dogs, cats, rodents, birds, amphibians (frogs) fish, insects (flies, beetles, bees, spiders,

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crickets), sea urchins, jellyfish, and probably others. For the study of developmental biology, not only the adult organism but also the developmental stages of the animals (e.g., tadpoles, larvae, pregnant mammals, and fertilized bird eggs) have been flown. Human research has, for obvious reasons, limitations-particularly in the field of developmental and reproductive biology. The reasons for using animals and animal cells in vitro in this field can be reviewed but are the same as for research on Earth. Use and care of animals for research in space must also be discussed, bearing in mind that space missions and the experiments conducted during them receive much publicity. The ethical aspects of animal research can be discussed either in this section, under human physiology (“Space Physiology”), or in a separate section on the ethics of human and animal research.

IV.

PROBLEMS IN SPACE LIFE SCIENCES RESEARCH

When describing results of spaceflown experiments, as for standard experimentation on ground, one should be able to discuss the results critically in the light of the limitations of space research. For biological and physiological experiments, the possibility of spaceflight artifacts should be considered. Factors to be taken into account are variation in microgravity levels, the simultaneous presence of microgravity and radiation, transportation of samples to and from the spacecraft on the ground, vibration and hypergravity during launch, lowered atmospheric pressure during extravehicular activities, and changes in ambient temperature in the orbital habitat. A.

Access to Spaceflight and to Microgravity

In order to understand the limitations and constraints of spaceflown experiments, the different possibilities of access to space must be reviewed in terms of duration, microgravity quality, orbit inclination, and whether the experiment is attended by the crew or not. The different possibilities are, in order of increasing duration, as follows: on Earth, drop towers and parabolic flights; in space, sounding rockets; and orbit spacecrafts such as biosatellites, space shuttles, and space stations. Each of these has its own specific advantages and limitations. Biological processes can last from milliseconds to years; obviously only those of very short duration (seconds) can be studied in drop towers, parabolic flights, and sounding rockets. The advantage of these techniques are relatively frequent opportunity and low cost. In physiology, neurosensory disorientation and blood redistribution (in lungs and brain) adjust quickly enough to be investigated during parabolic flights. These flights are also quite useful for testing equipment and procedures to be used in life sciences experiments during spaceflight.

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Orbital flights afford the opportunity for the study of both short- and long-lasting phenomena and for the adaptation of organisms to microgravity. However, the microgravity effects can be modified or be obscured by other spaceflight factors mentioned earlier. The distinguished types of orbiting flights are a satellite, a space shuttle, and a space station. Advantages of the latter two are the presence of crew members who can perform the experiments and tend to the animals and cell cultures. In experiment duration the space shuttle is limited to two weeks; orbiting biosatellites (like the Russian Cosmos) permit experiments of slightly longer duration, but lack crew intervention and on-board laboratory facilities. A space station provides all three: crew, laboratory facilities, and, in theory, experiment durations of a year or more. Exobiology research requires very diverse equipment, depending on the topic to be studied: dust collection systems for the study of interstellar dust particles; sun exposure facilities on orbiting spacecraft for studying the effects of solar radiation; radiotelescopes for the study of organic molecules in planet atmospheres; and planetary or cometary material analysis in situ or on Earth after sample collection and return. B.

Control Experiments

In general, control experiments (in which the factor to be studied is not operative, but all other conditions are equal to the experimental conditions) are critical for the interpretation of the results of life science experiments on Earth as well as in space. However, space experimentation poses its own specific problems for the design of a suitable control experiment. Consider the most common type of space experiment, one in which the effect of microgravity on a biological parameter is to be investigated. The most obvious choice is to conduct the control experiment on the ground, where there is constant 1-G gravity. However, the experiment must then be conducted in exactly the same equipment as in space and under the same conditions. Obviously some conditions cannot be parallelled on the ground: launch vibrations and hypergravity, stress to human or animal during launch and upon entering orbital microgravity, radiation during flight, stress and vibrations during reentry and landing. Others, such as temperature course and cabin atmosphere composition, can only be parallelled with considerable difficulty. These problems can be overcome by the use of an inflight 1-G centrifuge. However, first of all, there has to be one, and it must accommodate the same containers with the same provisions (food, water) as used for the stationary experimental specimens. The centrifuge must be large enough to limit the radial gravity gradient. This condition is relatively easily met for cell cultures, as is the case in the Biorack facility, which has been flown six times on space shuttle flights. For the International Space Station, a 2.5-m diameter centrifuge is planned, which will accommodate rats and plants. For humans no provisions have yet been made. An

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additional problem is that for cleaning cages and taking of blood samples the animals must come to microgravity for short periods of time. In human experimentation, the experimental person is commonly used as its own control by comparing the inflight values against prefight and postflight measurements. The limitations of such measurements should be discussed, because they are the most critical points to be taken into account when interpretating the experimental results. Another problem in human experiments is that countermeasures (various forms of exercise) against the effects of microgravity are commonly used by crew members, and can obscure the microgravity effects on various physiological systems. Complications can also result from bad sleep quality, nutritional state, and conflicting experiments being performed simultaneously. Examples of such interactions should be presented. In view of these problems and the relatively limited number of flight opportunities, various spaceflight simulation methods have been developed for humans as well as for animals, plants, and cell cultures. These will be discussed in the next section.

C . Spaceflight Simulation Some aspects of spaceflight can be simulated on Earth by means of various techniques: clinostat, freefall machine, magnetic levitation, head-down suspension, neutral boyancy in water immersion, bed rest, and confinement. It is important that the advantages and disadvantages be well understood to avoid any confusion about what these simulations can offer. Clinostat

This can be used for cell cultures and small plants that are made to rotate around a horizontal axis. The cells are then in freefall through the culture medium, although gravity is still present. Likewise, in whole plants on a clinostat, the amyloplasts in the gravitropic cells are kept from sedimenting, although there is no true microgravity. Clinostats should only be considered for the study of cell-surface interactions and cell-cell interactions, especially for three-dimensional tissue growth. The same principle has been used in bioreactors for cultivating tissues in three dimensions. It must be pointed out that effects in the clinostat are not directly related to microgravity except for one minor aspect, namely, the absence of contact of the sample with the container. In plant biology, slowly rotating clinostats have been used for over a century to study gravitropism, but again this involves only a change in the vector of gravity over time and is not in any way real microgravity. Another device that has been proposed instead of a clinostat is a random positioning machine and here also the same remarks apply.

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Freefall Machine

More recently, a freefall machine has been developed. The principle is to provide a vertical impulse (50 ms) after which the experimental samples can be in real microgravity for about 1 second. If desired, a 1-G control experiment can be performed. The effects of having alternating periods of hypergravity and microgravity is not yet known. Magnetic Levitation

This procedure has been used recently to levitate biological samples and small animals. There are nevertheless important differences between microgravity and magnetic levitation that must be explained in order to avoid confusion. One main difference is that microgravity acts on each molecule proportionally to its mass, but magnetic levitation is proportional to the diamagnetic properties of the molecules and is therefore mainly acting on water. The high magnetic field is another unknown parameter to be considered when using magnetic levitation for simulating microgravity. Head Down Suspension

In animal research the main simulation method is head-down suspension of rodents to study fluid shift, bone unloading, and muscle atrophy. Despite stress occurring as a side effect, this model has proven to be very useful. Bed Rest

Some aspects of spaceflight on humans can be simulated on Earth by means of hypokinesia through bed rest (with or without head-down tilt). This is the most frequently used simulation method for the study of fluid-shift, bone loss, and muscle atrophy in humans. Here again stress must be considered as a likely side effect. Fluid shift has also been studied in water immersion, but other drawbacks exist. Neutral buoyancy training for Extravehicular activity (EVA) can also be mentioned in this section. Isolation and Confinement

An important and unavoidable aspect of spaceflight, quite apart from the effects of microgravity, is the isolation and confinement. Experience from Antarctic wintering and nuclear submarine service has been studied. Special studies have been carried out in a simulated space station (see volumes 3 and 5 in this series on the ESA-sponsored ISEMSI and EXEMSI projects).

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D. Medical Care

The main topics in medical care in a space environment that need to be addressed are prevention of medical risk, diagnosis, treatment, and radiation health . Prevention of Medical Risks

The health and well-being of the crew members during a spaceflight greatly depend on risk assessment so that most risks can be prevented. Risk assessment not only applies to the inflight situation but also to the preflight and, to some extent, the postflight situation. The most effective way to prevent medical problems inflight is, of course, adequate crew selection and training. The physical/ medical requirements for the crew are dependent on the type of mission and the duties to be performed on board. They are therefore different for pilots, mission specialists (with EVA requirements), and payload specialists (scientists, no EVA requirements). They are also different for short- and long-duration flights. Training must also be mentioned because the physician inflight, if there is one, must receive specialized training, and one or two nonmedical crew members must be trained to serve as medical officers. Inflight medical prevention must include risk assessment in the spacecraft, that is actually determining the origin of medically-related problems which can occur. The risks can be subdivided as follows: primary medical risks that would occur independently of the mission, (e.g. cardiovascular accidents) risks due to the space environment: external hazards (micrometeorites); internal hazards (floating objects, microbial contamination); physiological changes as a consequence of microgravity (kidney stone, bone fractures); physiological changes as a consequence of radiation (increased probability of cancer); psychological problems (anxiety, somatic diseases) risks due to accidental events: malfunctioning of the closed environment and life support system (toxic gases, decompression); a solar flare (acute radiation sickness); during EVA (temperature, oxygen, pressure) risks due to specific activities: handling of specific hardware; EVA (cardiac arrhythmia, acute radiation disease due to solar flares) How to prevent risks becomes obvious once the risk assessment has been performed. However, special emphasis may be placed on physical exercise and psychological management to prevent physical and mental deconditioning. Prevention of longterm effects can be accomplished by a postflight rehabilitation program (including psychological rehabilitation in the Russian program), readaptation of the deconditioned body systems, and debriefings.

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Preventive medicine should include the selection criteria for astronauts, health monitoring in flight, and the study of the effects of the environment on human healr'h. This last point is partially addressed in environment control and life support systems involving barotraumatic risks, microbiological contamination, and food and gas toxicology risks. Radiation health in terms of protection and monitoring, also fits into this section. Preventive medicine may include development of countermeasures against microgravity effects as well as some aspects of crew training.

Diagnosis This section should include three types of diagnostic activities: 1. Monitoring the environment, such as the life support system, but also radiation monitoring and tracking of micrometeorites and space objects to prevent risks. 2. Medical monitoring of the crew members on a regular basis is necessary to record their medical status and to detect possible abnormalities at an early stage. The list of required instruments should be reviewed to see which can be shared with the biological and physiological experiments to be carried out during the mission. 3. Describing how the inflight capabilities (hardware) are chosen, which is done by ranking the most likely events that can occur, (e.g., foreign bodies in the eye, decompression-related disorders, etc.). The validity of extrapolating the probability of medical risks from analogous situations can be discussed here. Treatment

Before speaking about the possibilities of medical treatment, the capabilities of the on-board medical facility should be described. This will, of course, depend on the type of spacecraft and mission. The space shuttle carries only a few medical kits (pharmaceutical, surgical, and dental). However, on the International Space Station, in addition to these standard kits, there will be more sophisticated equipment present. There will, for example, be a multimedia telemedicine work station with a telemedicine instrumentation pack. In this section, it is interesting to dwell more on future improvements in the field of miniaturization of biomedical analyzers, monitoring equipment, medical imaging, computer diagnostic aids, fiber optic endoscopy for diagnosis and surgery, and telemedicine (diagnostic assistance, telesurgery). The implementation and improvement of the facilities must be discussed in the context of cost, engi-

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neering challenge, and weight-volume aspects. At this point, confidentiality issues and the need for encrypting information should be mentioned. The possibilities for inflight medical treatment should be described with examples of mild, intermediate, and severe medical problems. Because the medical treatment includes the use of pharmaceuticals, one should not forget to mention that drug metabolism and the pharmacodynamics of drug action may be changed due to the physiological changes in microgravity. It is very important to stress that clinical symptoms are often changed in microgravity (e.g., pulmonary oedema). In this section, attention should also be given to rescue operations during a mission abort. The type of instrumentation in a crew rescue vehicle from a space station is of particular interest. The ground segment of the medical aspects must be mentioned in terms of preflight medical monitoring, mission support during the flight, and preparation for emergency situations during launch and landing abort. Finally, medical care in space should not only be seen as a mission impact issue, but also as an ethical issue (How far can and should we go into medical prevention and care?) and as a social issue (Are medical or technical failures acceptable to public opinion?). Radiation Health

The introduction to radiation health should start with the main aspects of radiation biology (see section 1I.E.).In fact, the origins and physics of radiation and the its effects on the molecular and cellular levels and on the cellular repair mechanisms, must be understood before dealing with the effects of radiation on crew health. Space radiation health concern? the known effects of radiation on the human body as a whole. This can mainly be done by extrapolating the effects of radiation observed in animals on the ground. The field can be divided into two areas: (1) short-duration, acute effects and their treatment and (2) long duration, chronic effects (mainly cancer induction and prediction). One should also stress the lack of knowledge in this field and the potential future for pharmaceutical radiation protection. Space radiation health is essentially the field of radiation dosimetry and mapping, radiation prediction, and finally physical radiation protection by shielding or attenuation. The differences between low earth orbit radiation in an orbiting spacecraft and a mission outside the Van Allen belt, such as a Mars mission, should be emphasized.

E.

Psychology and Performance

These two topics can be grouped together, because they are closely related to each other. Psychological and sociopsychological issues start already during the selection process of astronauts. The “select in” and “select out” criteria can be reviewed as well as the differences between the Russian and the U.S. selection procedures in this respect. The importance of group interactions and the continu-

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ous psychological follow-up during training in the Russian program for longduration flights is also of interest. With the upcoming International Space Station, CI-ewcomposition in terms of gender and multicultural aspects will probably be an interesting topic to cover, especially after the various problems encountered during the long-duration flights of U.S. astronauts together with a Russian crew on board the MIR station. The effects of confinement and isolation on human psychology can be discussed together with spaceflight simulations (see section V.C.), namely, in a space station model. From such studies it has become clear that sociopsychological problems in crew members often result from inadequate training, not only of the crew but also of the ground support teams, and from lack of knowledge about psychological support. Human performance in space can be subdivided into three main areas: (1) human capabilities as determined by the human requirements and technologies needed for optimal efficiency of operation at the human-machine interface; (2) human behavior as addressing the requirements for interaction of humans among themselves. Note that changes in behavior during spaceflight can be mainly ascribed to the confinement and isolation conditions, but it can also be due to some extent to direct and indirect effects of microgravity; (3) human health, which can be addressed separately under medical care (section 1V.D.).

F.

Extravehicular Activities

EVA is probably the ultimate aspect of spaceflight. It is also a case study to implement many of the “pieces” and should therefore be taught after all other matters. EVA is in fact quite multi- and interdisciplinary. It involves the following:

1. physiology in terms of microgravity, hypobaric physiology, radiation health, and medical monitoring 2. the environmental control and life support system in terms of control of temperature and gas composition, and waste management 3. engineering in terms of overall safety, thermal insulation, and shielding against radiation and space debris 4. design and ergonomics for optimization of crew work (mobility and dexterity) 5. training, simulation, and psychological aspects In future exploration missions of the Moon and Mars, extrahabitat activities, the equivalent of extravehicular activities from an orbiting spacecraft, will be an essential part of the mission. The constraints of such activities can be addressed in this section.

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C . Environmental Control and Life Support Systems To ensure viability of the crew, spacecraft and space habitats require active control by means of an environmental control and life support system (ECLSS). This is a many-sided subject, which has many interfaces with space life sciences such as human physiology, operational medicine, psychology, human factors, biotechnology, and plant biology. ECLSS can be used as a good example for the importance of the interdisciplinary aspects in space life sciences. Design and implementation of an ECLSS is a very complex engineering task that also involves internal spacecraft architecture and ergonomics. The fundamentals of ECLSS should be taught to space life scientists without going into details about its technical aspects. The fundamental aspects concern management of atmosphere, water, waste, and food. The management of the atmosphere must include gas composition, detection of toxic gases, and control of pressure, temperature, and humidity. ECLSS should also be extended to the management of noise, vibrations, accelerations, hygiene, crew safety in terms of radiation and micrometeorite shielding, and fire detection and suppression. Since ECLSS is critical for survival and operational performance of the crew. it can also be linked to psychological support and countermeasures. The ECLSS requirements for extravehicular activities should also be addressed in this section. After the general aspects of ECLSS have been explained, one should also show that requirements are different depending on crew size, mission duration, resupply versus total autonomy, power availability, and gravity conditions (planetary versus orbital missions). The constraints of ECLSS must also be understood by space life scientists, so they will understand the potential effects of changes in the environmental parameters on the physiological system they wish to study. Biospherics can also be included in this section, while terraforming on Mars may be mentioned as the ultimate limit of ECLSS.

H. Spin-offs Spin-offs were expected in biotechnology in the 1970s and 1980s in various fields such as the production of specific proteins and their purification. Numerous reasons can be analyzed as to why this sector led to spin-off expectations that have not materialized:

1. industrial production trials before sufficient fundamental research has been carried out 2. lack of anticipation in the progress of technological developments on the ground 3. insufficient private investment in space research

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For a more positive view of the potentiality of space electrophoresis, the reader is referred to chapter 6 by Bauer and colleagues. The future for protein crystal growth may well be more promising, but the technological difficulties and the limited interest of commercial investigators are to be mentioned. The main spin-off from space research is actually taking place in technology transfer to the medical field. This can be illustrated by the following examples: I . non-invasive endoscopic surgery 2. digital imaging and image processing 3. pacemakers and implantable insulin infusion pumps

The subject of future spin-off areas is necessarily speculative and can also be discussed in the areas of space biology, physiology, and medicine. Potential spinoffs in space biology can be envisioned in cancer research by studying the effects of microgravity on cell interactions in three dimensions or by discovering new mechanisms of slowing down cell proliferation, a phenomenon observed in microgravity. In space physiology, there are expectations for ground applications in several pathological conditions such as motion sickness, balance impairment, cardiovascular deconditioning (mainly orthostatic intolerence), and bone loss in the elderly. For technical aspects in medicine, the spin-offs will probably result in the improvement of telemedicine (e.g., telemonitoring of the elderly) in space-driven miniaturization (implants) and use of virtual reality. A new approach is to promote the development of common devices or procedures for space and ground medical applications by using the very demanding space environment as a driver for technological advancement.

V.

INTERNATIONAL, INTERCULTURAL, AND INTERDISC1PLI NARY ISSUES

A common theme that presides over most of the significant space projects today is international cooperation. It is becoming increasingly important and even necessary for countries to work together in order to successfully launch any major space project. Closely associated with the idea of international projects is the issue of interculturalism. Because people from different nations with very diverse cultural backgrounds are now working together on space projects, it is of vital importance that everyone learn to not only understand each other but to respect each other’s differences, whether they be cultural, political, religious, or linguistic. Inherent in any large project is the involvement of people with diverse disciplinary backgrounds. The complex nature of these projects require that engineers, lawyers, medical doctors, scientists of various backgrounds, managers, and financial experts all work together toward a common goal. Each must understand the other so as to promote synergy and avoid conflicts.

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The importance of international, intercultural, and interdisciplinary issues is certainly not unique to the major space projects. In fact, it is a facet of many if not all space life sciences and medical projects. These issues should be understood and appreciated when teaching space life sciences, whether it be to life scientists or non-life scientists. It should also be understood that these three issues are all interconnected and have features that overlap with each other.

A.

International Issues

Even at the height of the Cold War, when the two super powers were competing with each other for space supremacy, the scientific communities from both sides were cooperating with each other. During a time when secrecy was the norm, scientists were taking part in a number of joint missions and programs. One aspect, which has distinguished the scientific community from most others, is that political barriers are often set aside when it is realized that all participants in joint projects benefit from the synergy of having minds from different walks of life working together. Although international programs have been important in many areas of scienctific research, this has been particularly true for space life sciences and medicine. There may be many ways to design and build a launch vehicle or satellite, depending on the country, but the human body and biological specimens react the same way in space regardless from which country they come. Although the approach to studying space life sciences may differ from country to country, the fundamental science behind them is the same. In the early days of manned spaceflight, the primary concern in space life sciences was the health and well-being of the astronauts or cosmonauts, both during and after flight. Results of experiments and anecdotal information from both the United States and the former Soviet Union were published and made available to all investigators for their use. Although it is true that not all information was freely distributed, especially concerning operational aspects, the overwhelming understanding was that scientific data would be shared. An excellent example of an international program in space life sciences is the Russian Bion program, which had its first launch in 1973 and still exists today after a quarter of a century and 12 missions, all of them having been successful. The program involves flights of biosatellites lasting from 5 to 22 days. Specimens that have flown aboard these biosatellites include plants, bacteria, and other cells in culture, insects, and animals. A total of 212 rats and 12 monkeys have been flown on the Bion missions. From its inception, it was decided that the Bion program would have three distinct features, which would always be present: (1) the project would be multiobject, implying that biomedical experiments would be carried out on a variety of plant and animal species; (2) it would be multisubject, referring to studies in a whole range of different biomedical disciplines such as physiology, gravitational biology, biochemistry, and genetics; (3) it would be

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multinational, making the Bion program truly an international endeavor from the start through the participation of western European nations and the USA. Today, the political climate has changed dramatically, but the emphasis on international cooperation and mutual understanding has not only survived, but increased. Many new players have joined the arena and the term “international project” has never been as telling as it is now. The motivation for carrying out international projects may sometimes be driven by political and economic factors, but the advantages from a practical and scientific point of view are numerous. A significant practical advantage is a reduction in cost for each individual participant. By avoiding unnecessary duplication of equipment, personnel, and procedures, each participant can benefit from the contributions of their partners. This is particularly true in physiology where all systems have to be understood as a whole. Additionally, different countries may have expertise in different areas that may positively impact the project. Scientists from different countries may have different ways of approaching a question and conducting research. Although this may appear to be a potential barrier in the beginning, it may in fact be mutually beneficial to learn and understand different ways of looking at the same question. Unfortunately, closely associated with any international endeavor are political and legal issues. Although this is related to some of the interdisciplinary issues that will be discussed later, one must understand the complex legal and logistical issues encountered with any international project. Whenever national borders are crossed, problems shipping equipment or biological samples can be encountered. Together with other issues associated with international projects, this can make them overwhelmingly complicated, and this too will be discussed later. International projects can sometimes be complicated by a certain degree of competition between members from different countries. Although this often arises from political motives at higher levels, it may directly or indirectly affect how individuals of different nationalities work together. Issues that come up include deciding who is in “charge” of the project, whose ideas or experiments take precedence when there is a conflict, how to organize the project, who gets to keep the data or see them first, or who is accountable if something goes wrong. It is important that a framework for resolving these matters be established very early on, so that all participants know exactly what their role and contributions are. B.

Intercultural Issues

Inevitably, in an international project comprised of individuals from different countries, there will be several intercultural issues. People of different cultural backgrounds differ not only with respect to interpersonal differences, but also in ways of conducting research, organizing a project, and managing a team. In addition, religious or political differences and language barriers may exist. All of these issues should be addressed when teaching about life sciences with reference to international projects.

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Interpersonal issues are probably the most significant intercultural issues that may arise. Special attention should be given to such potential problems, since the misunderstandings that may arise can be very harmful to a project. People of different cultures have different ways of interacting with others, especially when rank, seniority, or hierarchy are involved. When negotiating or debating, it should be considered that there may also be differences in methods such that what may be acceptable to some may be seen as offensive by others. The ways of organizing a project or of the management of personnel taking part in a project may also differ considerably from one culture to another. Some cultures adopt structures that are very rigid and hierarchical, while others may be much more informal. Although English is usually adopted as the working language in international projects, many participants may not have English as their first language. This can be a source of problems with regards to communication, leading to frequent and potentially hazardous misunderstandings. Special consideration should, therefore, be made when speaking with those whose first language is not English. Intercultural issues are particularly important for anyone teaching any international group, whether on space life sciences or any topic. Language barriers should also be considered when students and teachers may not understand each other as certain phrases or sayings are used. Differences in accent, interpretation of jokes, and styles of teaching must be taken into account. Classroom “etiquette” also differs with cultures. Some cultures look down upon students asking too many questions during class, while others encourage it. Some find it impolite and even disrespectful to question a teacher or professor, while others appreciate it. When teaching such a diverse group, it is important to be aware of these cultural differences so students are not adversely affected due to misinterpretation or misunderstanding of their behavior. The key to higher efficiency is to be aware of the fact that people of different cultural backgrounds have different waj s of doing things, have different expectations of others, and think and act differently. One must be careful not to assume that others understand one’s way of working and thinking. No one way is the “right” way, and standards may differ widely from one country to another, but as long as all are mutually aware of these differences and respect them, problems that can arise from a lack of this awareness can be avoided.

C.

Interdisciplinary Issues

When teaching space life sciences, one should emphasize the importance of having at least a basic understanding of other disciplines and how they relate to space life sciences. Few disciplines, if any, can exist independently of any others, and this is particularly true for space related disciplines. Research in space life sciences often involves exposing specimens to a microgravity environment or its simulation. This requires that the specimens be placed in hardware that is specially designed for such use. Particularly for hardware that

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must be space-flown, the requirements are often very stringent. The hardware requirements for biological specimens are also determined by the needs of the space life scientist. This is where engineer and space life scientist need to understand each other so that an engineer can grasp why certain requirements are needed and what the acceptable ranges are and the space life scientists can understand the limitations and needs from an engineering point of view. Through this mutual understanding an effective solution can be found. The harsh environment of outer space can have numerous effects on biological systems. Although microgravity is the most commonly observed factor, other factors must not be overlooked. A space life scientist should have a basic understanding of atmospheric or plasma physics as it deals with the elements of the space environment that can affect biological specimens and humans. Topics of concern include solar and terrestrial radiation, the Earth’s magnetic field, and of course, microgravity. Physics can explain many biological and medical phenomena and is necessary for all life scientists, not just those working in space life sciences. A good knowledge of physics will not only help in understanding a given spacerelated phenomenon, but may also help in developing better equipment for space life sciences research in the space environment. Topics of particular interest include the Coriolis forces in fluids, gravitropism in plants, and decreased physical loading effects on the musculoskeletal system. Legal and policy issues are becoming increasingly important for space life scientists. When working on an international project, complex issues in international law and policy may arise. Often, the scientists themselves must be involved in negotiating agreements between countries, necessitating some knowledge of the associated political and legal issues. National policies also have a great impact on the amount and type of research that is conducted in a given country. Support for space life sciences research is neither always stable, nor predictable. Space life scientists should understand the political climate as it relates to space life sciences research. This will allow them to anticipate trends with regard to funding and availability of research opportunities and, for example the use of animals for research. Another issue to be considered is that of intellectual property. Especially when dealing with international groups, the issue of who “owns” the rights to discoveries or findings that may arise from international projects can be very complex. On the other hand, lawyers and policymakers should have an understanding of what is being done in space life sciences and why. Too often, policies are made without proper regard for the impact they will have. An appreciation for the need and possible urgency of conducting research in space life sciences should be understood by policymakers before decisions are made. This not only favorably affects national policies, but it can also have major impacts on space projects, especially when it involves some aspect of space life sciences. Given the current climate in which space programs exist, the economic and financial considerations are of prime importance. The realities of limited funding

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and possible cutbacks in support for space life sciences research should be realized. This is very closely related to the policy issues that have already been mentioned. Space life scientists should understand the financial issues because they can significantly impact a given project or program in the shortterm or longterm. Scientists can also become intimately involved in the day-to-day budgetary matters of a project, so some knowledge of effective financial management is important. This is particularly important for expensive hardware development, where scientists are advising engineers or managers from industry or from space agencies. Space life sciences research is increasingly taking on an international flavor. Not only are more countries getting involved in space life sciences research, but more projects include contributions from more than one country. As scientists from various countries work together, however, many intercultural issues may arise. Although these can be potentially disruptive and counterproductive, this can be avoided if all participants have understanding and respect for each other’s cultural differences. The field of space life sciences does not exist by itself, but is highly integrated with other disciplines. Space life scientists and other scientists must have a basic understanding of each other’s discipline, because they will inevitably have to work together. Adequate knowledge of the problems of other disciplines will potentiate the output of a project.

VI.

TELE-EDUCATION AS A TEACHING TOOL

Tele-education can be defined as the use of telecommunication techniques for the purpose of providing education, training, and information over a distance. This is in contrast to distance education or distance learning that does not require the use of telecommunication-based tools. This section focuses on tele-education, even if some aspects are more general and apply also to distance learning. A.

General Needs for Tele-Education

Why tele-education? There are various needs for tele-education, depending on the context. In the case of specific or high level education, tele-education is useful in several ways. It may reinforce a particular content, if needed, focus on a particular segment of society, become diffused in a heterogeneous population, or overcome geographic isolation in order to receive state-sanctioned education-as in “open universities”. The main need for tele-education is to take advantage of a world of experts in a specific field. Other reasons for using tele-education are to resolve schedule conflicts between teachers, especially international experts, to save time, and to reduce the cost of education. One of the main goals of tele-education is to give students access to more experts. Traditionally the students are enrolled in a university and are educated by

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permanent faculty. With tele-education, they can interact with international experts in a specific field to deepen their understanding or to obtain another perspective on the subject. Tele-education, then, is needed because the expert cannot come over for just a few lectures or a question-and-answer period. Time and money are saved in direct costs such as travel expenses and fees and indirect costs such as time and schedule management for the expert or the organizers owing to the flexibility of expertise on “request”. An example is the tele-training in laparoscopic surgery by IRCAD (Strasbourg, France) to surgeons in all of Europe through the European TESUS Project (TEleSUrgical Staffs, Telematics’ project, European Commission, 1995-1997). In this type of tele-education, the remote expert does not replace the local lecturer, who introduces the subject to the students, moderates the question-and-answer session, and manages the follow-up with the students. Tele-education can also be used to communicate with a large public. The broadcast mode allows the reaching of a large audience not necessarily involved in specific fields. A typical example is a television educational program. An interactive mode, rather than the broadcast form, is more suitable for communication within an international community of space experts or life science experts and students. This mode allows students to interact with experts and thus to participate actively in the educational process.

6.

Tele-education Systems

Tele-education can use several diffei-ent tools, which can be combined in a system for a more efficient exchange of information and knowledge. A tele-education system can be based on live tools such as a video-conference over the digital technolory of the phone network (ISDN),2 and soon over ATM, the broadband technology of ISDN, and at a good quality over the Internet in the form of an audio teleconference’, e-mail, or an internet chat session4. A one-way medium such as television can be combined with a two-way medium in order to allow interaction. For example, some television programs are associated with a video-conference link to a remote studio or to a personal video-phone for a direct broadcast of an interaction between a person and the television studio. These tools could be combined with off-line and on-line non-live tools such as information on a World Wide Web server, CD-ROM, video tape, and viewgraphs to be downloaded across the Internet in order to permit local use. Several tele-education experiments in the world are based on Internet tools, particularly in North America. Some difficulties still exist due to both the impossibility to reserve bandwidth and to the general lack of available bandwidth^.^ In experiments performed at IRCAD, a video-conference over ISDN at 384 Kbps (kilobits per second) is of sufficient quality to provide remote access to live views of laparoscopic surgery. Over ATM at 2 Mbps, the quality is equal to that of a local transmission. At the International Space University (ISU), we experiment

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with the combination of a video with live sessions. For example, a video on “A Mission to Mars: Human Factors” describes the problems and potential solutions; :t is associated with question-and-answer sessions based on video-conferences and organized between experts who have participated in the video and students of the ISU’s Master of Space Studies program and from several affiliated universities. ISU also uses video-conferences for tele-lectures and for broadcasting events with interactive question-and-answer sessions. The aim of such tools is to allow interaction between students and remote teachers in order to preserve the vital aspects of the teacher-student relationship, such as answering questions, encouraging the students, and assisting them in understanding the lessons. Therefore, a tele-education system should be designed for an adapted educational environment. For example, in the European Project BIC (Blueprint for Interactive Classrooms, Telematics project, European Commission, 1996-1997), five environments are described: (1) an automated teaching presentation area with students at remote sites, (2) an automated teaching presentation area with local and remote students, (3) an area suited to group presentation and interaction, (4) a learning area for an isolated learner, and (5) a learning area for a group of learners. The use of such a system requires training, not only in how to use the technology, but also on strategies for teaching at a distance. Depending on the selected tool, it is desirable to match the approach with the technology,6 (e.g., for a tele-lecture presented by video-conference, viewgraph fonts should be larger than usual, and the lecturer should avoid moving around). Sometimes more preparation is required, especially when non-live media are used. Foe example, when combining an interactive question-and-answer session with a video tape, it is important to have at each site a moderator, who introduces the subject before watching the tape and then works with the students to prepare the questions, who is the moderator during the question-and-answer session, and finally, who works with the students in the follow-up. A general trend in all domains is specialization, especially in the area of high-technology, phenomenon that leads to a lack of local experts in a specific field. The tele-education approach solves the lack of experts and research in a specific field at a given university. It can give students access to more specialized experts and to different fields of expertise. It offers multi-specialist confrontations and inter-disciplinary interactions. Currently, tele-education is essentially used for tele-lectures and for interactive question-and-answer sessions. With the upcoming new tools and new network technologies, hands-on sessions will be possible. This is the case with tele-surgery, developed in the European project MASTER (Minimal Access Surgery by Telecommunications and Robotics, Eureka’s project, European Commission, 1994-1999). The current step is on remotely watching laparoscopic surgery for tele-diagnostic training and passive learning of laparoscopic surgery. The next step is to control the surgery remotely with the tele-assistance of an international expert.

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Tele-Education Applied to Space Life Sciences

Space life sciences education is a type of high-level education as shown in this paper; as such it is a suitable candidate for tele-education. It has manifold and essential international aspects, and there are few, if any, specialized experts available in each university. The field is mainly developed in the USA, Russia, and Europe, with three different types of expertise and approach. The experts are spread over the world, either as permanent faculty members in a university, or members of a space agency or industry. Tele-education is the only tool that can provide an adequate and complete expertise to students in this field. It allows access to expertise but also interaction between several space life sciences experts of different nationalities and cultures thus providing different perspectives. Tele-education helps in solving the problem of the availability of specialized experts and gives students access to very busy people like astronauts who cannot spend too much time abroad. Moreover, tele-education is the only way to manage the scheduling of an interaction between several experts to allow students to participate in a confrontation on a space life sciences topic or in a multidisciplinary discussion. In summary, tele-education allows students to have access to more space life sciences content, to different perspectives, to more confrontations, to interdisciplinary points of view, and soon not only to verbal tele-interaction but also to physical telemanipulations.

VII.

EXISTING TEACHING PROGRAMS A.

NASA-Sponsored Programs

Nearly every space agency has an education or information center where documents on space life sciences can be obtained. NASA is the agency providing by far the greatest amount of educational materials. NASA’s Central Operation of Resources for Educators (CORE) 7 has been established for the national and international distribution of NASA-produced audiovisual educational material. NASA’s Education Division established the NASA Teacher Resource Center Network.8 Every NASA research center has a “Teacher Resource Center” or “Teacher Resource Laboratory”. They can provide teachers with a variety of educational materials such as text, audio, visual, and computer generated data. There is also on-site training for students and teachers. The NASA Ames Research Center has developed a six-week, hands-on science training program for teachers wanting to produce lesson plans focusing on space life sciences, code-named STELLAR9 (Science Training to Enhance Leadership and Learning through Accomplishments in Research). The lesson plans are available on Spacelink, while hands-on science training programs exist at NASA Ames

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Research Center (six-week training in space life sciences) and at John F. Kennedy Space Center (summer program for undergraduate college students, called “Space Life Sciences Training Program”). I’ Much information can be obtained through the Internet. NASA Spacelink’ offers a wide range of materials for educators and students, such as computer text files, software, and graphics related to the NASA space program. Eight NASA Specialized Centers of Research and Training (NSCORT)12 are or have been sponsored in the following space life sciences fields: gravitational biology, bioregenerative life support, radiation health, vestibular research, plant sensory systems, exobiology, and physiology. The primary goal of these centers is to develop research programs, but some of them have also initiated courses. At the North Carolina State University, for example, interactive courses over video network are organized between several affiliated universities. These examine the biology of plants, animals, and humans as related to gravity and the spaceflight environment. Workshops for high school teachers to promote space biology research and teaching are also 0 r g a n i ~ e d . There l~ is also the QuestI4 server of NASA Ames Research Center supporting on-going educational projects. Finally, realtime information on current events in space research can be obtained by watching NASA Television.” B.

Universities and Institutions

Many teaching activities in the field of space life sciences exist in universities and institutions. The following list is not exhaustive and will only give a few examples.

1. At the University of Texas Medical Branch (UTMB),16 the aerospace residency program includes many aspects of physiology, biomedicine, history, and behavior related to space. 2. A collaboration between Stanford University and NASA has led to a very complete course adapted by the U.S. Air Force Academy.17 The courses introduce environment and exploration of space as related to life science. 3. At the University of Toulouse Medical School” in France, a diploma of space biology, physiology, and medicine is offered to graduate students. 4. A one-to-two-week Biology in Space program” is sponsored by the European Union (ERASMUS Program) at a different location every year. 5. The International Space University2’ offers essentially an interdisciplinary, intercultural, and international approach of teaching all aspects related to space, including space life sciences. This is provided by a 10-week Summer Session Program changing place every year and a Master of Space Studies program (1 1 months) including lectures, seminars, workshops, a placement period, and individual and team projects.

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Some useful textbooks and reviews are listed in references 21-25, and additional detailed scientific publications in references 26-33.

VIII. CONCLUSIONS AND SUMMARY Space life sciences is not really a new life sciences discipline such as immunology was some decades ago and it may never be so. Rather it is a field that will provide each existing life sciences discipline with new and more information gathered from space research. In fact, the danger is that space research will be confined in a separate discipline, and thus it will be cut off from classical ground research. Conversely, scientists should increasingly consider spaceflight as a tool and should integrate the findings of space research into their traditional disciplines. A brief survey of topics and main findings in the various subdisciplines of space life sciences is provided. This is followed by a discussion of typical problems encountered such as access to space, controls, ground-based simulations, medical care in space, extravehicular activity, and environmental control and life support. As many space life sciences courses are initiated around the world either by space agencies or universities or jointly, there is a need to consider the international, intercultural, and interdisciplinary aspects of such programs. It is argued that the growing knowledge derived from space research should be integrated into the regular teaching of life sciences rather than leaving it confined to a separate field. Teaching of space life sciences is a prime candidate for the application of the new techniques of “cyberspace education”, where interactive learning and globalization of the learning process will take a leading place. The experts and student body are dispersed over many nations, research is of necessity conducted on a basis of international cooperation. The conduct of tele-education is discussed and existing information sources and courses are listed.

REFERENCES AND NOTES References on Tele-Education 1.

2. 3.

4. 5.

GATES: Global Access Tele-health and Education System, Summer Session Design Project, International Space University, 1994. Portway, P.S., Lane, C. Guide to Teleconferencing & Distance Learning. 2nd ed., Applied Business TeleCommunications, 1994. Showalter, R.G. Instructional Audio Teleconferencing: Strategies for Encouraging Faculty to Teach by Telephone. In: 19th Annual Pacific Telecommunications Conference, 1997. Wulf, K. Training Via the Internet: Where Are We? Training and Development, (5):50-54, 1996. Tiffin, J. The Coming of the Virtual College. Proceedings of the 19th Annual Pacific Telecommunicutions Conference, 19-22 January, 1997.

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6. Thach, C., Murphy, K.L. Training Via Distance Learning. Training & Development, (l2):44-46, 1995.

Useful Addresses for Existing SLS Teaching and Related Documentation 7. NASA CORE, Lorain County Joint Vocational School, 15181 Route 58 South, Oberlin, OH, 44074, USA. 8. Education Division, NASA, Mail Code FE, Washington, DC 20546-0001, USA. Website: http:/ /www.gsfc.nu.sa.gov/planetary/NTRC.HTLM 9. STELLAR Program, NASA/ARC, Moffett Field, CA, 94035-1000, USA. Website: http://stellar.arc.nasa.gov E-mail: [email protected] 10. SLSTP, Florida A&M University, College of Pharmaceutical and Pharmaceutical Sciences, Tallahassee, FL, 32307, USA 11. website: http://spacelink.msfc.nasa.gov 12. Life and Biomedical Sciences and Applications Division, NASAIHQ, Washington, DC 20546000 I , USA. 13. NSCORT Associate Director, Department of Botany, NCSU, Campus Box 7612, Raleigh, NC 27695-76 12, USA. E-mail: Christopher-brown @ncsn.edu website: http://www2.ncsu.edu/ncsu/ cals/nscort/ 14. website: http://quest.arc.nasa.gov 15. NASA TV, NASA/HQ, Code P-2, Washington, DC 20546-0001, USA. website: http:// www. hq.nasu.gov/office/pao/Television/ntvtext3. htlm 16. E-mail: CoulterGR@DFB@USAF 17. E-mail: [email protected] 18. E-mail: phdupui @cerco.ups-tlse.fr 19. Prof. L.G. Briarty, Life Science Department, Nottingham University, Nottingham, Endland, NG7 RD. E-mail: [email protected]. 20. Website: http://www.isunet.edu/

Reference Texts for Teachers 21. Churchill, S.(Ed.). Fundamentals ofspace Lcfe Sciences. Krieger Press, 1997. 22. Moore, D., Bie, P., Oser, H. (Eds.) Biological and Medical Research in Space: An Overview of Life Sciences Research in Microgravity. Springer-Verlag, New York, 1996. 23. Nicogossian, A.E., Huntoon, C.L., Pool S.L. (Eds.), Space Physiology and Medicine, 3rd ed., Lea & Febiger, Philadelphia, 1995. 24. Lujan, B.F., Ronald, W.J. (Eds.) Human Physiology in Space. NIH, Bethesda, MD, 1994. 25. Bonting, S.L. (Eds.) Advances in Space Biology and Medicine, vols. 1-7, JAI Press, Stamford, CT, 1991-1999.

Additional Scientific References 26. International Workshop on Cardiovascular Research in Space. In: Medicine and Science in Sports and Exercise, 28:SI-S112, 1996. 27. International Workshop on Muscle Research in Space. In: International Journal of Sports Medicine, 18:S266-S333, 1997. 28. International Workshop on Plant Biology Research in Space. Planta, 203:s 1-S219, 1997. 29. International Workshop on Bone Research in Space. Bone, 22:S71-S161, 1998. 30. Internatianal Workshop on Radiation Research in Space. Radiation and Environmental Biophysics, (Suppl.), 1999.

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3 I . International Workshop on Neuroscience in Space. Brain Research Reviews, 28:Sl-S232, 1999. 32. International Workshop on Molecular and Cell Biology in Space. FASEB Journal, 13:SI-S177, 1999. 33. lntcrnational Workshop on Integrative Physiology i n Space. European Journal of Physiology, (Suppl.), 1999 (In press.)

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Adductor 1ongu.r muscle, 33 Albrecht-Buehler, G., 198 Algal bioreactors, 133 Allen, John, 149, 156 Anemia, and space missions, 118, 222 Anti-gravity muscles, 33

Biocosmos missions, and postflight tissue collection delay, I7 Bioregenerative life-support system (see Controlled Environment Life-Support System; CELSS) Bone calcium loss and spaceflight, 50, I03 Catecholamines, and the sympathetic system, 1 19- 120 Chorella, 133, 150 Chufa nut sedge ( C y p r u s esculentus), 153 Controlled Environment Life-Support system (CELSS), 133 BIO-Plex, 156- 157 BIOS-3, 150- 156, 159 Biosphere-2, 148- 150 buffering capacity, 156 components, 136-137 development challenges, 137-138 in Earth orbit, 140-141 economics, 135- 136 on Mars, 138-139 and microfloral instability, 154- 155 on the Moon, 139-140 247

partial closure and resupply vs. deadlock substances, 150, 155, 157 research sites, 135 Convective flow stable and Grashof number (GR), 190 unstable and Raleigh number (Ra), 190 Cortisol effect, 73-75 Cultivation in space, 141-146 Cyanobacteria, and nitrogen fixing, 133, 137 Doubly labeled water (2H2'80) expendi ture measure, 77 Electrophoresis (space-based), 164, 186 advantages and limitations, 191-192 column, 166- 170, 183 commercial feasibility, 172, 183, 199 continuous flow (CFES), 183- 184, 198-199 early experiments, 164-166 French device (RAMSES), 178-183, 184 isotachophoresis (ITP), 166 Japanese Free Flow unit (FFEU), 177-178, 184, 208 NASA program, 192 need for two-step standardization process, 193-194

248

Octopus Continuous Flow device, 202-205, 208 physical parameters, 188-192 PSCF, 170, 172 sedimentation, 183, 186, 189-190 Soviet systems, 170, I84 United States Commercial Electrophoresis Program in Space (USCEPS), 199-202, 208 wall vs. bulk effect, 169 Endocrinology axes review, 121 - 122 (see also Hormones; Space physiology) Ethylene and fungi, 147-I48 as plant growth inhibitor in space, 147,157 Extensor digitorum longus (EDL) muscle, 34 Flexor muscles, 33 Gender and cortisol effect, 74 and spaceflight, 109 Glycolytic metabolism adaptation, 35, 42,54-55 Gravitaxis vs. geotaxis, 218 Ground-based simulations of spaceflight, 32, 59-60 bedrest, 5 1 , 69-70, 103, 119, 227 Clinostat, 226 drop towers, 224, 227 head down suspension, 227 isolation and confinement, 227 magnetic levitation, 227 parabolic flights, 224 protein loss possibilities, 60 water immersion, 1 16 Growth hormones, 110, 196 insulinlike growth factor (IGFI), 103, 110-1 1 1

INDEX

Harvest index, 133 and effective CELSS, 136 Hormones, 100,101 as “endogenous drugs”, 115 limiting factors of space endocrinology, 120-121 plasma hormone measurements sampling method, 70 pulsality and microgravity studies, 108 regulation of bone turnover on the ground, 102-103 regulation of bone turnover in space, 103-104 and use of radioimmunoassay techniques, 185 Hypergravity, 2 18 Hypoandrogenism in space, 104, 109 “Hyposympathetic condition” of microgravity challenge, 120 Hypothalamic-pi tuitary-adrenal (HPA) axis, 61, 70-72, 101 regulation on the ground, 104- 105 regulation in space, 105-106 Hypothalamic-pituitary-gonadal axis regulation on the ground, 106-108 regulation in space, 108- 109 Hypothalamic-pi tui tary-somato-mammotropic axis regulation on the ground, 109-111 regulation in space, 11 1-1 13 Hypothalamic-pituitary-thyroid axis regulation on the ground, 113 regulation in space, 1 13- 114 Hyperthyroidism, rats and spaceflight, 113-114 Ilynia-Kakueva, E.I., 54 Insulin, 72-73, 118-119 International Space Station “coupled technology” goal, 187 crew composition issues, 23 1

lndex

new opportunities for research, ix, 100,215,225 and space life sciences research, 214 Laron’s syndrome, I I 1 Life and Microgravity Sciences (LMS) shuttle mission, 77-78 exercise requirement, 81-82, 83 Lower body negative pressure (LBNP) regimen, 42 Mars exploration four gravity transitions, 50 requirements for energy balance, 84 Medical care in a space environment diagnosis, 229 drug resistance, 1 15 radiation health, 230 risk prevention, 228-229 treatment, 229-230 wound healing, 83 Melatonin, 100 Methionine, 87-88 Microgravity and onset of edema, 41-43 “quality”, 2 18 and study of muscle biology, 44 vs. zero-gravity, 193 Muscle atrophy and microgravity in rodents, 2, 22, 54-56 literature summary, 3- 13 postflight secondary changes, 39 simple deconditioning, 33-35 Muscle atrophy and reloading in humans, 103 lack of controlled testing of exercise, 69 noninvasive research, 32, 56-59 pathological effects, 36 possible limitations to length of spaceflights, 52 preservation of function, 38-39

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reactivation of Earth-gravity motor skills, 39-40 susceptibility to structural damage, 42-43 vs. “remodeling” muscle, 5 1 Muscle fiber evolution of typing procedures, 19 fast vs. slow twitch, 33, 54-55 transformation, 38

N glycine measurement of protein synthesis, 61-62,64-67 NASA’s Central Operation of Resources for Educators (CORE), 241-242 Nitrogen balance and insulin, 72 mission specific, 83 Nutrition astronaut needs, 85-88 and energy deficit, 77-82 and fluid loss, 118 and future studies, 117 vegetarianism and successful CELSS, 154 PGE-M (see Prostaglandins) PGF-M (see Prostaglandins) Phytotron, 150 Plants assimilation quotient, 154 cultivation in space, 141 male sterility in space, 146, 147 Plasma amino acid distribution method, 86 Prostaglandins, 75-77, 88 Protein crystal growth vs. pharmaceuticals production as commercial space application, 219, 233 Protein metabolism, 52-54 and insulin, 72 metabolic stress remonse. 61-67

250

myofibrillar protein breakdown assessment, 67-68 nitrogen balance measure, 56-57 Protein turnover process, 52 benefits, 53 energy costs, 54 Psychology and performance in space environment, 230-23 1 psychological effects vs. endocrine reactions, 101 Renin-angiotensin-aldosteronesystem (RAAS) regulation on the ground, 1 14- 1 15 regulation i n space, 1 IS- 1 18 Rodent myology, 2 cage effects, 18 issues for future research, 21 -22 major investigative groups, 15,2021,23 postflight skeleton muscles examined, 16-17, 19 Rodent vs. human response to spaceflight, 59 Soleus muscle and bedrest, 35 and exercise, 38 as “model”, 18- 19, 34 Space biology, 2 17 biotechnology, 2 1 8 cellular and molecular, 21 8 developmental, 219 and effects of weak electromagnetic fields, 220 exobiology, 220 plant physiology, 2 19 radiation biology, 219-220 Space life sciences educational resources, 241 -243 physics requirement, 237 radiation and vacuum as well as microgravity, 214

INDEX

research problems, 224-226 three main thrusts, 21 6 (see also Space biology; Space physiology) Space physiology, 221 animal physiology and biology, 223224 cardiovascular and circulatory functions, 221-222 endocrinology, metabolism, and regulatory physiology, 222 immunology, hematology, and microbiology, 222-223 musculoskeletal system, 221 -222 neuroscience, 222 respiratory function, 221 Space research control experiments, 225-226 cooperation, 2 1, 233-234 costs, 2 historical phases, 217 intercultural issues, 235-236 interdisciplinary issues, 236-238 need for countermeasure protocols, 32 and pathophysiology of aging, 122 Space station (see International Space Station) Spacelab Life Sciences missions contradictory evidence of muscle protein breakdown, 69 exercise requirements, 79-80, 84,85 (SLS-2) pivotal experiment, 55-56, 57 Spaceline database, 20 Specific tension ratio, 35 Stokes law, 189 Stress and “acute-phase protein” synthesis, 61 “stress reaction”, 104 Substrate cycle, 54 “Svet” plant growth chamber, 141

hdex

Tele-education as a teaching tool, 23824 1 Tihcalis anterior muscle, 33 Tsiol kovsky, Konstatin Edwardovich, 132 United States Space Shuttle, inflight tissue collection capability, 18.32

251

Waste recycling, physiochemical and plant based techniques, 132, 158 Water-electrolyte-regulating peptides, 114-115, 117

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