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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


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


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


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


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


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


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


Synchronous ( 5 )

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

BIOS-2 Chamber


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


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


BIOS-2 Chamber


BIOS-2 Chamber

group of 10 66x22~16em

BIOS-2 Chamber


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


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 )


? 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


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.


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


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


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


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


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


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


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.

REFERENCES I . Fejtek, M., Wassersug, R. Effects of laparotomy, cage type, gestation period and spaceflight on abdominal muscles of pregnant rodents. Journal of Experimental Zoology, 284:252-264, 1999. 2. llyin, E.A., Oganov, V.S. Microgravity and musculoskeletal system of mammals. Advances in Space Researrh, 9(1 1):(1 1)l l-(lIj19, 1989. 3. Roy, R.R., Baldwin, K.M., Edgerton, V.R. Response of the neuromuscular unit to spaceflight: What has been learned from the rat model. Exercise and Sports Science Reviews, 24:399425, 1996. 4. Tischler, M.E., Slentz, M. Impact of Weightlessness on muscle function. ASGSB Bullelin, 8(2): 3-81, 1995. 5. Souza, K., Hogan, R., Ballard, R. (Eds.) Life Into Space: Space Life Sciences Experiments, NASA Ames Research Center 1965-1990, Government Printing Office #1995-685-928 (NASA Reference Publication 1372), Moffett Field, CA, 1995. 6. Ilyina-Kakueva, E.I., Portugalov, V.V., Krivenkova, N.P. Space flight effects on the skeletal muscles o f rats. Aviation, Space, and Environmental Medicine, 47(7):700-703, 1976. 7. Ilyina-Kakuera, E.I., Portugalov, V.V., Krivenkova, N.P. Effects of space flight factors on skeletal muscles o f rats. Kosmicheskaya Biologiya i Aviakosmicheskaya Meditsina, 1:20-25, 1977. 8. Savik, Z.F., Rokhlenko, K.D. Ultrastructure of blood vessels and muscle fibers of rat skeletal muscles after flights aboard Cosmos-605 and Cosmos-782 hiosatellites. Kosmicheskaya Bio[ogiya i Aviakosmicheskaya Meditsina, 15(5):78-83, 198 I , 9. Nesterov, V.P., Tigranyan, R.A. Effects of' space flight factors on electrolyte composition of rat skeletal muscles. Kosmicheskaya Biologiya i Aviakosmicheskaya Meditsina, 13( 1):66-68, 1979. 10. Oganesyan, S.S., Eloyan, M.A. Cathepsin activity of skeletal muscle and myocardial myofihrils after exposure to weightlessness and accelerations. Kosmicheskaya Biologiya i Aviakosmicheskava Meditsina, 15(6):38-42, 1981. I I , Portugalov, V.V., Petrova, N.V. LDH isozymes of skeletal muscles of rats after space flight and hypokinesia. Aviution, Space, and Environmental Medicine, 47(8):834-838, 1976. 12. Kamryan, V.A., Rapoport, E.A., Goncharova, L..4., Bulycheva, S.Ya. Effect of prolonged weightlessness o n metaholism of proteins in red and white skeletal muscles o f rats. Kosmicheskaya Biologiya i Aviakosmicheskaya Meditsina, 6:19-23, 1977. 13. Baranski, S., Marciniak, M. Morphometric ultrastructural analysis of muscle fibers and neuromuscular junction of rats kept under conditions of weightlessness for 21 days. Materia Medica Polona, 4(76):258-262, 1990.

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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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DANNY A. RILEY

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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.


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 . ~ ~ , ~ ~

36

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.


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.

38

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


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


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-


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.


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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DANNY A. RILEY adaptation to weightlessness and readaptation to one-g: An overview. Experimental Brain Resecrrch, 64:201- 298, 1986. Zhou, M.-Y., Klitgaard, H., Saltin, B., Roy, R.R., Edgerton, V.R., Gollnick, P.D. Myosin heavy chain ihoforms of human muscle after short- term spaceflight. Journal qf Applied Physiology, 78:1740-1744, 1995. Baldwin, K.M., White, T.P., Arnaud, S.B., Edgerton, V.R., Kraemer, W.J., Kram, R., RaabCullen, D., Snow, C.M. Musculoskeletal adaptations to weightlcssness and development of effective countermeasures. Medicine cind Science in Sports and Exercise, 10:1247-1 253, 1996. Baldwin, K.M., Herrick, R.E., McCue, S.A. Substrate oxidation capacity i n rodent skeletal mubcle: Effects of exposure to zero gravity. Journal qfApplied Physiology, 75:2466-2470, 1993. Bat-anski, S., Baranska, W., Marciniak, M., Ilyina-Kakueva, E.1. Ultrasonic (“ultrastructural”) investigations of the solens muscle after space flight on the Biosputnik 936. Aviation Space & Environmental Medicine, 50:930-934, 1979. Ilyina-Kakueva, E.I., Portugalov, V.V., Kirvenkova, N.P. Space flight effects on the skeletal muscle of rats. Aviation Space & Environmental Medicine, 47:700,703, 1976. Martin, I.P., Edgerton, V.R., Grindeland, R.E. Influence of spaceflight on rat skeletal muscle. Journal ofApplicd Physiology 65:23 18-2325, 1988. Riley, D.A., Ellis, S., Slocum, G.R., Satyanarayana, T., Bain, J.L.W., Sedlak, F.R. Hypogravityinduced atrophy of rat soleus and extensor digitornm longus muscles. Muscle & Nerve, 10:560568, 1987. Riley, D.A., llyina-Kakueva, E.I., Ellis, S., Bain, J.L.W., Slocum, G.R., Sedlak, F.R. Skeletal muscle fibcr, nerve, and blood vessel breakdown in space-flown rats. FASEB ./ournu/, 4:84-91, 1990. Riley, D.A., Ellis, S., Giometti, C.S., Hoh, J.F.Y., Ilyina-Kakueva, E.I., Oganov, V., Slocum, G.R., Rain, J.L.W., Sedlak, F.R. Muscle sarcomere lesions and thrombosis after spaceflight and suspen\ion unloading. Joiirnul of Applied Physiology, 73:833-S43, 1992. Riley, D.A., Thompson, J.L., Krippendorf, B.B., Slocum, G.R. Review of spaceflight and hindlimb suspension unloading induced sarcomere damage and repair. Basic & Applied Myology 5:139-14S, 1905. 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., DeBruin, J.A. In-flight and postflight changes in skeletal muscles of SLS-1 and SLS-2 spaceflown rats. Journd of Applied Physiology, 81:133-144, 1996. Widrick, J.J., Knuth, S.T., Norenberg, K.M., Romatowski, J.C., Bain, J.L.W., Riley, D.A., Karhanek, M., Trappe, S.W., Trappe, T.A., Costill, D.L.. Fitts, R.H. Effects of a 17-day spaceflight on contractile properties of human soleus muscle fibres. Journal of Physiology (London), 516:915-930, 1999. Swachenko, 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-S 165, 1992. Tidball, J.G., Quan, D.M. Reduction in myotendinous junction surface area of‘rats subjected to 4kiay spaceflight. Journal $Applied Physiology 7359-64, 1992. Tischler, M.E., Henriksen, E.J., Munoz, K.A., Stump, C.S.,Woodman, C.R., Kirby, C.R. Spaceflight o n S T S 4 8 and earth-based unweighting produce similar effects on skeletal muscle of young rats. Journal ojApplied Physiology, 74:2161-2165, 1993. Bigard, A.X., Lienhard, F., Merino, D., Serrurier. B., Guezennec, C.Y. Effects of growth hormone o n rat skeletal muscle after hindlimb suspension. European Jo~rrnalqfApplied Physiology 69:337-343, 1994. Caiozzo, V.J., Haddad, F., Bakcr, M.J., Herrick, R.E., Prietto, N., Baldwin, K.M. Microgravityinduced transformations of myosin isoforms and contractile properties of 5keletal muscle. Journal ($Applied Physiology, 81:123-1 32, 1996.

Spaceflight arid Gravity Loading Effectson Skeletal Muscle 27

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Diffce, G.M., CaioLzo, V.J.,Herrick, R.E., Baldwin, K.M. Contractile and biochemical properties of rat soleus and plantaris after hindlimb suspension. American Journcil of Physiology, 260: C528-CS34, I99 1 . Dilfee. G.M., Haddad, F.. Herrick, R.E., Baldwin, K.M. Control of myosin heavy chain expression: Interaction of hypothyroidism arid hindlimb suspension. American Joirrnul of Physiology, 261:Cl099-CI 106, 1991. Krippendorl, B.B., Riley, D.A. Distinguishing unloading- versus reloading-induced changes in rat soleus musclc. Muscle Ce Nervr, 16:99-108, 1993. Krippendoid, B.B., Riley, D.A. Temporal changes in sarcomere lesions of rat adductor longus muscles during hindlimb reloading. The Anatomical Record, 238:304-3 10, 1994. McDonald, K.S., Blaser, C.A., Fitts, R.H. Force-velocity and power characteristics of rat soleus muscle fibers after hindlimb suspension. Journal ofApplied Physiology, 77: 1609-16 16, 1994. McDonald, K.S., Dclp, M.D., Fitts, R.H. Effect of hindlimb unweighting on tissue blood flow in thc rat. Journal oj Applied Physiology, 72:2210-2218, 1992. Riley, D.A., Slocuni, G.R., Bain, J.L.W., Sedlak, F.R., Sowa, T.E., Mellender, J.W. Rat hindlimb unloading: Solcus histoeheinistry, ultrastructure, and electromyography. Journul ofApplied Phy.siolo,qy, 69:SX-66, 1990. Widrick, J.J., Fitts, R.H. Peak force and maximal shortening velocity of’ soleus fibers after nonweight-hearing and resistance exercise. Journal ofApplied Physiology, 82: 189-1 95, 1997. Widrick, J.J., Romatowski, J.G., Bain, J.L.W., Trappe, S.W., Trappe, T.A., Thompson, J.L., Costill, D.L., Riley, D.A., Fitts, R.H. Effect of 17 days hedrest on peak isometric force and maximal shortening velocity of human soleus fibers. American Journal qfPhy.siology, 273:C 1690C 1699, 1997. Rilcy. D.A., Bain, J.L.W., Thompson, J.L., Fitts, R.H., Widrick, J.J., Trappe, S.W., Trappe, T.A., Costill, D.L. Disproportionate loss of thin filaments in human soleus muscle after 17-day bedrest. MI4.Yck & Nerve, 21: 1280-1289, 1998. Stauber, W.T., Clarkson, P.M., Fritz, V.K., Evans, W.J. Extracellular matrix disruption and pain after eccentric muscle action. Journd qfApplied Physiology, 69:868-874, 1990. Larsaon, L., Li, X., Berg, H.E., Frontera, W.R. Effects of removal of weight-bearing function on contractility and myosin isoform composition :n single human skeletal muscle cells. Pf2uger.s Archive.s--Ei~roprcinJournal of Physiology, 432:320-328, 1996. Metzger, J.M., Moss, R.L. Shortening velocity in skinned single muscle fibers-Influence offilament lattice spacing. Biophysics Jounzal, 52:127-13 I, 1987. Howells, K.F.. Jordan, T.C., Howells, J.D. Myofibril content of histochemical fibre types in rat skeletal muscle. Actu Histochemicn Bd. 63: 177-182, 1978. Kugelberg, E., Thornell, L.-E. Contraction time, histochemical type, and terminal cisternae voltime of rat motor units. Mii.sclr & Nerve, 6:149-153, 1983. Stick, C., Grau, H., Witzleb, E. On the edema-preventing effect of the calf muscle pump. Europ’an Journul ofApplied Physiology, 59:39-47. 1989. Olsaewski, W., Engeset, A,, Jaeger, P.M., Sokolowski, J., Theodorsen, L. Flow and composition of leg lymph in normal men during venous stasis, muscular activity, and local hyperthennia. Acttr Physio/ogii,cz Sctindinuvica 99: 149-1 5 5 , 1977. ular localization of albumin and IgG in normal tonic and Stauber, W.T., Ong, S.-H. Extra ph;isic muscles of the rat. Histochemical Journal, 13:1009-1015. 1981. Roer, R.D., Dillaman, R.M. Decreased femoral arterial flow during simulated microgravity in the rat. Journal ofApplied Physiology, 76:2125- 2129, 1994. Wilson, L.B., Dyke, C.K., Parsons, D., Wall, P.T., Pawelczyk, J.A., Williams, R.S., Mitchell, J.H. Effect of skeletal musclc fiber type on the pressor response evoked by static contraction in rabbits. Journzal qfApplied Physiology. 79: 1744-1752, 1995.

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Warren, C.L., Hayes, D.A., Lowe, D.A., Williams, J.H., Armstrong. R.B. Eccentric contractioninduced injury in normal and hindlimb-suspended mouse soleus and EDL muscles. Journal of Applied Physiology, 77:1421- 1430, 1994. Gibala, M.J., MacDougall, J.D., Tarnopolsky, M.A., Stauber, W.T., Elorriaga, A. Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise. Journal of Applied Physiology, 78:702-708, 1995. Thompson, J.L., Balog, E.M., Fitts, R.H., Riley, D.A. Five myofibrillar lesion types in eccentrically challenged, unloaded rat adductor longus muscle, a test model. Anatomical Record, 254:39-52, 1999. Russcll, B., Dix, D.J., Haller, D.L., Jacobs-El, J. Repair of injured skeletal muscle: A molecular approach. Medicine and Science in Sports and Exercise 24: 189-196, 1992. Rezvani, M., Ornatsky, O.I., Connor, M.K., Eisenberg, H.A. Hood, D.A. Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse. Medicine and Science in Sports and Exercise, 28:79-84, 1996. Campbell, K.P. Three muscular dystrophies: Loss of cytoskeleton- extracellular matrix linkage. Cell, 80:675-679, 1995. Matsumura, K., Campbell, K.P. Dystrophin-glycoprotein complex: Its role in the molecular pathogenesis of muscular dystrophies. Muscle & Nerve, 17:2-15, 1994. Petrof, B.J., Sharger, J.B., Stedman, H.H., Kelly, A.M., Sweeney, H.L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proceeding of the National Acudemia VfSciences, U.S.A.,90:37 10-3714, 1993. Stone, M.H. Muscle conditioning and muscle injuries. Medicine and Science in Sports and Exercise, 22:457-462, 1990. Danowski, B.A., Imanaka-Yoshida, K., Sanger, J.M., Sanger, J.W. Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes. The Journul of Cell BioZoRy, 118:1411-1420, 1992. Strauh, V. Campbell, K.P. Muscular dystrophies and the dystrophin-glycoprotein complex. Current Opinion in Neurology, 10:168-175, 1997. Stauber, W.T., Fritz, V.K., Burkovskaya, T.E., Ilyina-Kakueva, E.I. Effect of injury on mast cells of rat gastrocnemius muscle with respect to gravitational exposure. Experimentul und Molec.ular Pathology, 59:87-94, 1993. Taipale, J., Keski-Ojh, J. Growth factors in the extracellular matrix. FASEB Journal, 11:s1-59, 1997. Oaborn, M.J. (chair), Committee on Space Biology and Medicine, Space Studies Board, National Research Council. A Strategyfor Research in Space Biology and Medicine in the New Centurv. National Academy Press, Washington, D.C., 1998. Fritz, D., Danko, I., Roberds, S.L., Campbell, K.P., Latendresse, J.S., Wolff, J.A. Expression of deletion-containing dystrophins in mdx muscle: Implications for gene therapy and dystrophin function. Pediutric Research, 37:693-700, 1995. Haagstrom. J.E., Sebestyen, M.G., Ludtke, J.J., Fritz, J.D., Wolff, J.A. Complexes of non-cationic lipoaomes and histone HI mediate efficient transfection of DNA without encapsulation. Riochimica Riophysica Acta, 1284:47-55, 1966. Linderman, J.K., Gosselink, 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 Journal o j Physiology, 267:R35&R37 I , 1994. Tidball, J.G., Spencer, M.J. PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle. Journal of Cell Biology, 123:627-635, 1993.

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

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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

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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) bpcarotu; 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-

Crowing Crops for Space Explorers

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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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-


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-

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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


159

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

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19 20 21 22 23 24 25

26 27

28

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33. 34. 35.

36.

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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

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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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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,

'*


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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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

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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).


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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

4000

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FRACTION NO.

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

175 N

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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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IOHANN BAUER et al.

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,


177

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f

f

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a,

-0

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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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IOHANN BAUER et a/.

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

Electrophoresis in space

179

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)

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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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JOHANNBAUER et al.

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


181

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.

JOHANN BAUER et al.

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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

183


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.

JOHANN BAUER et al.

184

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


185

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.


187

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-


189

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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IOHANN BAUER et al.

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


191

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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JOHANN BAUER et a!.

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.,


193

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

JOHANNBAUER et al.

194

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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JOHANN BAUER et al.

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.

197


.* *

--

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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JOHANN BAUER et al.

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-


199

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.

201


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:

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