(Advances in Space Biology and Medicine 6) Sjoerd L. Bonting (Eds.) - Elsevier Science (1997)

ADVANCES IN
SPACE BIOLOGY
AND MEDICINE
Editor: SJOERDL. BONTING

Goor, The Netherlands
VOLUME 6 1997
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LIST OF CONTRIBUTORS
August0 Cogoli Space Biology Group

ETH Technopark
Zurich, Switzerland
Marianne Cogoli-Greuter Space Biology Group

ETH Technopark
Zurich, Switzerland
). Darginaviciene Institute of Botany

Lithuanian Academy of Sciences
Vilnius, Lithuania
A.D. Eggorov Institute for Biomedical Problems

Moscow, Russia
A.I. Grigoriev Institute for Biomedical Problems

Moscow, Russia
A. Cue11 lnstitut de Medecine et de

Physiologie Spatiales
Toulouse
France
G. Houin Laboratoire de PharmacocinCtiqueet

Toxicologie Clinique
Hopital Purpan
Toulouse, France
Akerni Izurni-Kurotani Space Utilization Center

Institute of Space and Astronautical Science
Kanagawa, Japan
Richard Jennings NASA-JohnsonSpace Center

Houston, Texas
vi i
... LIST OF CONTRIBUTORS
Vlll
Nick Kanas Department of Psychiatry

University of California, San Francisco
San Francisco, California
lrena Konstantinova Institute for Biomedical Problems

Moscow, Russia
Ludmila Kornilova Institute for Biomedical Problems

Moscow, Russia
lane M. Krauhs KRUG Life Sciences Inc.

Houston, Texas
Carolyn 5. Leach NASNJohnsonSpace Center

Houston, Texas
A. Merkys Institute of Botany

Lithuanian Academy of Sciences
Vilnius, Lithuania
Yoshihiro Mogami Department of Biology

Faculty of Science
Oc hanomizu University
Tokyo, Japan
Makoto Okuno Department of Biology

Faculty of General Education
University of Tokyo
Tokyo, Japan
A. Pavy-Le Traon lnstitut de Medecine et de

Physiologie Spatiales
Toulouse, France
M. Pujos lnstitut Europ4en de T6IPrnCdecine

Hopital Purpan
Toulouse, France
S. Saivin Laboratoire de Pharmacocinetique et

Toxicologie Clinique
Hopital Purpan
Toulouse, France
List of Contributors ix
Steven H. Schwamkopf Lockheed Missiles and Space

Corporation, Inc.
Palo Alto, California
Scott M. Smith NASA/Johnson Space Center

Houston, Texas
Gerald Sonnenfeld Carolinas Medical Center

Charlotte, North Carolina
C.Soulez-LaRiviere European Space Research and Technology

Centre
Noordwijk, The Netherlands
Gerald R. Taylor NASA-JohnsonSpace Center

Houston, Texas
Luzian Wolf European Space Agency

ESTEC
Noordwijk, The Netherlands
Masa m ichi Yarnashita Space Utilization Center

Institute of Space and Astronautical Science
Kanagawa, Japan
INTRODUCTION TO VOLUME 6
The sixth volume in this series, Space Biology andMedicine, is a regular volume
again with contributors from all spacefaring nations. Although all space agencies
must currently operate under severe budgetary restraints, progress in the field of
space biology and medicine continues. The preparations for the International Space
Station, in which Russia is going to participate with the United States, Europe,
Japan, and Canada, are also continuing. For the longer term, studies for a Lunar
Station are in progress. The contributions to this volume are witness of all these
activities.
Two chapters deal with the effects of weightlessness on the immune system.
Taylor and colleagues review the effects in vivo, indicating a reduction of the
immune function in space. The blunting of the immune function after short-term
flights resembles that after acute stress on the ground, while long-term effects
compare to those caused by chronic stress. Cogoli and Cogoli-Greuter describe
studies on single cells, which show that proliferation and cytokine expression of
T-lymphocytes are reduced in microgravity, possibly through a non-equilibrium
thermodynamic effect.
Preparation for long-term space missions is the express purpose of four contri-
butions in this volume. Kanas considers the usefulness of space simulation studies
by means of extended isolation and confinement on Earth, and points to be
examined in future projects of this kind. Volumes 3 and 5 in this series were
dedicated to two ESA projects of this nature. Grigoriev and Egorov describe a
xi
xii INTRODUCTION TO VOLUME 6
medical monitoring system for long-term missions. Schwartzkopf reports on the

design of life support systems using plant cultivation for food and oxygen regen-
eration, with particular reference to a future Lunar base. Wolf describes a small-
scale bioregenerative system based on an algal bioreactor.
The use of medicinal drugs by astronauts is the subject of two chapters by
Pavy-Le Traon, Saivin and colleagues.The resistance of bacteria to antibiotics can
be changed in weightlessness, and no suitable drug against bone demineralization
in space is available. The pharmacokinetics of drugs is also changed, mainly due
to the fluid shift in space. Smith and colleagues provide a comprehensive review
of our present knowledge of the regulation of body fluid volume and electrolyte
levels and the hormonal regulation mechanisms involved, The effects of weight-
lessness on the function of the vestibular system, which are the cause of space
motion sickness in astronauts during the first week in space. are reviewed by
Kornilova.
Finally there are two chapters on the effects of gravity on non-human creatures.
Izumi-Kurotani describes behavior and stature of frogs during spaceflight, as well
as some histological and biochemical changes in organs and tissues after return to
Earth. Merkys and Darginaviciene have studied the mechanism of the plant
gravitropic response (spatial orientation along the gravity vector) in space and on
Earth, and conclude that flows of calcium ions and the growth hormone indole
acetic acid in opposite directions appear to be involved.
It is the editor's hope that this volume will serve the purpose of the series to
review and summarize the findings of space biology and medicine for the benefit
of all those interested in this field of science.
Sjoerd L. Bonting
Editor
Chapter 1
CHANGES IN THE IMMUNE SYSTEM

DURING AND AFTER SPACEFLIGHT
Gerald R. Taylor, lrena Konstantinova,

Gerald Sonnenfeld, and Richard Jennings
I. Introduction ..... ... .. . . ... ....., . ... . ... .. ... .. 2

LI. Immunology Studies in Space and on the Ground . . . . . . . . . . . . . . . .' 3
A. Spaceflight Studies Involving Human Subjects . . . . . . . . . . . . . . . 3
B. Spaceflight Studies Involving Animals . . . . . . . . . . . . . . . . . . . . I8
C. Ground-based Analogs of Spaceflight , , , . . . . , , . . . . . . . . . . . 19
III. Probability of Inflight Infectious Disease . . . . . . . . . . . . , . . . . . . . . 23
IV. Stress and Mission Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
A. Acute Stress. . . . . , . . . . . . . . . . . . . . . . . . . . . . , . . . . . 2 6
B. Chronic Stress . . . . . . , , . . . . . . , . . . . . . . . . . . . . . . . . 2 7
V. Conclusions and Summary . . . . . . . . . . . . . , , . . . , . . . . . . . . . 2 8
References . , . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . . . . . , 2 8
Advances in Space Biology and Medicine

Volume 6, pages 1-32
Copyright 0 1997 by JAI Press Inc.
All rights of reproduction in any form reserved.
ISBN:0-7623-0147-3
1
2 TAYLOR, KONSTANTINOVA, SONNENFELD, and IENNINCS
1. INTRODUCTION
Spaceflight represents an unusual occupation that demands uncommon activities
to be performed in a unique environment. This exceptional combination of envi-
ronment and activity has been shown to cause measurable changes in the immune
mechanism of humans and animal surrogates.' The effect of spaceflight on the
human immune system has long been studied so that some fundamental problems
of space biology and medicine have been defined. These problems include the
significance of Earth gravity and stress reactions for cellular relationships under-
lying an immune response, for intracellular processes leading to activation of
lymphocytes and expression of the receptor apparatus of cells, and intersystemic
relations between the neuroendocrine and immune systems. Successful resolution
of these problems is necessary to accommodate the ever increasing duration of
manned space missions, which is only possible with continued monitoring of many
physiological functions, including the immune system.2
The human immunology studies have generally involved in vitro analysis of
samples collected before or after, and rarely during, spaceflight. Only on very rare
occasions has immune competence been measured within the subject in flight.
Immunological analyses have been incorporated in both the United States and
Russian space programs, although these studies have never reached the highest
priority in either. Many of the appropriate tests have not yet been satisfactorily
conducted, even though both countries have been actively involved in space life
sciences research for more than thirty years. This situation has resulted from the
relatively low priority given to in flight immunology research, combined with the
inherent difficulties associated with conducting research concomitant with a space-
'-'
flight.
In their December 1985report entitled "ResearchOpportunities on Immunocom-
petence in Space",The Federation of American Societiesfor Experimental Biology
(FASEB) analyzed the status of spaceflight immunology research at that time?
They observed that for a variety of reasons, the national programs, designed to
investigate immunologic aspects of manned spaceflight, had been inadequate.
However, they observed that a number of immunology experiments, some of which
were of high quality, had provided useful data that suggested some concepts for
future research planning. Nevertheless, competentjudgment of whether space-re-
lated immunologic changes have finite clinical or operational implications was, at
that time, hampered by lack of knowledge. They concluded by enumerating the
most significant elements of a productive space flight immunology program." In
the years since this national report, considerable information has been gathered in
conjunction with both the Russian and US space programs.
immune System Changes in Spaceflight 3
II. IMMUNOLOGY STUDIES IN SPACE AND ON THE

GROUND
A. Spaceflight Studies Involving Human Subjects
Long-duration results have mostly derived from the Russian space program.'
Only three U.S. missions, with 9 astronauts total, have materially exceeded two
weeks duration. These were the 28-, 58-, and 84-day visits to the U.S. Skylab.s
Conversely, the Russian program has successfully maintained cosmonauts in space
for up to one year. Whereas the Russian program has emphasized long-duration
activities in their Salyut and MIR space stations, the U.S. Program has conducted
many more short-duration flight^.^ Similar activities have been conducted in these
various missions, regardless of flight duration or parent national program. Most
immunological studies have involved in vifro analyses of blood samples collected
as soon as possible after landing, with the results being compared with the preflight
values for that individual.
Cellular immune responses of U.S. and Russian crew members have been studied
by various methods for more than two However, there remains a
paucity of reliable information from which to draw conclusions. Considerable
immunological testing was performed following the eleven U.S. Apollo missions,6
the three US. Skylab flights,' and the U.S./Russian Apollo-Soyuz mission." In
addition, post flight alterations in the in v i m response of cosmonaut lymphocytes
were reported for the Soyuz 6, 7, 8, and 9 flights, for the two Soyuz visits to the
Salyut 4 Space Station, for Soyuz 24, Salyut 5, Soyuz 26 and 27, and for two visits
to Salyut 6.9*i2-'4Because of small sample size, mission anomalies and constraints
on analytical conditions, the resulting data were suggestive but generally not
conclusive.
In the U.S. space program, immunological investigations commenced with the
Apollo series (1 968 to 1972). The duration of the Apollo flights ranged from 143
hours (Apollo 13) to 302 hours (Apollo 17). Postflight studies failed to demonstrate
any alterations in RNA or DNA incorporation in response to phytohemagglutinin
(PHA). However, as shown in Table I, a marked increase in the peripheral
neutrophil count, and a trend towards a reduced lymphocyte count was reported 2
hours after landing. These values returned to preflight levels within 24 hours after
landing.6
Three crew visits to the US. Skylab were conducted between May, 1973 and
February, 1974 and lasted 28, 59.5, and 84 days. Following these three visits of
astronauts to the U.S. Skylab, the postflight hnctional capacity of crew lympho-
cytes, measured in terms of DNAproduction in response to PHA, was ~nchanged.~
However, postflight RNA production was reported to be depressed, as shown in
Figure 1.
The data illustrated in Figure 1 demonstrate that the stimulation ofRNAsynthesis
by phytohemagglutinin (PHA) was nearly abolished in all six flight subjects
4 TAYLOR, KONSTANTINOVA, SONNENFELD, and JENNINGS
Table 1. Summary of Apollo Results

Postflight, Mean f SO
Preflight
Parameter MeanfSD R+ZHrs R+lDay R+7Days R+l4Days
White cells 7000 f 1800 8900 f 3000 7300 & 1900 6500 f 1700 6500 f 2200
Neutrophils 3900 f 1 1 00 +
6200 f 2600 4000 1 1 00 3500 f 1300 3900 f 1800
Lymphocytes 2bOO f 700 +
2300 1300 2700 f 1100 2400 f 800 2300 f 800
Notes: The preflight mean represents the average of approximately 99 determinations ( 3 per crew member).
The postflight means are average of 33 determinationsor less (1 per crew member).
Units in each case are standard with respect to routine hematology parameters and represent
cells/mm3
Source: After K i m & ' 201
followingthe Skylab2 and 3 missions. However, a similar depression was not noted
following the longest (84 days) mission. It is likely that this is due to differences
between the postflight activities of this last Skylab mission and is not a function of
mission duration. The Skylab data, Table 2 and Figure 2, also demonstrate the trend
towards postflight increased neutrophils and decreased lymphocytes in the periph-
eral circulation, first noted for the Apollo missions.
After the joint U.S.-USSR Apollo-Soyuz Test Project (ASTP) flight, variable
lymphocyte responses to a variety of mitogens. as well as absolute leukocytosis,
were reported." However, because all three US.crew members were exposed to
a toxic level of nitrogen tetroxide upon landing, and subsequently received gluco-
corticoid therapy, it is not possible to attach any spaceflight-related importance to
these data.''
Response of lymphocytes to Mitogenic Challenge
In the Russian space program, alterations in the in v i m response of cosmonaut

lymphocytes to mitogenic challenge were reported after the Soyuz 6, 7, 8, and 9
flights.I6 Each of these flights had a crew of two, except Soyuz 7, with a crew of
3. All flights were just under 5 days in length, except S o p 9 which lasted I7 days
and 17 hours. Tritiated uridine uptake was estimated by photographic film exposure
with variable results.
Subsequently, a decreased PHA-reactivity was found in 33 out of 46 Russian
cosmonauts after prolonged spaceflightsof 30 to 366 days on Salyut 4,6,7 and the
Mir orbital station. The average percentage of lymphocytes labeled with 3H-uridine
+
after 24-hour cultivation with PHA was 20.4 1.06%, 30 days prior to flight, and
12.9 f 1.08% the day after landing, as shown in Figure 3. These depressed values
returned to normal 7 days after landing. In another study, a moderate decrease in
the number of cells positive for the marker was noted in 5 of the 8 crew members
figure 1. RNA syqthesis rates in lymphocytes, cultured with and without PHA, obtained p m the Skylab crew and control groups. Cells
were pulsed with H-uridine at 23 h and harvested at 24 h after initiation of the cultures.
Table 2. Summary of Skylab Immunology Results

Postflight Day
Preflight
CrewMember Cells MeanfSD R+O R+l R+4 R+7 R+l3
uo/lab 2
Commander WBC 7020 f 590 6800 7600 6500 6700 6000
Neutr. 3010f350 3880 4480 2290 2880 3300
Lymph. 3490 f 350 2788 3040 2860 3350 2400
Scientist-Pilot WBC 54aof 1150 6300 6300 5000 4800 3900
Neutr. 2660 f 440 3840 3280 2350 2110 2070
Lymph. 2420 f 770 2020 2580 2300 2300 1640
Pilot WBC 5020 f 950 6700 6000 5600 5800 3800
Neutr. 2800 f 300 5630 3660 3800 3600 2200
Lymph. 1750 f 620 1010 2280 1510 1800 1400
Skylab 3
Commander WBC 5770 f 970 9700 7300 5800 7100 5000
Neutr. 3030 f 650 7370 4230 2900 4260 2750
Lymph. 2470 f 730 1750 3060 2730 2 700 2050
Scientist-Pilot WBC 5400 f 880 12000 9200 5500 400 5700
Neutr. 3300 f 780 9240 61 60 3300 700 3600
Lymph. 1930 f 450 2040 2940 1760 2500 1940
Pilot WBC 41 00 f 290 6500 5600 4400 4600 3500
Neutr. 2560 f 254 4420 3530 2330 1770 2900
Lymph. 1360 f 400 201 0 2010 1940 1930 1770
Skylab 4
Commander WBC 4440f305 6400 5400 5000 5200 3400
Neutr. 2590 f 41 0 4800 3290 2900 2960 1970
Lymph. 1510f200 1340 1780 1700 1870 1220
Scientist-Pilot WBC 4940f500 loo00 7500 6200 41 00 4400
Neutr. 2940 f 460 8100 4880 4530 2500 2950
Lymph. 1720f300 1400 2030 1360 1440 1280
Pilot WBC 6260f118013200 6500 6200 5800 5200
Neutr. 3860 f 1340 11 220 4550 3470 3360 3070
Lymph. 2090 f 290 1580 1560 21 70 2080 1820
Nofe: All numbers represent celldmm3.

Source: After Kimsey’
after a stay of 60 to 90 days in space. Despite identical conditions, there were no

deviations in the other crew members.’
The data summarized in Figure 4 show that the PHA response decreased in 20
of 26 cosmonauts after flights of 112 to 175 days. Five of these individuals showed
a profound decrease, only 0 to 3.3% of cells being labeled with 3H-uridine.After
the six longest flights (2 11-366 days) the changes were found to be moderate: no
/mmune System Changes in Spaceflight 7
5 -
0 cmnda
0 h d b l PuCl
A Pm
i'-
0 I I I
-20 -10 0 0 10 x)
Pf*M PmlilQhl
T i m . day
10 20
0 - a-20 .10 0 Podfilm
Figure 2. The white cell, neutrophil, and lymphocyte counts of the Skylab-3 crew
during the preflight and postflight examination p e r i ~ d s . ~
8 TAYLOR, KONSTANTINOVA, SONNENFELD, and IENNINGS
figure 3. PHA reactivity of lymphocytes in cosmonauts after prolonged spaceflights

(30-366 days). n = 46. Here and in Fig. 4 the shaded areas represent the allowable
range of the norm. L + 1 = 1 day after landing2
deviations in 4 out of 10 crew members, and a moderate decrease in the other 6

cosmonauts. The average postflight response of 29 subjects, who were in space for
7 to 10 days, was unchanged from the preflight ~ a l u e . ~ ” ~
lymphocyte Responses After Multiple Missions
Multiple examination of 12 cosmonauts participating in two or three long-term

space missions, demonstrated that this parameter remained normal and quite
constant in each person during periods of 2 to 8 years between space flights. The
figure 4. Average PHA reactivity of “T” lymphocytes in cosmonauts after (landing +

1 day) prolonged short-term spaceflightson Salyut and Mir orbital stations.2
Immune System Changes in Spaceflight 9
following example exemplifies these results. One cosmonaut participated in an

extended-duration mission in 1978, a short-term flight in 1980, and another
extended mission in 1987. In 1980,29.2% ofthe cells were labeled with 3H-uridine
preflight, while in 1987the corresponding value was 27.2%. A lower value of 11.8%
was recorded for this cosmonaut prior to the first flight in 1978,but this lower value
was associated with a “severe dysbacteriosis as revealed by microbiological intes-
tinal studies.”’
Studies were conducted on Russian crew members participating in more than one
flight to evaluate whether or not there exists a trace response following the first
extended space travel and, if so, how it manifests itself during repeated exposure
to spaceflight.2In one study of 12 cosmonauts, it was shown that on the day after
termination of the first extended mission, there was a decrease of PHA-reactivity
among all subjects. In this case the change ranged from moderate to highly severe.
In succeeding flights, six cosmonauts of this group again had a decrease of the
measured parameter on the first postflight day. The magnitude of change was
similar, despite the fact that the interval between the first and second flights ranged
from 2 to 4 years.
One of these six cosmonauts participated in three extended space missions. The
duration of the missions was 237 days in the orbital station Salyut-7, followed by
flights of 125 and 169 days in the Mir space station. During all three missions
similarly low parameters were recorded on the first postflight day, 11.1 YO,16.0%
and 13.4%, respectively, which a week later had increased to 24.6%. 20.0% and
25.0%, respectively.’
The postflight pattern of the other six cosmonauts, who participated in two or
three prolonged missions, was different. Their PHA-reactivity was decreased only
after the first flight. No correlationbetween reactivity, flight duration, or the interval
between the first and succeeding flights could be determined. For instance, in one
cosmonaut, after the first flight of 65 days aboard the Salyut-7 station, a decreased
PHA-reactivity was revealed on the first day after flight, and this reduced level was
also observed a week later. However, his PHA-reactivityremained normal after two
succeeding missions of 151 and 176 days on board the Mir station, which were
separated by a 3-year interval.*
Similar changes, i.e., a decreased PHA response only after the first flight and no
response to subsequent missions, were noted in the other five persons who made
two long-term missions. Among them, one cosmonaut flown on the Mir station
should be especially mentioned. After his first flight of 151 days there was a
significant postflight decrease in PHA-reactivity, to 10.6%.However, after his
second flight, 3 years later, of 310 days, there was no postflight decrease in
PHA-reactivity of T-lymphocytes? The authors suggest that these results indicate
the possibility not only of adaptation of the immune system to the flight environ-
ment but also a peculiar memory habituation to these conditions. They further
propose that analysis of the responses to repeated spaceflights has revealed two
possible reactions: the first reaction manifesting itself in uniform shift dynamics,
whereas the second response results in the development of a training effect from
the first flight.2
Changes in Humoral Blood Components

Serum complement levels have been measured after many Russian space flights.
Postflight values showed various changes compared to preflight averages. C3 levels
were significantly higher following the 16-, 18-, and 49-day Salyut flights. C4
levels were generally unchanged postflight, except that the 49-day Salyut 5 flight
resulted in significantly higher postflight C4 values, as well as a large increase in
serum immunoglobulin IgA, IgG, and IgM
Numerical Changes in Cells of the immune System

Extensive comparisons of preflight and postflight immunological parameters
were conducted with the first 41 US.Space Shuttle astronauts, who were in space
from 54 hrs to 244 hrs (2.3 to 10.2 days duration). This study, summarized in Table
3, demonstrated unequivocally, for the first time within the U.S. space program,
that the absolute number of lymphocytes in the peripheral circulation, the ability
of these cells to respond to mitogenic stimulation, and the number of eosinophils
in the peripheral circulation, were typically decreased after flight. Conversely,there
was an almost universal doubling of the absolute neutrophil number. Often there
was a major change in the CD4/CD8 ratio, resulting from an increase in the Helper
Table 3. Summary of Postflight Changes in Shuttle Crew Peripheral Blood Cells

Parameter N NPI NPD APC
Lymphocyte number 41 9 31 - 13.3
Lymphocyte stimulation 41 5 36 - 25.7
Neutrophil number 41 40 1 + 102
Eosinophil percent 41 4 35 NA
Pan T lymphocyte number 11 6 5 + 1.6
Pan B lymphocyte number 11 4 7 + 9.7
Pan monocyte number 11 3 7 -11.6
T helper cell number 11 a 3 + 11.1
T suppressor cell number 11 5 5 - 2.3
T4n8 ratio 11 7 4 + 13.4
Notes: N = number of crew members
NPI = number of postflight increases
NPD = number of postflightdecreases
APC = average postflight change in percent
NA = omitted as ratio of two small numbers is not a meaningful value
Source: From Taylor and Dardanoa

tmmune System Changes in Spaceflight 11
(CD4) lymphocyte Additional data from 11 of these crew members

indicatea postflight decrease in circulatingmonocytes and B-lymphocytes. Further,
the reduced T-lymphocyteblastogenesis was shown to correlate with the decreased
monocyte count. Monocytes serve a critical role during lymphocyte activation as
potent immunoregulator cells through release of cytokines. So these findings
suggest a possible mechanism for blunted in vitro mitogen-induced blastogene-
sis.'s3
More recently, an additional group of 30 US Shuttle astronauts were evaluated
using similar methods." The resulting data, presented in Table 4, confirm the
customary granulocytic increase and lymphocytic decrease within the peripheral
circulation postflight. However, contrary to previous findings, this study reported
a 52% increase in the postflight monocyte population. This increase was borne out
by a significant increase in monocytes (P < 0.0 l), which was derived from analysis
of isolated peripheral blood mononuclear cell fractions. The authors speculate that
the apparent discrepancy may be the result of differences in mission length. The 30
subjects that demonstrated a postflight increase in peripheral blood monocytes were
in space for 4 to 5 days, while other studies of crew members that were in space for
6 to 8 days showed a postflight decrease in peripheral blood monocytes."
These results suggest that the monocyte population may move between compart-
ments as the mission progresses up to 8 days. This compartmental shift may in fact
Table 4. Effect of Spaceflighton Peripheral Blood Leukocytes

of 30 United States Astronauts
Launch Launch Landing
Parameter - 10 Days - 2 Days landing Day + 3 Days
Total leukocytesa *
5800 200 5600 i 200 7000 f 200' 5680 ? 200
Granulocytesa 3200+116 3 0 2 0 i 112 4970 f 140' 3080 f 112
Lymphocytesa 2262 i 58 2240k 112 1680 f 70' 2 1 3 0 i 112
Monocytesa 133 i 7 190+22 245 f 28' 190 28 +
Monocytes (CD14+)b 13kl 12i1 21 f 1 13+1
*
T inducer (CD4+, L e ~ - 8 + ) ~ 32 + 2 34 i 2 23 f l., 31 f 2
T cytotoxic (DCD8+,CDll bTb 18il 16i 1 12fl 17f2
b
T helper (CD4+, Leu-87 5i1 7 i1 7fl 7+1
T suppressor (CD8'; CD11 b+)b 3i1 3fl 3kl 2f1
NK cells (CD16' or CD56+)b'C 911 9 f l 3fl 5+1
6 cells (CD19+)b 751 6 i1 6 f l 7+1
Notes: "Data are mean i SE of cells/mm3 determined by slide whole blood differential counts
bData are mean 5 SE of the percentageof MNC which express specific cell-surface antigens
'NK assay performed on samples from 10 astronauts
-
'P < .01. Landing versus launch 10 days and landing + 3 days
"P < 05. Landing day versus launch - 10 days and landing + 2 and + 3 days
Source: After Meehan et al.lU

12 TAYLOR, KONSTANTINOVA, SONNENFELD, and JENNJNCS
be preceded by related neuroendocrine changes. Meehan and co-workers recognize

that the increased monocyte count is inconsistent with their finding of an increase
in glucocorticoids, since the latter should be accompanied by a decrease in periph-
eral blood monocytes.I * Likewise, there was no postflight increase in the percent-
ages of insulin receptor-positive cells and IGF-I receptor-positive cells, as would
be expected with increased monocytes. Therefore, these investigators may have
sampled the population between a change in the neuroendocrine affector, and the
immune cell response. In addition to the interesting monocyte findings, this study
confirmed the previously shown postflight reduction in the number of T-inducer,
T-cytotoxic, and NK cells in the peripheral circulation.
Production of Interleukin-2, Interferon-2, and Interferon-y
In vivo T cell proliferation requires interferon-gamma (IFN-y) and probably

interleukin-1 (IL-l).I9 In addition, proper secretion and activity of interleukin-2
(IL-2) is necessary for feedback control between lymphocytes.20Antigen present-
ing cells (APCs), which are mostly macrophages, produce molecules that are
involved in the activation of lymphocytes, particularly T cells. Best characterized
is IL- 1 that has many other actions as well. Both resting T- and B-lymphocyteshave
receptors for IL- l.19 All APCs have major histocompatibility complex (MHC) class
I1 antigens on their surface. Expression of these antigens is not a fixed process, but
is under complex regulation. Class I1 antigen expression of monocytes, macro-
phages, endothelial cells, and astrocytes requires the presence of inducing signals
such as IFN-y, which is produced by activated T cells and natural killer (NK) cells.
A degree of immune activation, therefore, leads to the production of IFN-y, an
increase in APC function, and the potential to activate T cells further. Thus IFN-.I
acts as a positive feedback signal.”
Significantdecreases in interleukin production (especially IL-2) and IFN-y have
been reported in cosmonauts as well as in rodents after spaceflights of up to I68
days.10,16.21-23 Add‘itionally,
. significant decreases in IFN-alB and IFN-y with a
decreased ability to elaborate IFN-a have been reported in blood collected post-
flight from 6 out of 9 Russian cosmonauts after 7-8 days in space.
The mitogen-induced production of interleukin 2 (IL-2) was shown to decrease
perceptively, compared to preflight levels, in 12 of 13 cosmonauts studied on the
first postflight day after Russian space missions of 2 to 12 months duration. The
data presented in Figure 5 show that although the magnitude of the change vaned
among subjects, a reduction of about 50% was observed in two out of two
cosmonauts after a flight lasting two months, and in three cosmonauts out of seven
following flights lasting 4 to 6 months. The decrease in JL-2 production was even
greater in three other individuals remaining in flight up to six months, and in all
three cosmonauts following the two longest flights of 11 and 12 months2
The in vitm production of EN-2 and IFNy by blood lymphocytes, in response
to Newcastle disease virus and Con A. was studied in sixteen individuals before
2 12 month
(flight tlrn)
100 -r
90-
00 -
70 -
60-
%
50 -
40 -
30-
20 -
10 -
Figure 5. Interleukin-2 (IL-2) production by lymphocytes of 13 cosmonauts after
prolonged spaceflights (biologicaltests with CTLL cell line).2
and after prolonged flights. The IF"-a levels after landing did not differ from
preflight values in eleven cosmonauts, although this factor increased noticeably in
three and decreased in two subjects. The production of IFN-y was unchanged in
six, increased in five, and decreased in five individuals. The authors thus concluded
that there is no correlation between interferon production and the flight duration,
ranging from 125 to 366 days?
It is interesting to compare these data with the effect of short-term flights of 7 to
8 days, which represents the acute period of adaptation to the absence of terrestrial
gravity. After one week stay in microgravity the production of IFN-I/ had decreased
in all 17 cosmonauts investigated in this group, while the production of IFN-ahad
decreased only in seven individuals and remained unchanged in eight. In no case
was an increase in the formation of IFN-a or IFN-y observed after brief flights.
This result led the authors to conclude that stressful demands associated with the
first seven to eight days in microgravity are characterized by the suppression of the
capacity of the lymphocytesto synthesizeinterferons, particularly IFN-y. However,
an increase in flight duration to 366 days leads to quite different results. In this case,
decreased, increased, and unchanged production of IFN-y was observed with equal
frequency. These data demonstrate that the synthesis of IFN-y is more labile in
extreme flight conditions, whereas the synthesis of IFN-a is more resistant,
consideringthat IFN-alevels remained unchanged in 11 out of 16 cosmonauts after
prolonged flights, and in 8 out of 17 cosmonauts after short flights.*
CyiotoxicActivity of Natural Killer Cells
The cytotoxic activity ofNatural Killer cells was tested on the basis ofthe amount
of non-degraded 3H-RNAthat remained in the target cell (Human K562 cell line)
after contact with NK cells according to a method developed and studied in detail
by Rykova et al.24as a modification of the Hamaokaz5technique.
This activity was studied in 33 cosmonauts that participated in spaceflights of 60
to 366 days duration. The data summarizedin Figure 6 indicate that the cytotoxicity
index (CI) averaged 37.4 k 3.4 before flight for the entire group, decreased to 27.0
f 5.0 one day after landng, and remained low (23.5 f 4.2) one week after landing.
Normalization took place subsequently in some of the cosmonauts so that 14 days
after flight the CI group average was 3 1.4 f 7.8.* By analyzing the individual CI
values obtained after flight, the authors observed a decrease in this index below the
lower limit (CI = 20) of the normal range in 18 of the 33 cosmonauts examined on
the first day after landing. This decrease was characterized as moderate in four
individuals (CI = 15.6 to 18.4), large in seven crew members (CI = 5.0 to 9.4), and
largest in 7 cosmonauts (zero in five of these 7 subjects and 4.5 in the other two).’
An effort was made to determine if there was any correlation between the
magnitude of the postflight reduction in NK cell activity and the flight duration.
The data from all cosmonauts examined after prolonged flights were divided into
two groups, depending on the length of time on board the orbital station, 65 to 177
days, and 2 11 to 366 days. The authors indicated that three variants were observed
in the first group.2 First, a decrease in the CI on the first day after landing, with an
increase toward the norm in the following examination on the 7th day was seen in
four cosmonauts. Second, absence of a decrease in the CI on the first day after
landing with a significant fall in the CI on the seventh day was noticed in seven
I
I
I
I
I T
1
I
#- 30
I
I
I
c
G- I
I T
I
I
I
I
.
20 I
I
I
IL+l
L+7 L+14 dsyr
postlligM
Figure 6. Cytotoxic activity of natural killer (NK)cells in cosmonautsbefore and after

spaceflights lasting 60-366 days (n = 33).2
cosmonauts.Third, a decrease in the CI on both the first and seventh days postflight
was observed in eight cosmonauts. They further reported the same three variants
of NK activity in ten cosmonauts in the second group (flights lasting 2 11 to 366
days), indicating that an increase in the flight duration from six to twelve months
does not lead to an increase in the magnitude of the changes in NK cytotoxic
activity.
This study also investigated NK cell activity in 28 cosmonauts following short
term flights (7 to 8 days). The CI group average was 24.5 f 5.1 on the first day after
landing, with a decrease in this index being observed in 16 individuals. This
decrease was significant in 9 cosmonauts (CI from 0 to 5), suggesting an enhanced
sensitivity of NK cells to the factors associated with the acute period of adaptation
to extreme flight conditions.2
f imitations of Postnight and In Vitro Analyses
Our present insight concerning immunological changes due to spaceflight is

nearly completely based on postflight analysis of astronauts compared to their
preflight values. This is inevitable, because it is usually operationally impossible
to collect data in any other way. However, reliance on postflight analyses presents
two serious problems of data interpretation. First, and most important is the fact
that the samples must be collected after the subjects have endured the stressful
conditions associated with landing and with readaptation to terrestrial conditions.
Thus, one cannot adequately separate inflight effects from landing effects on the
immune Secondly, the length of time elapsing between landing of the
spacecraft and sample collection or medical analysis varies with each flight. The
latter problem has been alleviated somewhat with the advent of the U.S. Space
Shuttle program, since the shuttle vehicle normally lands on a pre-deterrmined
runway. This is a great improvement over previous programs, where the actual
landing of a capsule could be many miles from the anticipated site leading to a wide
variation in the time elapsing between landing and sample analysis.
In a few cases, an attempt has been made to avoid the use of postflight samples
by collecting samples during flight, and either analyzing them inflight or postflight.
In the case of postflight analysis, unpredictable sample storage conditions seriously
compromised the results. Inflight sample analysisis compromised by the variability
of the analytical conditions,especially cell culture, between flights.2G32When the
inflight analytical or storage conditions are different from those used to produce
the ground-based control values, a valid comparison of the inflight data with the
control values is impossible.
Another limitation is that the tests have typically been conducted in vitro, which
requires an extrapolation in determining the degree to which crew members are
immunocompromised. Although this is no different than the situation one is
typically confronted with in health care, it has made statements about the clinical
importance of the noted immune changes more difficult to interpret. In the next
16 TAYLOR, KONSTANTINOVA, SONNENFELD, and JENNINGs
section the use of an in vivo test is described, which circumvents the extrapolation
inherent in the in v i m tests.
Delayed-type Hypersensifivity Skin Tests
Recently the effect of spaceflight on the ability of the human cell mediated
immune (CMI) system to function normally has been determined inflight by means
of an in vivo test, the Merieux Multitest Cell-Mediated Immunity (CMI) test.33This
allowed an inflight evaluation of the ability of U.S.Space Shuttle crew members
to mount a delayed-type hypersensitivity response. This test employs a plastic skin
puncture device that simultaneously injects seven different glycerinated recall
antigens and a glycerin control in a standard pattern. Reactivity,reliability, repeat-
ability, and safety of the antigens and the application technique have previously
been established through extensive field evaluation^.^^ Concentrations were se-
lected such that each was the lowest possible which still produced the maximal
incidenceof positive DTH reaction in a representative population of normal healthy
adults. This procedure is highly suitable for inflight use, because the incidence of
extensive skin reactions is low, thus allowing multiple tests with application sites
to be placed only 20 mm apart. The sensitivity of the test for detecting hypoergy or
anergy is maximized by using a minimal concentration of each antigen. The
delayed-type hypersensitivity(DTH) response to common recall antigens has thus
been established as a simple, yet effective, method for evaluating in flight-mediated
hyp~ergy.~~
The CMI mechanism was evaluated in ten astronauts, as shown in Table 5, by
measuring their inflight DTH response to the common recall antigens of Tetanus,
Diphtheria, Streptococcus,Pmteus, old tuberculin, Candida, and Trichophyton.”
For all subjects, except crew member 2, the cell-mediated immune system showed
fewer antigen responses inflight than preflight. It should be noted that crew member
2 was on the shortest flight tested. Crew member 4, who was aboard the 5-day flight,
was the only subject who demonstrated anergy during spaceflight. Inflight data
were also analyzed according to the total value, in mm. of the mean induration
diameters of all the positive reactions for a particular subject. This is referred to as
the reaction score. In all but two cases (crew members 1 and 2) the reaction score
was decreased during flight. Again, the two subjectsthat registered an increase were
aboard the shortest mission. These results demonstratethat hypoergy was the least
during the shortest (4 day) mission, whereas the 5-day mission resulted in the
greatest change.
These data suggest that on day four of a Space Shuttle mission the cell-mediated
immune system is measurably degraded and that between day 5 and day 10 the
depression becomes maximal, after which the CMI mechanism begins to adjust to
the new conditions.These findings would tend to support the previously discussed
monocyte data, because monocyte control also appears to change considerably
between day 4 and 5 of spaceflight. This similarity of results is very useful for
Table 5. lnflight Changes in DTH Reactions of Ten United States Space Shuttle
Crew Members
Positive Reactions (number) Reaction Score imm)
Mission
Subject Length (days) Preflight lnflight Preflight lnflight
1 4 6 5 31.5 32.7
2 4 4 5 16.0 18.3
3 4 6 4 37.1 18.3
4 5 2 0 7.0 0.0
5 5 5 2 22.8 11.0
6 5 5 3 26.0 10.5
7 10 5 3 19.5 11.5
8 10 5 3 21 .o 12.0
9 10 3 2 10.5 8.5
10 10 4 3 23.0 13.5
Note: DTH = Delayedtype Hypersensitivity skin test
Source: From Taylor and Ianneyl3
developing an explanation of the mechanism of immune depression early in the

mission, since cells of the macrophage lineage are generally considered to be the
main antigen-presenting cells in the DTH reaction.
The delayed-type hypersensitivity response of six Russian crew members was
evaluated on four Mir space station missions of 132, 177, 145 and 310 days
duration.2The skin-test was applied before, during and after the mission. The data
illustrated in Figure 7 show that scores before and after the mission were found to
be above the lower ‘warning’level of 10 mm in three out of six cosmonauts, whereas
the scores of the other three totaled only 6 mm or 9 mm nine days after landing.
The authors considered values below this ‘warning’ score suggestive of an in-
creased risk for deteriorated cell-mediated immunity, as defined by B ~ c k l e yIn .~~
two of these subjects the score remained below the warning level (10 mm) when
measured at 59, 155, and 240 days in space. In both of these cases a recovery to
normal was noted 8 days after landing.2
The authors note that the most important finding of this study is that in 3 out of
6 cosmonauts a dramatic decrease in delayed type cutaneous hypersensitivity was
noted during long-term spaceflight. They fkther note that the decrease in one
cosmonaut on mission day 59 followed a period ofsevere physical snd psychologi-
cal distress associated with performance of a series of three prolonged extravehicu-
lar activities one week before skin testing.2
25
-
€20
5
t
H 15
3 10
F;
pdllght lnfiiglt m portnrgm
figure 7. Total size of delayed-type hypersensitivity (DTH) reactions in six cosmo-

nauts (A through F) before, during, and after flights on the orbital MIR station. F95 =
95th day of flight (the day the test was done).2
B. Spaceflight Studies Involving Animals
Mammalian surrogate studies have long been used to simulate or mimic some of
the effects of spaceflight on the immune system of humans. These studies have
included both inflight and postflight analyses of animals flown on a variety of
manned and unmanned vehicles. In most cases rodents were used because of their
small size and the availability of extensive background information. However,
some work has been conducted with other animals, such as Rhesus monkeys and
dogs.
In studies conducted with rats, exposure to prolonged spaceflight resulted in
hypoplasia of lymphoid organs and alterations in mitogen-induced blastogenesis.
Also, IFN-y, but not interleukin-3 production, was reduced significantly in spleen
cells from rats that had been in space for 7 days during a U.S.Spacelab flight.36
Studies on Russian unmanned BioSputnik (Biosatellite)flights indicated postflight
shifts in populations of T-lymphocytes, helper cells, suppresser/cytotoxic T cells,
and interleukin-2 receptor bearing T-lymphocytes in rat spleen cells. Postflight
analysis of bone marrow cells of flight rats, as compared to ground controls,
revealed a large number of myelogenous cells bearing surface immunoglobulins.
In addition, bone marrow cells from flight rats were inhibited in their ability to form
colonies in the presence of colony stimulating factors(M- and GM-CSF), indicating
a lack of division, and possibly maturation, on the part of the precursor cells. Other
studies indicated that spaceflight resulted in decreased rat natural killer (NK) cell
and cytotoxic T-lymphocyte a ~ t i v i t y . ~ ' ~ ~ - ~ ~
Additional studies by Nash and colleagues 40i41 indicate that spaceflight did not
affect production of interleukin-2 or blastogenesis from lymph node cells. This is
in contrast to earlier studies, which indicated that spaceflight did inhibit these
actions of spleen cells.’ These data may indicate that spaceflight could have
different effects on different compartments of the immune response.
Studies carried out on rats flown aboard the Space Shuttle have confirmed that
leukocyte numbers and leukocyte subset distribution is altered by spaceflight.
Taking these findings together, it is now clear that spaceflight results in marked
changes in immune responses in laboratory animals, which could lead to alterations
in resistance to infection and tumors.The actual resistance studies remain to be
performed.
C. Ground-based Analogs of Spaceflight
Ground-based model systems have long been employed to obtain the information
required to develop spaceflight immunology studies as well as to supplement both
human and animal inflight experiments. In the case of research with humans these
model systems have included paradigms involving bed rest, academic or psycho-
logical stress, physical stress, hypobaric or high altitude stress, and confinement.
Animal models have centered around ground-based antiorthostaticand orthostatic
suspension, hypobarism, and Confinement.
Bed Rest
Bed rest has been extensively used to simulate some of the physiological
consequences of spaceflight. Thls paradigm seems to be especially useful in the
study of muscle deconditioning, and when employed with a head-down tilt can be
used to simulate cephalad fluid s h i f ? ~ . ~In’ contrast,
~ the use of bed rest has not
been widely used to evaluate immunological dysfunctions. However, clinical
observationsof bed-ridden patients have demonstrated reductions in IgG level and
phagocytic activity with concurrent increases in the incidence and quantitation of
pathogenic microorganisms. Perhaps the most ambitious of the non-clinical bed
rest studies involved nine subjects maintained in a head-down hypokinetic envi-
ronment for 370 days. This model demonstrated decreased Natural Killer cell
f i n ~ t i o n ?‘decreased
~ antiviral imm~nity’?~ increased numbers of T and B lym-
phocytes in the peripheral and decreases in blast transformation!6
These and other limited immunological measurements that have been conducted
on bed rested subjects, have demonstrated that the validity of bed rest as a model
for studying the effect of spaceflight on the immune system has not yet been proven.
Academic Stress
A particularly useful model system is academic stress. In one study designed to

evaluate the psychological stress associated with major academic examinationsby
first-year medical students, a significant depression of PHA-stimulated thymidine

uptake was observed in subjects exposed to a stressful environment. On the other
hand, no correlation could be made between depressed blastogenesis and the
relative numbers of mononuclear cell ~ubsets.~’
In a more recent study, the relationship between stress, immune function, and
illness in 96 first-year U.S. Air Force Academy cadets was investigated. This study
was the first attempt to examine simultaneously in virm immune function, Epstein-
Ban virus (EBV) reactivation, and risk of infectious illness in otherwise healthy
subjects exposed to a moderately severe environmental stressor. Another unique
aspect of this study was the use of two independent measures of illness. Reduced
in vitm PHA-induced lymphocyte transformation was reported to be associated
with the stressor. However, the results failed to confirm the hypothesis that the
moderate, stress-induced immune declines were useful in predicting subsequent
risk of illness during the test period!8 The investigators indicate that this finding
was unexpected because previous research had suggested a linkage between EBV
reactivation and academic examination s t r e s ~ . ~ ~ * ~ *
Physical Exercise
Physical exercise is perhaps the most widely used activity employed during
spaceflight to counter physiological decrements in crew members. The United
States and Russian space programs have used treadmills, bicycle ergometers,
resistive exercisers, and jogging in an attempt to diminish inflight changes such as
muscle atrophy and bone loss, and to minimize postflight orthostatic int~lerance.~’
However, it has long been known that prolonged moderate-to-extremeexercise may
have a depressive effect on the immune m e ~ h a n i s m . ~ *Several
- ~ ~ studies have
demonstrated that acute, prolonged endurance exercise, such as running, can
produce immune system effects similar to those reported after spaceflight. These
include an increase in total neutrophils in the peripheral circulation,with concomi-
tant decreases in natural killer cell activity, blast transformation ability, neutrophil
bactericidal activity, IL-2 production activity, and the numbers of cytotoxic/sup-
presser lymphocytes. Therefore, it is clear that the emphasis on inflight exercise
could exacerbate the noted immune system degradation. Accordingly, appropriate
use of exercise during spaceflight is very important to mission planners both to
limit negative effects on the immune system and to reduce the amount of crew time
devoted to inflight exercise activities.
Dysbarism
Another model of use to spaceflight planners is dysbarism. In the early days of

spaceflight, low pressure studies (i,e., less than one atmosphere) were of great
importance because several of the space vehicles (e.g., the Mercury, Gemini,
Apollo, and Skylab vehicles in the U S . program) maintained an environment with
an internal pressure considerably below one a t m o ~ p h e r e Although
.~~ modem
spacecraft typically maintain a one-atmosphere internal environment, this pressure

is routinely lowered for extravehicular activities (EVA). In addition, a Lunar
outpost, or Mars excursion, may similarly be designed with a reduced-pressure
environment.s63s7Hypobaric studies on Earth have been conducted either at high
altitude” or in decompression~harnbers.’~.~~ The latter techniquehas the advantage
of convenience combined with the ability to alter the partial pressure of oxygen in
addition to reduced pressure.
In one chamber study, 15 men and 13 women were exposed for 6 hours to
simulated EVA (100% oxygen pre-breathing at 1 atmosphere, followed by simu-
lated EVA activity at 0.33 atmosphere) in a decompression chamber. There was no
change in mononuclear cell phenotypes or in PHA-stimulated thymidine uptakeam
However, Giron demonstrated that mice maintained at a pressure equivalent to
6,000 m altitude while breathing 43% oxygen had a decreased resistance to
mengovirus infection.“ In another study spleen weights and antibody titers were
somewhat depressed in mice immunized with Brucellu abortus antigen and imrne-
diately placed in a 100% oxygen environment.62
In an actual high altitude (hypoxia) study, all of the 235 subjects maintained at
3600 m above sea level for 150 days displayed a significant decrease in circulating
B- and T-lymphocytes and reduced blast transformation during the first few days
of exposure.” These alterationswere found to persist in subjects in which mountain
sickness continued, but nor in those recovering from hypoxia symptoms.
Confinement
Confinement of individuals in small or restrictive spaces with isolation fiom

normal sensory affectors, has been shown to result in alterations to the immune
system with potential clinical consequence^.^^ These studies have involved a wide
range of environments, such as Antarctic expedition^:^ cave space-
flight6’ and other extreme environment^.'^ In the early days of space travel
confinement was a very useful model, as the habitation modules were very small
and movement was extremely restricted. Moreover, early crews consisted only of
one or two individuals in a single capsule. However, modem spacecraft are larger,
accommodatea larger crew complement, and provide for recreational and exercise
activities. Future missions to the Moon or Mars may require isolation of individuals
for long periods of time in a relatively small living area.’?’’ Accordingly, it is
important to understand the long-term effects of this restrictive environment on the
efficacy of the human immune mechanism.
Animal Suspension
A useful model that simulates some of the effects of spaceflight on animal

systems was developed in 1979,66which has subsequently been used by several
investigators to study changes in rodent immunity. In this model, the animal is
suspended with the rear limbs unloaded and unrestrained, whereaz the fiont limbs
22 TAYLOR, KONSTANTINOVA, SONNENFELD, and JENNlNGs
touch the surface below. The animals are generallysuspended with a 10 to 20 degree
head down tilt. This situation, in which the front limbs are exercised maximally but
the rear limbs are not, is considered by some investigators to be the kind of activity
that approximates human arm and leg movement during space flight. In fact,
animals thus suspended are reported to demonstratesome of the same physiological
changes seen in astronauts, including a cephalad fluid shift, a negative balance of
water, nitrogen and potassium, and increased metabolic
Additionally, SoMenfeld,6* and G o ~ l d , ~have
’ found in suspended
female SwissWebster mice a reduced IFN-dp production that correlates with
increased susceptibility to viral infection. These mice are normally resistant to
infections with the diabetogenic strain of encephalmyocarditis virus (EMC-D
virus), while males are susceptible to the virus.70After 4 days of antiorthostatic
suspension, the majority of suspended female mice showed abnormal glucose
tolerance tests, indicating successful viral infection. Control, normally caged mice,
and control, orthostaticallysuspended mice, did not show any evidence of infection.
These data indicate that a model of spaceflight conditions can yield increased
susceptibility to infection, which raises the question whether spaceflight itselfcould
similarly yield enhanced susceptibility to infection.
In more recent suspension studies, Nash and co-workers found no changes in
interleukin-2 receptor expression and in interleukin-2 production after suspen-
~ i o n . These
~ ’ data indicate that not all cytokine activities are affected in a similar
fashion by suspension.
Fleming and others showed that in suspended mice neutrophil and macrophage
activation were inhibited after antiorthostatic suspension in a fashion independent
From steroid hormone level^.^*-^^ However, in suspended rats Miller and associ-
a t e observed
~ ~ ~ no effect of suspension on neutrophil function. These data indicate
that there may be species differences in the effect of suspension, and possibly
spaceflight, on immune responses.
The results of the above studies indicate that suspension is a useful model for the
effects of spaceflight on many aspects of immune responses and resistance to
infection. However, it is not a perfect model, particularly for non-functional aspects
of immune responses, and this should be taken into considerationwhen interpreting
experiments.
Lymphocytes From tested rats tend to produce less IL- 1, IL-2, and IFN-dP as
compared to controls, whereas interferon-y production tends to in~rease.2~ This
suggests that some of the immunologic effects are related to restraint rather than
hypokinesia, hypodynamia, or antiorthostasis.However, studies with mice indicate
that whereas the ability to produce IFN-a@ is reduced in antiorthostatically
suspended animals, orthostatically suspended mice retain that ability.69This sug-
gests that the suspended mouse is a more appropriate model than the suspended rat
for studies of the immunologic effects of spaceflight on the human body.
In one study the recruitment of neutrophils and monocytes and the functional
changes induced in these cells was studied during s ~ s p e n s i o n .No
~ ~difference
-~~ in
/mmune System Changes in Spaceflight 23
phenotypic makeup of inflammatory cells was reported, although the superoxide

response was impaired, and corticosterone levels were elevated, indicating that
stress may be a factor in the suspension model. This conclusion is supported by the
fact that the mass of the spleen was decreased in antiorthostatically as well as
orthostatically suspended mice.
111. PROBABILITY OF INFLIGHT INFECTIOUS DISEASE

Alterations in the human immune mechanism, and in microorganisms potentially
capable of causing an infectious process, have been studied from the early days of
manned spaceflight. Although results have often been incomplete, and sometimes
ambiguous, one can draw several general conclusions. Reports from the US.and
Russian space programs list major depressions in the ability of blast cells to
transform in response to mitogenic challenge,8730 a loss of cytokine production or
fun~tion,~’ major changes in peripheral or splenic immune cell populations.“’
alterations in natural killer cell activity and response to colony stimulating factor.”
and depressions in the delayed-type hypersensitivity response.33Microbiological
changes include a “simplification” of crew autoflora, characterized by a significant
reduction of saprophytes, with a relative increase in the incidence of potentially
pathogenic microorganismson body surface^,^' a buildup of yeasts and filamentous
fungi within the space cabin,79microbial contamination between crew members,*’
and increased ‘pathogenicity’of certain species following spaceflight.”
These studies indicate that spaceflight can be expected to result in a blunting of
the human cellular immune mechanism concomitant with a relative increase in the
numbers and distribution of potentially pathogenic microorganisms. This combi-
nation would seem to increase the probability of infectious disease events during
spaceflight. In fact, there was a very high inflight infectious disease incidence
reported in the early days of the U.S. space program before adoption of a preflight
health stabilization program. For example, during the first seven flights of the
Apollo program illness events were not uncommon, with crew members experi-
encing generalized upper respiratory problems, influenza, viral gastroenteritis,
rhinitis, pharyngitis, and mild dermatologic problem^.^^*^^ Some long-duration
Russian spaceflights have been curtailed due to infectious disease eventsaX3
Apollo 13 was an especially important mission from the infectious disease point
of view.X4First, one of the crew members was removed from flight just days before
launch after being exposed to an active case of measles. Second, an active Pseudo-
monus aeruginosa urinary tract infection developed from the latent state in one
crew member. It will be rememberedthat the Apollo 13 vehicle was damaged while
traveling towards the moon and that the remainder of the mission was characterized
by unusually high stress. Given what we know about the blunting of immune
capability during spaceflight there is no doubt that stress exacerbated this infec-
ti~n.~~
Preflight crew isolation was initiated after the Apollo 13 mission, and has
continued in one form or another as an integral part of the U.S. space program.85
This procedure was designed to allow the autoflora to equilibrate at a level
consistent with confinement and to allow contracted infectious agents to demon-
strate themselves before flight. It is likely that this procedure contributed to the
significant reduction in microbial problems that occurred during, or immediately
after U.S. Space flights subsequent to Apollo 13.'*3We do not have reliable data to
show the effect of spaceflight on the immune system prior to Apollo 14, before the
preflight health stabilization program was initiated. However, data collected sub-
sequent to Apollo 13 have shown changes in immunologicalparameters that would
be of greater concern without the intervention of this effective countermeasure.
Among the nine astronautsvisiting the U.S. Skylab, the mean scores for gingival
inflammation and dental calculus approximately doubled over preflight values
during their 28 to 84 days in space, although in no case was there a noted dental or
oral disability. It is noteworthy that these responses occurred during a time when
careful oral hygiene was maintained by the affected astronauts.86The Russian space
program has provided anecdotal reports of several inflight infectious disease
problems. These include, but are not limited to, a 5 to 8% incidence of skin
infections, two urinary tract infections, and an incidence of gastroenteritis possibly
as high as 35%.
Prior to 1974, Russian and U.S. studies of the effects of spaceflight on the
infectious disease process were conducted independently. However, in 1975 the
U.S. and Russian space agencies took advantage of the joint Apollo-Sop Test
Project (ASTP) mission to conducted the first concerted effort to evaluate compo-
nents of the infectious disease process in ~paceflight~~ by measuring inflight
alterations in three areas. These were: (a) the composition of the microbial popu-
lations inhabiting the crew members and spacecraft, (b) the ability of each crew
member's defense mechanism to resist infection, and (c) the ability of certain
microorganisms to originate infections. This study was unique in that two crews
(two Russians in the S o y craft and three Americans in the Apollo craft) visited
each other during the flight. The two crews were shown to differ microbiologically
and immunologically.This provided an opportunity to study inflight cross contami-
nation by means of specific, naturally occurring, marker microorganisms. Addi-
tionally, this offered the first opportunity for a comparative assessment of
simultaneous immunological changes among two different crews.*' There were no
inflight infectious events and the small crew size precluded identification of major
immunologicalor microbiological events originatingfrom interactionsbetween the
crews. However, this activity was successful in that it provided the first opportunity
for the analytical methods ofthe two countriesto be standardized, adding materially
to the cross utilization of data between the United States and Russian space
pr~grams.~
The launch of Apollo 9 was delayed three days due to crew member illness and
other factors. Since the introduction of the Health Stabilization Program, only one
other flight has been delayed by crew member illness. This speaks well of the
preflight safeguards, when one considers the opportunity for infectious disease.
This is illustrated by the fact that after Apollo 13 the United States space program
flew in space 12 Apollo, 9 Skylab, 3 ASTP, and 352 Space Shuttle crew members,
a total of 376 persons, without a single mission launch being delayed because of
infectious disease.
The United States Health StabilizationProgram has several facets that contribute
to reduced pre- and in-flight infections. These include: (1) limiting contact with the
crew during the week preceding flight to ”primary contacts” with a true need for
access, (2) education of crews and primary contacts in disease prevention, and (3)
an active crew immunization program with antibody verification. The program is
designed to assure immunity for viral illnesses with long incubation periods and to
reduce the spread of viral illnesses with short incubation periods. In addition to
assuring adequate antibody titers for common viral illnesses such as rubella,
rubeola, and varicella, influenza vaccinations are encouraged on a seasonal basis.
If inadvertent contact is made with an ill primary contact, an aggressive attempt is
made to identify the cause of the illness. If influenza A is suspected, the crew is
offered amantadine prophylaxis.
The United States Health Stabilization Program does not include a total quaran-
tine, as crew members are exposed to hundreds of contacts during the week before
flight. The immediate families of crew members are flown on National Aeronautics
and Space Administration (NASA) aircraft from the Johnson Space Center (JSC)
near Houston, Texas to the Kennedy Space Center (KSC) near Orlando, Florida,
and are not initially exposed to large numbers of individuals outside the NASA
community. However, the extended families, friends, and guests usually fly from
Texas to Florida on commercial aircraft and are thus exposed, in a confined
environment, to many potentially infected individuals. If the launch is delayed for
more than three days, crew members are allowed to be with their immediate family,
who can become vectors for illnesses originating in the large number of guests and
extended family with whom they are in contact.
It is especially difficultto prevent the spread of illnesses that derive from viremia
before symptom presentation. For this reason, and those mentioned above, approxi-
mately one in four Space Shuttle flights have at least one crew member affected by
a minor infectious illness. Documenting each illness as a viral or bacterial infection
is difficult due to the impossibility of medical examination or performance of
laboratory tests during the flight. In addition, inflight neurovestibular disturbances
often lead to symptoms such as nausea. vomiting, and reduced appetite, identified
as “space motion sickness,” that are difficult to distinguish from the symptoms of
certain infectious diseases. Also, the fluid shifts that commonly occur during flight
often result in facial fullness, headache, and nasal stuffiness, which symptoms are
difficult to separate from coryza1 symptoms found in upper respiratory infections.
Crew members have experienced diarrhea on several U.S. spaceflights. As with
the “space motion sickness” and fluid shifts. it is generally difficult to equate the
inflight diarrhea with an infectious disease event. Moreover, diarrhea is common

before flight and is believed to be due either to a change in water supply and food
consumption in quarantine or to stress related increased gastrointestinal motility.
On at least two occasions,a U.S. crew member who had diarrhea inflight developed
diarrhea during a subsequent flight. Occasionally, one member of a US.crew will
have diarrhea preflight and another member will develop the symptom inflight.
This circumstance suggests the possibility that an infectious agent could be in-
volved.
One flight launched in January was associated with one crew member and several
ofhis family members, who were ill during the quarantineperiod. The crew member
had minor symptoms and was on antimicrobials at the time of launch. During the
first few inflight days, several other crew members became ill with fever, chills,
myalgia, cough, and pharyngitis typical of influenza. This particular flight is a clear
example of inflight spreading of illness carried from Earth. More recently, multiple
upper respiratory illnesses were experienced. These events occurred early in the
flight and could be due either to exposure or to declining immune competence.
IV. STRESS AND MISSION DURATION

It has been frequently that the human immune system responses to
long-term space missions (one month to one year) can be quite different from those
to short missions (two weeks or less). It has also been suggested that spaceflight-
induced immune system changes resemble those occurring in terrestrial stress
situation^.'^.^^'^ Therefore, it is of value to compare immune system changes
during short space missions with those observed in connection with acute stress,
and those observed after long-term space missions with changes due to chronic
stress.
A. Acute Stress
Most of the recent work has involved correlating stress and immune responses
in selected groups of people without adequate controls, but there have been
relatively few studies employing controlled acute laboratory stressors." Neverthe-
less, some conclusions can be drawn from the latter type of studies. In one study
immune changes under controlled stress conditions were compared to immune
changes in situations of uncontrolled stress.x9Immune activity was not significantly
altered by exposure to acute uncontrollable stress, but subjects in the controllable
chronic stressor group exhibited measurable immune changes. These included
significant decreases in (1) lymphocyte proliferation in response to the mitogen
Con A, and (2) the percentages of monocytes present in the peripheral circulation.
This finding is especially relevant to the condition in which space travelers find
themselves, as they are required and trained continually to control stressful situ-
ations. Therefore, they appear to be in the high risk group due to the requirement
for stress control. Indeed, a blunting of the lymphocyte proliferative response to

mitogens and major alterations in the numbers of monocytes in the peripheral
circulation are two of the most reproducible changes associated with spaceflight.
Therefore, the spaceflightresponses are compatible with those resulting from acute
stress on the ground.
Another controlled laboratory study with humans showed that the acute stress
effects on blastogenesis may occur very quickly.% In this example, blastogenic
response was significantlyreduced immediately after the subjects watched a 7 min
combat surgery videotape. Individuals in the experimental group, who were labeled
as ‘high reactors’ on the basis of their large changes in systolic and diastolic blood
pressure while viewing the film,also showed a significantly blunted lymphocyte
proliferative response to Con A that was absent in the ‘low reactors’ and controls.
These findings suggest that physiological as well as psychological factors may
influence the way in which the immune system of individuals responds to stressful
situations. The data also suggest that individualswith greater sympathetic reactivity
to a stressor will show larger decreases in immune function. This may explain why
some individuals will react more dramatically than others to the same stressor.88
The pattern displayed by crew members of both Russian and United States space-
flights appears to match this relationship.
Several animal surrogatetests have been conducted to model acute stress-derived
immune changes in humans.**These animal models can be useful, because the
experimentalvariables such as life history, diet, and living conditions, can be better
controlled. However, interspecific differences in perception limit the utility of
animal model systems for studying the relationships between spaceflight-associ-
ated cognitive and emotional variables and the observed immune changes.
B. Chronic Stress
In other studies the effects of several model systems of chronic stress on immune
fimction have been observed.88The results indicate that the depression of immune
system activity during chronic stress may manifest itself in a variety of ways,
depending upon the perception of the person being stressed. In one example, lonely
students were found to have a significantly lower NK cell activity and a signifi-
cantly depressed T lymphocyte proliferative response to PHA. Certain interven-
tions, such as hypnosis or relaxation training, acted as buffers to reduce distress and
to block stress-related decreases in immune function.”
One of the few controlled long-term studies demonstrated that during a period
of bereavement, lymphocyte proliferation in response to mitogenic challenge does
not diminish all at once, but gradually decreases over the first 6 weeks, while
recovery is also slow. It was further noted that 14 months after the death of a spouse
the mitogen response of the bereaved subject had not yet returned to normal.”
The immune system responses to long duration spaceflight have yet to be
critically evaluated in terms of how well they mimic these reactions to chronic
stress. Preparations for hture flights will require a better understanding of this
parameter. It is hoped that the current space exploration climate, in which the United
States and Russia are planning cooperative, joint activities, will facilitate inflight
chronic stress studies.
V. CONCLUSIONS AND SUMMARY

The results of immunological analyses before, during, and after spaceflight, have
established the fact that spaceflight can result in a blunting of the immune mecha-
nism of human crew members and animal test species. There is some evidence that
the immune function changes in short-term flights resemble those occurring after
acute stress, while the changes during long-term flights resemble those caused by
chronic stress.
In addition, this blunting of the immune hnction occurs concomitant with a
relative increase in potentially infectious microorganisms in the space cabin envi-
ronment. This combination of events results in an increased probability of inflight
infectious events. The realization of this probability has been shown to be partially
negated by the judicious use of a preflight health stabilization program and other
operational countermeasures. The continuation of these countermeasures, as well
as microbial and immunological monitoring, are recommended for continued
spaceflight safety.
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Chapter 2
ACTIVATION A N D PROLIFERATION OF
LYMPHOCYTES A N D OTHER
MAMMALIAN CELLS IN
MICROGRAVITY
August0 Cogoli and Marianne Cogoli-Greuter
I. Introduction . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 3 4
11. Cell Proliferation . . , . . . . . . . , . . . . . . . . . . , . . . . . . . . . . . 3 5
A. Experiments in Centrifuges. . . . . . . . . . . . . . . . . . . . . . . . . . 36
B. Experiments in Clinostats . , . , . . . . . . . . . . . . . . . . . . . . . . 39
C. Experiments in Orbit . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 39
D. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3
111. Signal Transduction. Genetic Expression and Metabolism . . . . . . . . . . . . 46
A. Experiments in Centrifuges . . . . . . . . . . . . . . . . . . , . . . . . . . 4 6
B. Experiments in Clinostats . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7
C. Experiments in Parabolic Flight . . . . . . , . . . . . . . . . . . . . . . . 4 9

Copyright Q 1997 by JAI Press Inc.
ISBN:0-7623-0147-3
33
34 AUGUST0 COGOLI and MARIANNE COGOLI-GREUTER
D. Experiments in Sounding Rockets . . . . . . . . . . . . . . . . . . . . . 50

E. Experimentsinfibit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
F. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
IV. Morphology and Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
A. Experiments in Centrifuges . . . . . . . . . . . . . . . . . . . . . . . . . 62
B. Experiments in Clinostats . . . . . . . . . . . . . . . . . . . . . . . . . . 63
C. Experiments in Parabolic Flight . . . . . . . . . . . . . . . . . . . . . . . 64
D. Experiments in Sounding Rockets . . . . . . . . . . . . . . . . . . . . . 64
E. Experiments in Orbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
F. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
V. Theories and Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
A. Biophysical and Thermodynamic Theories . . . . . . . . . . . . . . . . . 69
B. Direct Effect: Gravity Receptors . . . . . . . . . . . . . . . . . . . . . . 70
C. Non-Equilibrium Thermodynamics: Bihrcation Theory . . . . . . . . . . 71
D. Indirect Effect: Physicochemical Effects . . . . . . . . . . . . . . . . . . 72
E. Experimental Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
VI. ConclusionsandSummary.. . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
1. INTRODUCTION
The surprising results of an experiment conducted in 1983 in Spacelab-I with
human lymphocytes’ has triggered a wide range of investigations of the effect of
microgravity on single mammalian cells in culture. The activation of T-lympho-
cytes upon exposure in culture to the mitogen concanavalin A was depressed by
more than 90%, compared to synchronous controls on the ground. The results were
confirmed on subsequent Spacelab flights, in which I-G reference centrifuges
provided a reliable inflight control. Since then, several experiments with lympho-
cytes and other cells have clearly shown that gravity can influence important
cellular mechanisms like proliferation, differentiation and genetic expression.
These effects may have little consequence for the immune system function of
humans in space. Although a marked depression of T-lymphocyte activation is
reported in 56% of space crew members during and immediately after flight,2this
appears to be the effect of neuroimmunological interactions due to the psychologi-
cal and physical stress of spaceflightrather than to the exposure to microgravity of
the cells of the immune system. The impact of spaceflight on the immune system
of humans in space is not discussed in this chapter, but is reviewed elsewhere
(Taylor et al., this volume; Gmiinder and C~goli’~).
The purpose of this chapter is to give a comprehensive and updated report on the
behavior of lymphocytes and related cells cultured in microgravity. In this context,
the results of several experiments with other mammalian cells are also discussed.
T-lymphocytes and related cells have been chosen by several investigators as a
Activation and Proliferation of 1ymphocytes 35
suitable model to study cell differentiation and genetic expression following

transduction of an external signal.
Current studies of cell biology in microgravity have two main objectives: (i) basic
science, i.e., the study of significant changes of cellular behavior in the absence of
gravity. These studies may help to increase our understanding of the complicated
systems regulated by cytokines which are expressed by genes under different
control. In fact, in microgravity,the one or the other gene may be switched off, thus
permitting to identify the sequence of events leading to cell differentiation or to
signal transduction, (ii) medical diagnostics, i.e., the use of cefluiar systems to
assess the status of certain physiological conditions of humans in space. A third
goal, namely bioprocessing, i.e., the use of microgravity to produce substances of
pharmaceutical interest, will probably be very difficult to achieve. It is doubtful
that the, yet theoretical, gain would balance the cost of the operations in space.
This review discusses experiments conducted in hypergravity in centrifuges, in
simulated microgravity in clinostats, and in real microgravity in parabolic flights,
sounding rockets, and in manned as well as unmanned orbital flights. The results
are presented in relation to the respective cellular functions and the investigated
structures. Long-lasting processes (hours or days), such as cell division, differen-
tiation and, sometimes, genetic expression can be studied only in orbiting labora-
tories. Fast events, like binding of ligands to the cell membrane, signal transduction,
cell movements and morphological aspects can be investigated in sounding rockets
(7-12 minutes), parabolic flights (serial episodes of microgravity of 15-30 seconds
each), and even in drop towers ( 3 4 seconds). On the ground, there is no limit to
the exposure time when centrifuges or clinostats are used. In the clinostat, which
is a useful tool to verify or prepare experiments in space, microgravity is simulated
through compensation of the gravity vector by rotation. Real microgravity is
attainable only under free fall ~ o n d i t i o n s . ~ ~
Most of the experiments described here have been carried out with peripheral
blood lymphocytes, monocytes or with cell lines derived from peripheral blood
lymphocytes, monocytes or hybridoma cells. These experiments appear here under
the heading “Cells of the immune system.” In addition, it is important to notice that
mitogenic activation of T-cells always takes place in the presence of monocytes as
accessory cells.
The terms microgravity, 0 G, and weightlessness have the same meaning and are
used interchangeably in this chapter. As a matter of fact, the gravity level aboard a
space laboratory usually ranges between lr2and G, depending on the
activities of the crew and the noise (vibrations, shocks) of the equipment. In an
unmanned satellite the G-level may be as low as 10-4 G.
II. CELL PROLIFERATION

Cell proliferation or division is the result of a complex series of processes which
constitute the cell cycle. In eukaryotes, the cell cycle is subdivided into four discrete
stages: the GI period, the S phase, the G2 period and the M period. In the synthetic
phase S, replication of DNA and synthesis of histone proteins occur, The S phase
is preceded by G 1 and followed by G2, two ‘gap’ periods as far as DNA synthesis
is concerned. Cell division occurs in M, the mitotic period. Under certain circum-
stances, e.g., when nutrients are depleted, the cell cycle can come to a stop at a so
called GO stage. Other cells, like lymphocytes, remain in the quiescent GO status
until a stimulating signal, an antigen or a mitogen, triggers the onset of the cell
cycle. The regulation and timing of the cell cycle phases varies from cell to cell.
While the S, G2 and M periods show little variation for mammalian cells (7-1 0
hours, 2-4 hours, and 1-2 hours, respectively), G 1 may vary from 1 hour to many
days or even be absent. Whereasyeast cells can double their number within 2 hours,
mammalian cells may require a minimum of 1&20 hours. For example, HeLa cells,
a cell line derived from a human cervical carcinoma, take approximately 16 hours
to double their number.
Methods commonly used to measure cell proliferationare: (i) electronic counting
of cells with a Coulter Counter or microscopic counting with a Neubauer chamber
(hemacytometer), (ii) spectrophotometric measuring of the optical density of a
culture suspension and converting the optical density to the cell number, (iii)
measuring the DNAcontent by chemical means, and (iv) measuring the rate of DNA
synthesis by incorporation of radiolabeled thymidine into DNA. Especially when
cell aggregation interferes with counting, the rate of synthesis of DNA can thus be
related to cell proliferation. For instance, the mitogenic response of lymphocytes
is measured by means of the 3H-thymidinepulse-labeling index, which under these
conditions indeed reflects the rate of DNA synthesis. Immunologiststhus often use
the term ‘proliferation’ to indicate the rate of DNA synthesis in lymphocytes
exposed to mitogens. This may not apply to all systems or conditions. Data obtained
in microgravity with the 3H-thymidine pulse-labeling procedure, must be inter-
preted with great caution, because microgravity or other space flight factors may
change the labelling of the precursor pool. Independent assays are required to
confirm the data.
A. Experiments in Centrifuges
Cell proliferation and other cell functions have been studied under hypergravity
conditions. The reason is that if the transition from 1 G on Earth to 0 G in space
produces alteration of cellular behavior, then the transition from 1 G to hyper4 in
a centrifuge would also be expected to cause changes. Since experiments in
centrifuges can be performed easily and inexpensively, it is logical to carry out such
experiments before going to microgravity. There is, however, an obvious and
fundamental qualitative difference between the transition from 1 G to hyper-G and
that from 1 G to 0 G. Experimentswith single cells in centrifuges are reviewed here
only as a complement to investigations in space, microgravity remaining the
Activation and Proliferation of 1ymphocytes 37
interesting environment virtually not reproducible on Earth. Nevertheless, several

interesting results were obtained in hyper4 between 2 and 70 G.
Cells of the Immune System
Gravitationaleffectswere first discoveredin murine spleen lymphocytesexposed

to 2 G and activated with the T-cell mitogen concanavalin A6 Studies with human
lymphocytes purified from peripheral blood and cultured in the presence of conca-
navalin A at 10 G showed a 20-30% increase in the proliferation The effect
was not due to an increase of hydrostatic pressure, because 1-G controls in which
parafin was overlayered on the culture medium failed to show the effect. When
the lymphocytes were kept in whole blood cultures, obtained from fresh blood
diluted I :10 with culture medium, the increase in proliferation rate at 10 G could
be 300% higher than in 1-G control^.^ Interestingly, the effect also occurred when
the cultures were first centrifuged for 72 hours without mitogen and then cultured
at 1 G with concanavalin A. It could be shown that the increase at high G was
attributable mainly to the concomitant activation of B-cells by concanavalin A
molecules attached to the erythrocytes which are present at 1-5% in purified
cultures and in overwhelming amounts in whole blood cultures.""' It is known
that when concanavalin A is 'presented'to B-lymphocytesattached to a solid matrix
these cells are activated. Perhaps this effect is enhanced at 10 G; however, there is
not yet a definitive explanation, and it also remains to be clarified why the effect
occurs at high-G and not at I G.
HeLa Cells
The proliferation rate was increased by 14% and 30% at 10 G after 24h and 48h
of culture, respectively. Cell counts and radioactive thymidine incorporation data
correlated fairly well.' In other studies, conducted by Japanese scientists, it was
seen that at 35 G proliferation was 6 0 4 0 % higher than at I G after 4 days. At 18
G and 70 G the increase was 20% and 30% of the respective controls. It could be
shown that the change was due to a shorter G1 phase, 10.4 hours at 1 G and 8.0
hours at 35 G. Thus, the cell generation time was 22.4 hours at 1 G and 19.5 hours
at 35 G.I2.l3In a further study the proliferation rate increased by 34% after 72
hour^.'^ In contrast to the response of MC3T3-El cells (see below), no effect of
indomethacin, a suppressor of prostaglandin E2 synthesis, was detected on the
proliferation of HeLa cells.
Friend Cells
An experiment conducted at 10 G with this murine erythroleukemic cell line in

the presence of dimethylsulfoxide, which induces hemoglobin expression, showed
after 6 days an increase of 35% in the cell number, while glucose consumption
remained ~nchanged.~
38 AUGUST0 COGOLl and MARIANNE COGOLI-GREUTER
Other Mammalian Cells
Reports on other mammalian cells exposed to hypergravity conditionshave been

reviewed by Cogoli and Gmiinder.’ Cells like V-79 (Chinese hamster), sarcoma
Galliera (rat), CEF (chicken) showed increases in proliferation rate between 20%
(sarcoma) and 70% (V-79) compared to the controls. Another erythroleukemiccell
line, K-562 cells (human), showed no change of the proliferation rate at 10 G.
Three cell lines (MC3T3-El from neonatal mouse calvaria, HeLa from human
cervical carcinoma, and JTC-12 from monkey renal tubules) were investigated in
hypergravity by Sat0 et al.I4 Cell growth was determined in cultures kept at 5, 10,
20, and 40 G for 72 hours, respectively. The effect of conditioned medium on
MC3T3-EI cells was studied as follows: cells were incubated at 40 G for 24 hours,
the supernatant was used as conditioned medium for stationary cultures for 48
hours. The effect of indomethacin on all three cell lines was studied during 48 hours
at 40 G.The effect ofprostaglandin E2 was studied on MC3T3-E 1 cells for 8 hours.
Proliferation of MC3T3-El cells was depressed (-8%) after 72 hours at 10 G. At
20 G and 40 G it was increased by 11 and 27% respectively. In JTC-12 cells,
centrihgation at 40 G for 72 hours caused an 8% increase in proliferation.
Conditioned medium favored slightly but significantly more (+ 16%) growth of
MC3T3-El cells at 1 G, suggesting that humoral factors may play a role in
gravitational effects. Indomethacin significantly suppressed proliferation of
MC3T3-El cells at 1 G as well as at 40 G. At 40 G the suppression brought
proliferation to the level seen at 1 G without indomethacin, i.e., the hyper-G effect
was abolished by indomethacin. Indomethacin also abolished the 40-G effect in
JTC-12 cells. The indomethacin and prostaglandin E effects seem to be related to
humoral factors. Other authors reported that low prostaglandin E concentrations
( lOW7M) suppressed DNAsynthesis,whereas a high concentration (1 O”M) activates
it. However, the concentrationsused by Sat0 et al.I4 were lower than those used by
the other authors for MC3T3-El cells. In addition, the amounts of prostaglandin E
measured by Sat0 et ai.l4 at 20 and 40 G are not consistent with those of the other
authors. It is concluded that some other unknown factors must be involved. These
data need further verification.
Hypergravity (5 G) stimulates proliferation, possibly through enhanced produc-
tion of prostaglandin E2, but suppresses differentiationof osteoblast-like cells. The
signalling pathway involved was independent of the activation of protein kinase C
and the production of cyclic nucleotides, and distinct from the pathway through
which insulin-like growth factor I stimulates proliferationofthese cell^.'^ Nakajima
provided further evidencethat hypergravity enhancement of the proliferation of the
same osteoblast-like cell line is mediated by a prostaglandin E2-mediated mecha-
nism.I6 In this study cells were exposed to 5 and 18 G for 1 to 3 days, or cultured
at 5, 10, 20, and 40 G. Migration was increased at 18 G, and proliferation was
enhanced at 20 and 40 G, but reduced at 10 G.
Activation and Proliferationof Lymphocytes 39
In conclusion, the data obtained in hypergravity strongly suggest that alterations

of the gravitational environment have an important impact on several cell functions
and thus justify the investment of resources to conduct similar studies in space.
B. Experiments in Clinostats
Cultures of purified as well as of whole blood lymphocytes in the presence of

concanavalin A were placed on the fast rotating (90 rpm) clinostat, and the rate of
DNA synthesis was measured by means of the 3H-thymidinepulse-labeling index.
A reduction of proliferation by 50% was o b ~ e r v e d . * ~The
” ~ ’data
~ were confirmed
by two independent qualitative analyses. First, blast cells (e.g.. activated lyrnpho-
cytes) appeared in large amount in the 1-G controls, whereas none or very few were
seen in the clinostated cultures. A quantitative analysis was not possible, since cells
clustered in the typical aggregates formed by the polyvalent intercellular binding
of concanavalinA to the alpha-glycosides on membraneproteins. Second, activated
lymphocytes were identified in large number in electronmicrographsof the I-G
controls, whereas few or none were found in the clinostated cultures.
Other Mammalian Cells
Proliferation did not change in the erythroid leukemic human cell line K-562
exposed to hemin.” This was expected since hemin is known to trigger the synthesis
of hemoglobin with concomitant halt ofproliferation. Conversely,proliferation was
increased in Friend cells exposed to dimethylsulfoxide.l o
C. Experiments in Orbit
Extensive studies on several space flights were conducted in the last decade with
lymphocytes and monocytes from human peripheral blood as well as with derived
cell lines.* Activation by concanavalin A of T-lymphocytes, isolated from human
peripheral blood, was carried out for the first time in space in an experiment in
Spacelab-1 in 1983.’.19Unexpectedly, it was discovered that activation, measured
as the rate of DNA synthesis by means of the 3H-thymidinepulse-labeling index,
was inhibited by 93%, compared to synchronous I-G controls on the ground,
despite the fact that the cells formed aggregates in microgravity. The results were
confirmed by qualitative light and electron microscopic analysis. Unfortunately, a
1 -G control in space could not be run.However, the results were confirmed in two
experiments performed in Biorack in Spacelab D-1203’ which included 1-G con-
trols on board. A synopsis of the gravitational effects detected in lymphocytes is
given in Figure 1.
200
z
0
t-
3 150
F
0
a
LL
0
100
I-
Z
W
0
[II
W
a 50
0
+MC -MC -MC +MC -MC +MC -MC -MC -MC
09 og .029 lg lg lg lg lg log
SPACE SPACE CLINOSTAT SPACE SPACE GROUND GROUND BALLOON CENTRIFUGE
Figure 1. Summary of gravitational effects on T-lymphocytes. Effects of spaceflight (0

G space and 1 G space), hypergravity (10 G centrifuge), simulated low-G (0.02 G
clinostat), and cosmic radiation (1 G stratospheric balloon) on mitogenic activation
of T-lymphocytes by concanavalin A. The experiments were conducted either with
resuspended (- MC) or microcarrier-attachedcells (+ MC). The bars give the percent
of activation (measured on day 3 after exposure to concanavalin A at 37°C as counts
per min of 'H-thymidine incorporated into DNA) compared to the relative control (set
at 100%) on the ground (1 G ground) without MC beads. Because the number of
independent experiments shown in the diagram varies from 1 to 20, standard errors
are not given here (for a statistical analysis see Refs. 1, 7, 17, 18, 21, 22).
A step towards understanding the strong inhibition of activation in microgravity

was taken in an experiment conducted on Spacelab SLS-1 in 1991.2223 Lympho-
cytes and monocytes were attached to Cytodex-l microcamers prior to exposure
to concanavalin A in microgravity. Activation was more than doubled compared to
the flight and ground controls at 1 G. As described below, analysis of cytokine
secretion permitted to establish that the lack of activation in free-floating cells is
due to the failure of monocytes to deliver interleukin-I as the second signal required
for T-lymphocyte activation. Unexplained remains the fact that attached T-cells
double their activation in microgravity when sufficient interleukin- 1 is available.
Proliferation of hybridoma cells, which are derived from the fusion of myeloma
cells and B-lymphocytes, has been determined in two Spacelab flights. In both
experiments the results at 0 G were compared to in flight 1-G controls. AM2 mouse
Activation and Proliferation of Lymphocytes 41
hybridoma cells, cultured in Biorack in D-1, did not show differences in cell counts
after 5 days in space.24
The cell line 7E3-N, which produces monoclonal antibodies against a
Iipopolysaccharide-bindingprotein, was cultured in Biorack in IML- 1.25 Since
these cells are derived from lymphocytes, it was thought that hybridoma cells might
also be gravity sensitive. As shown in Fig. 2a, the number of cells after 4 days in
culture (independent duplicates) increased to 9.8 x 105/mlin the 0-G cultures and
to only 7.0 x 105/mlin the 1-G inflight controls. This difference is significant (P=
0.05), while that between the 1-G inflight controls and the ground controls was not
significant. The 40% increase of the proliferation rate (f = 0.05) at 0 G was
confirmed by the 3H-thymidine incorporation data (Figure 2b). Conversely, no
important differences were seen after 2 days of incubation. This suggests that
microgravity effects become noticeable only after exposure to 0 G for more than
two days.
The proliferation rate ofT-leukemiacells was determined after an eight-day flight
in the Chinese scientific satellite 90105.26Due to the low number of cells, these
were cultured for one month after flight. Preliminary data show that the incorpora-
tion of ’H-thymidine was 16% lower in the flight samples than in the ground
controls. If this is true, it appears that the depression of the growth rate induced by
microgravity is a long-lasting effect.
friend Cells
Friend leukemia virus transformed cells were chosen for their interesting prop-
erty of differentiating along erythroid lines in the presence of dimethylsulfoxide.
In fact, Friend cells are a widely used in vztro model of murine erythropoiesis.The
major objective of the experiment was to test the hypothesis that, in analogy with
another in vitro differentiating system, namely T-lymphocytes exposed to mitogens,
important cellular hnctions would change in microgra~ity.~~ The number of cells
per ml of culture fluid after 140 hours of incubation in the presence of dimethyl-
sulfoxide amounted to 12 x lo5 in 0-G cultures and 1-G control cultures and to 10
x lo5 in 1-G ground control cultures, which difference was not significant. The
cells grew normally from a starting concentration of 0.75 x lo’ cells per ml.
W138 Human fmbryonic Lung Cells
These cells were cultured for 28 days in Skylab in sophisticated incubation

chambers with automatic medium supply, fixation and time-lapse cinematography.
No effect was observed on the lag phase and on the growth rate, but the glucose
consumption of the cells was 20% higher in the flight cultures compared to the
ground controls.27
42 AUGUST0 COCOLI and MARIANNE COGOLI-GREUTER
A 2 days
4 days
og lg lg 1.49
SPACE SPACE GROUND GROUND
-
-0
4000
B 2 days
3500 4 days
\
v
;
5000
.-
0
c.
p
L
2500
-2
0
.-
.-
v
2000
3
I-
I
r, 1500
og 19 lg 1.49
SPACE SPACE GROUND GROUND
Figure2 Proliferation of Hybridoma cells in Spacelab IML-1. Cultures of 0.75 x lo5

cells ml-' were incubatedfor 2 and 4 days inflight at 0 G (Ogspace)and 1 G (1gspace),
respectively. Identical controls were incubatedon the ground (1g > 1.4g ground). After
incubation the cells were treated with 10% dimethylsulfoxide and frozen. (A) Cell
counts, and (B) 'H-thymidine incorporation were determined after flight. (Redrawn
from Ref. 25.)
Activation and Proliferation of lymphocytes 43
HeLa Cells
Cultures were exposed successively to five to six orbital flights in Russian Zond
satellites. While the duration of the flights and the intervals between them are not
specified, no alteration of the mitotic index was detected.2x
Hamster Kidney Cells
The hamster kidney cells, attached to Cytodex 3 microcarrier beads, were grown
in a miniaturized cell cultivation instrument (Dynamic Cell Culture System),either
in perfision or batch culture.29After 7 days of incubation,higher cell concentrations
were found in the perfusion chambers than in the batch cultures in microgravity as
well as at 1 G. However, no differences were found between the 0-G cultures and
their I -G controls.
18 Rat Myoblast Cells
These skeletal muscle cells provide an interesting model system to study the
influence of microgravity on the relationship between muscle cell proliferation and
differentiation. Activation of muscle-specific genes during myogenesis is coupled
to withdrawal of proliferating myoblasts from the cell cycle. L8 rat myoblasts were
grown on collagen-coated microcarrier beads in the Space Tissue Loss Flight
Module A for 9 days on a recent Space Shuttle flight.30The behavior of the cells
was analyzed later on the ground and compared to a 1-G ground control performed
3 weeks after the space experiment with exactly the same culture duration and
temperatureprofile. No differences were found in the proliferation rate ofthe flown
cells compared to the ground controls.
D. Conclusions
The data on cell proliferation obtained from experiments in centrifiges. clinostats

and in microgravity are summarizedin Table 1. The data from the space experiments
can be divided into two categories: first, experiments conducted only once and
poorly supported by ground studies in centrifuges and clinostats; and, second,
experiments systematically performed in space, including on-board I-G controls
and with solid ground data support. The most valid conclusions about 0-G effects
on cell proliferation can be drawn from the second category:
1. Changes in the proliferation rate of single cells in microgravity are now well
documented. However, there is not a general pattern of behavior, occasion-
ally opposite effects are observed (e.g., T-lymphocytesvs. hybridoma cells),
and in the majority of cases there is no effect.
2. The fast rotating clinostat delivers results which are in fair agreement with
those obtained in space as seen in the case of lymphocytes.
Table 1. Cell Proliferation; M a i n Results Obtained
Cell Type Effect Remarks Refs.
Hypergravity
Human lymphocytes Activation with concanavalin A: 2&30% increase 7-1 1
in culture of purified cells, >300% increase in
whole-blood cultures, T and B cells are activated
HeLa cells (human) Increase of proliferation rate at different g-levels: 7,12-14
14-30% at 10 G, 20-30% at 18 G and 70 G,
60-8096 at 35 G; the 30% increase at 70 G is
due to a shorter G1 phase
Friend leukemia-vi- 35% increase in cell number after an incubation of 7
rus transformed 6 days at 10 G in the presence of
(murine) d imethylsulfoxide
MC3T3-E1, neonatal 8% decrease of cell proliferation at 10 G , 117'0 14-16
* mouse calvaria increase at 20 G,27% at 40 G; effect at 40 G is
abolished by indomethacin
JTC-12 monkey renal 8% increase of cell proliferation at 40 g; effect 14
tubules abolished by indomethacin
Hypogravity
Human lymphocytes Reduction of proliferation by 50% in cultures of 8,11,17
purified and whole-blood lymphocytes in the
presence of concanavalin A
K-562 (human) No effect on proliferation after exposure to hemin 18
Friend leukemia-vi- Increased proliferation after exposure to 10
rus transformed dimethylsulfoxide
(murine)
Microgravity
Human lymphocytes 90% reduction in activation by concanavalin A of Result of 5 experiments in Spacelab, 3 with inflight 1,19-23
lymphocytes and monocytes in suspension 1 G control.
Human lymphocytes 100°/~increase in activation by concanavalin A Spacelab, inflight 1 C control 22,23
with lymphocytes and monocytes attached to
microcarrier beads
AM2 murine hybri- No difference in cell number after 5 days in space Spacelab, inflight 1 C control 24
doma cells
7E3-N hybridoma 40% increase of proliferation rate after 4 days at 0- Spacelab, inflight 1 G control 25
cells G, no increase after 2 days
Friend leukemia-vi- No difference in proliferation rate Spacelab, inflight 1 C control 25
rus transformed
(murine)
WI 38 human em- No effect on growth rate Skylab, automatic medium supply 27
bryonic lung cells
HeLa cells (human) No alteration of mitotic index Satellites 28
Hamster kidney cells Cells attached to microcarrier beads, no alteration Spacelab, inflight 1 G control 29
of proliferation rate
L8 rat mvoblast cells Cells grown on collaaen-coated microcarrier Space Shuttle, ground control after flight in
performed bea’bs, 30 no cha&e in proliferation rate. accordance to flight data.
46 AuGUSTO COGOLl and MARIANNE COGOLI-GREUTER
3. Among mammalian cells, the lymphocyte/monocyte system appears to be

the most dramatically affected. Interestingly enough, Friend cells, when
induced to differentiateby dimethylsulfoxide,are not sensitiveto microgravity,
although an increased proliferation was found in clinostat experiments.
Ill. SIGNAL TRANSDUCTION, GENETIC EXPRESSION

AND METABOLISM
After the discovery of gravitational effects on the proliferation of single cells, the
next step was to deepen the investigations to genetic and metabolic effects at the
molecular level. This section deals (1 ) with effects on the expression ofcell-specific
products, like cytokines as well as on the early expression of genes like c-fos, c-jun,
and c-myc, (2) with effects on the transfer of genetic material between cells, and
(3) with effects on the consumption of nutrients as well as on the secretion of waste
products. All these effects are brought in relationship with signal transduction
within the cell. Normally, biochemical signals are received via recognition by and
binding to specific cell membrane receptors. Therefore, the experiments on the
binding of ligands in microgravity are described in this section. Since the process
takes few seconds, experiments were conducted also in parabolic flights with
episodes of microgravity of 15-30 seconds.
The gene products of the c-fos and c-jun proto-oncogene family are known for
their prominent role in cell proliferation and differentiation. Their expression is
usually rapidly induced by growth factors and can be induced also by a variety of
agents that by-pass the receptor and mimic the partial activation of signal transduc-
tion pathways. Examples are phorbol esters, calcium ionophores (e.g., A 23 187)
and agents that raise the intracellular concentration of cyclic adenosine monophos-
phate (e.g., forskolin). Lymphocytes and monocytes, epiderrnoid cells A43 1 and
HeLa cells are the most extensively studied systemswith respect to signal transduc-
tion and genetic expression under changed gravitational conditions.
HeLa Cells
The induction of the proto-oncogene c-myc in response to hypergravity was

determined in cells grown in monolayer and centrifugedat 18,35, and 70 G at 37°C
for 15-360 minutes. Induction of c-myc mRNA was determined by electrophoresis
of the extracted RNA, followed by hybridization with 32P-labeledc-myc probes and
densitomeuy of Northern blots. Elevated levels of c-myc mRNA were observed at
all hypergravity values tested. The effect was most pronounced at 35 G, where after
30 and 120 minutes ofcentrifugationthe c-myc mRNAlevels were 3.0- and 3.8-fold
higher than in the control, respectively. The c-my mRNA level remained high
(3.3-fold that of the control) even after 360 minutes exposure to 35 G.I3
In a later study conducted at 35 G Kumei et aL31observed that the production of

inositol 1,4,5-triphosphate(ITP) had increased 1.5-foldafter 2 minutes and 2. I -fold
after 5 minutes. The intracellular level of cyclic adenosine monophosphate (CAMP)
decreased by 11% after 10 minutes and by 16% after 20 minutes at 35 G . Also
determined was the phosphorylation of proteins immunoprecipitatedby antibodies
for microtubule-associated proteins. Phosphorylation of a 1 15-kDA detergent-
insoluble protein was enhanced by 100% after 5 minutes exposure to hypergravity,
but returned to the control level after 80 minutes. Phosphorylation of a 200-kDA
detergent-soluble protein was observed after 20 minutes exposure to 35 G. These
results suggest that ITP and CAMPmay act as second messengers in hypergravity
signal transduction. Phosphorylation of proteins immunoprecipitatedby antibodies
for microtubule-associated proteins in both detergent-soluble and insoluble fiac-
tions suggests that cytoskeletal structures may be influenced by g r a ~ i t y . ~ '
Erythropoietic Cell 1ines
K-562 cells were exposed to hemin and Friend cells to dimethylsulfoxideat 10

G." The K-562 cells showed depression of hemoglobin production, but the glucose
consumption and the proportion of hemoglobin-producingcells did not change. In
Friend cells, glucose consumption was reduced, but the hemoglobin production
remained unchanged.
A43 1 Cells
At 10 G a slight increase (+18%) of c-fos expression induced by epidermal

growth factor, without change in constitutiveexpression, was found by de Groot et
More details are provided in the next section.
a1.32-33
K-562Cells
Human K-562 cells were cultured in the fast rotating clinostat in the presence of
hemin. A 10% decrease in glucose consumption and a 50% decrease in hemoglobin
production (measured in the supernatantsof lysed cells) were observed, while the
number of hemoglobin-producing cells (stained with benzidine) remained un-
changed. '
A43 1 Cells
The induction of so-called 'immediate early genes', like c-fos, is the earliest
detectable indication of a normally functioning signal transduction cascade in the
cell nucleus. The epidermal growth factor-induced expression of c-fos proto-
oncogene in A43 1 cells was studied in the clinostat (60 rpm).32.33
At 1 G a 20-fold
increase of the c-fos mRNA level was seen after 10 minutes exposure to epidermal
48 AUGUST0 COGOLl and MARIANNE COCOLI-GREUTER
growth factor, while after 30 minutes it reaches a maximum of 50-fold. The

induction is ‘transient’, returning to pre-stimulation levels after 2-2.5 hours. With
a more sensitive method, induction can be detected after only 3-6 minutes. With
cycloheximide, the epidermal growth factor effect is longer-lasting as a resuIt of
blocking the expression of proteins responsible of the repression of c-fos transcrip-
tion and degradation of c-fos mRNA. The induction of c&s is temperame
dependent: there is no induction at 4”C, maximum induction is found at 30-37°C.
Various agents like 12-O-tetradecanoyl-phorbol- 13-acetate @horbol ester), cal-
cium ionophores (A 23 187, ionomycin), and mitogenic neuropeptides (bradykinin,
histamine, bornbesin) can induce the c-fos gene in A43 1 cells. The efficienciesare
different at ‘excess concentrations’. 12-O-tetradecanoyl-phorbol-13-acetate and A
23 187 mimic aspects of the signaling cascade initiated by epidermal growth factor
or mitogenic neuropeptides.
Constitutivec-fos mRNA levels did not change in the clinostat. Epidermal growth
factor-induced c@s expression was slightly depressed (-20%). Based also on
results obtained in the centrifuge, in which the opposite effect was found, it is
concluded that the results clearly show that c-fos expression induced by epidermal
growth factor in A43 1 cells is sensitiveto gravity changes.“The induction is a rapid
nuclear response following activation of the signal transduction cascade by ex-
tracellular factors, and is therefore a good indicator to study the influence ofgravity
changes on this p r o c e ~ s . ” ~ ~ , ~ ~
In another study the same team,34*35 managed to show, by coupling c-fos serum
response element with chlor-amphenicol acetyl transferase gene and transfecting
into A431 cells, that the reduction of c-fos gene expression is caused by a specific
decrease in serum response element activity in microgravity. To identify which
subset of signal transduction is affected in microgravity, induction of c$os expres-
sion was tested in the clinostat in the presence of epidermal growth factor,
12-0-tetradecanoy1-phorbol-13-acetate(activator of protein kinase C ) , A 23 187
(by-passing protein lipase C) and forskolin (activator of protein kinase A), respec-
tively. Expression of c-fos induced by epidermal growth factor and 12-O-tetrade-
canoyl-phorbol-13-acetate were reduced by 25% and 30%, respectively. No
significant difference from the 1-G control was found with A 23 187 or forskolin.
The question, whether microgravity just delays c-fos expression or really de-
presses it, was addressed in experiments conducted in the ~ l i n o s t a t and ~ ~ .in~ ~
sounding rockets (see section I11 D). In the clinostat experiments incubations were
performed for 10,20,30,60,and 90 minutes. Expression reached a maximum after
30 minutes, while after 90 minutes it decreased by 20%. The ratio of expression at
1 G to that at 0 G remained greater than 1 throughout the incubation, showing that
simulated microgravity really depresses rather than delays the response. However,
the effect remained low.
C. Experiments in Parabolic Flight
Inflammatory Cells
In a device specially developed for experiments lasting only a few seconds, it

was possible to show that the production of superoxide anion induced by phorbol
ester (phorbol myristate acetate) in peritoneal murine neutrophils was enhanced
four times in mi~rogravity.’~
Rat Osteosarcoma
A rat osteosarcoma cell line (ROS 17/2.8 osteogenic cells) was exposed to
gold-labelled epidermal growth factor at 37°C during parabolic flight for 20
seconds.” Epidermal growth factor was added 3 seconds before the onset of
microgravity, glutaraldehyde was added immediately before the end of the micro-
gravity period. Gold particles were counted in electronmicrographs. No difference
in binding of epidermalgrowth factor was observed in microgravity.Internalization
of epidermal growth factor via receptor-mediated endocytosis was observed during
the 25 minutes flight (8 parabolas). Receptor-mediated endocytosis is an active
mechanism closely related to the cytoskeleton.
In another experiment with the same cell line, the effect of ‘gravitational
stimulation’-continuous exposure for 18 minutes to 5 parabolas consisting of 5
periods of microgravity and 10 periods of hypergravity (1.8 G ) - o n receptor-
mediated endocytosis was studied.38Three phases of receptor-mediated endocy-
tosis were determined: binding, clathrin-mediated internalization of the ligand-
receptor complex, and recycling or degradation via endosomes and lysosomes.
Membrane binding (1st phase) of epidermal growth factor was reduced in ‘grav-
ity-stimulated’cells (-1 8.7%). No differencewas seen for the coated structures(2nd
phase). Increased cytoplasmic labelling (2 1%) was seen in the ‘gravity-stimulated’
cells (3rd phase).
Blood Platelets
The protein kinase C signal transduction pathway has been studied on the protein
kinase Cdependent phosphorylation of the 40K protein plekstrin. Phosphorylation
occurs within a few seconds upon activation of 32P-labeledplatelets with thrombin
or phorbol ester (phorbol myristate a~etate).’~Under these conditions the 40K band
was clearly visible 10 seconds after activation on sodium dodecyl sulfate-polyacryl
gel electrophoreticautoradiographs.Quantitativeanalysis of the radiolabeled bands
from samples exposed to 1 G, 1.8 G and microgravity did not show significant
differences.
50 AUGUST0 COGOLl and MARIANNE COCOLI-GREUTER
Gap Junction Channeling
Gap junctions isolated from chicken heart were inserted in liposornes containing
fluorescein isothiocyanate-labelled microperoxidase and incubated at 1 and 0 G
with Azure-C and H,O, as substrate!' Oxidation of Azure-C was followed spec-
trophotometrically at 612 nm. No effect was observed during 20 seconds of
microgravity.
Rhizobia
In an experiment on the binding of lectin to the cell membrane, no effect of

microgravity on the interaction between lectin and rhizobia was observed4' Al-
though this experiment did not involve mammalian cells, the data are of great
interest for the interpretation of lectin-cell membrane interactions.
Antigen-Antibody Binding
The effect of microgravity on the binding of alpha-fetoprotein to immobilized

monoclonal antibodies was determined.42Microgravity lasted 20 seconds, alpha-
fetoprotein was mixed with monoclonal anti-alpha-fetoprotein bound to latex
microparticles. Bound alpha-fetoproteinwas determined with monoclonal antibod-
ies anti-alpha-fetoprotein marked with alkaline phosphatase using nitrophenyl-
phosphate as substrate. No effect of microgravity on binding was observed.
D. Experiments in Sounding Rockets
The hypothesis that changes in membrane hnction and, consequently, in the

availability of membrane glycoproteins to concanavalin A may occur in micro-
gravity, was tested in two experiments conducted on sounding rockets.43 The
instruments provided automatic injection of fluorescent-labelled concanavalin A,
followed by fixation at given times with parafomaldehyde during seven minutes
in microgravity. Both experiments showed that binding of the mitogen to the
membrane was not affected, only a slight delay of patching and capping was
observed.
A43 1 Epidermoid Cells
Important studies were carried out by Dutch investigators with A431 cells
exposed to epidermal growth factor on the MASER 3 and MASER 4 sounding
rocket flights. In MASER 3, the expression ofc-fos and c-jun was measured, while
the activity of the serum response element was determined in the clinostat (see
section I11 B). Expression of c-fos gene was depressed by 50%. The same was true
for that of cjun, which is not surprising considering the fact that the product of
cjun forms a heterodimeric complex with the product of c - ~ o s Expression

.~~ of
beta-2-microglobulin gene, a gene not modulated by epidermal growth factor, was
unchanged, indicating that the effect on proto-oncogenes is specific. Serum re-
sponse element is present in the 5’ regulatory region of the c-fos gene.
In MASER 4,the protocol was essentially the same as that in MASER 3, except
that incubation with epidermal growth factor, 12-0-tetradecanoyl-phorbol- 13-
acetate, A23 187and forskolin were performed in real microgravity on the sounding
rocket and not in the clinostat. The expression of c-fos, induced by epidermal
growth factor and by 12-0-tetradecanoyl-phorbol- 13-acetate was decreased by
47% and 26%, respectively. No effect was detected with A 23 187 or forskolin on
the expression of c-fos or beta-microglobulin.Similar results were obtained with
c-jun, where epidermal growth factor and 12-0-tetradecanoyl-phorbol-13-acetate
depressed expression by 56% and 5 1% respectively, while no effect was seen with
A 23 187. Forskolin was not tested in this case, because it does not induce the
expression of
E. Experiments in Orbit
The first to study cultures of lymphocytes in space were Talas et al.44 They
discovered that the polynucleotide-induced production of interferon-alpha by hu-
man lymphocytes, cultured on the Soviet spaceship Salyut 6 , was increased by
500% compared to the ground controls.
Dramatic effects on ’H-thymidine incorporation were discovered in T-lympho-
cytes, as discussed in section I1 C. The experiment conducted in Spacelab SLS-1
initiated a systematic determination of the production of cytokines by free floating
as well as by microcarrier-attached cells, which contributed to the understanding
of the nature of the effk~ts.’’~~
To facilitate the understanding of the following discussion, a simplified version
of the activation mechanism of T-lymphocytes is presented in Figure 3. Electron-
micrographs of the cells attached to microcarrier beads are shown in Figure 4.
In resuspended cells the production by monocytes of interleukin-1, which is
believed to be the second signal required for T-cell activation,was nearly abolished
(Fig. S), as was also the case for the incorporation of 3H-thymidinein T-lympho-
cytes. This indicates that the function ofthe accessory cells in deliveringthe second
signal for activation failed. Conversely, the increased activation of T lymphocytes
on microcamers is accompanied by marked increases of the production of inter-
feron-gamma (Fig. 6 ) and of interleukin-2,2.5-fold and 2-fold compared to the I-G
inflight controls and the ground controls.23In addition, the pattern of tumor necrosis
factor production by monocytes was different fiom that of interleukin-1 (Fig. 7).
In conclusion, the data fiom the experiment on mitogenic activation of the
T-lymphocyteimonocytesystem support the following hypotheses: ( 1) interleukin-
52 AUGUSTO COGOLl and MARIANNE COGOLI-GREUTER
1st Signal 2nd Signal 3rd Signal?
MITOGEN OR @ IL-1 IL-2

ANT IGEN
- s
YEYBWE
CLYCOPROTEIN
,
.
A23187 I PHOREOL ESTERS
IONOPHORES f
CP
Figure 3. Simplified mechanism of activation of T-lymphocytes. Three signals are

thought to be required for T-cell activation:
1st signal: Binding of the mitogen to membrane glycoproteins (MGP), induction of
Protein C (G) to activate phospholipase C (PLC). Release of calcium ions from the
endoplasmatic reticulum. Expressionof the oncogenes c-fos and c-mvc. This pathway
can be by-passed by treatment with calcium ionophore.
2nd signal: Monocytes (as accessory cells) are triggered to secrete IL-1 with conse-
quent activation of protein kinase C (PKC) upon interaction with IL-1. Probably, there
is a synergistic effect with the activation of PLC, production and insertion of IL-2
receptors (IL-2 R) in the cell membrane. Amplification of the synthesis and secretion
of IL-2.
3rd signal: Full activation of T-lymphocytes is achieved by interaction between IL-2
and its receptor. While the need of the 1st and 2nd signals for full activation is well
established, the requirement of the 3rd signal is controversial.
2 is produced independently of interleukin-1 in concanavalin A-induced activation,

(2) interleukin-1 production is low in cultures of free cells at 0 G, but this effect is
reversed by attachment of the cells to microcarrier beads, (3) the expression of
interleukin-2 receptors depends on interleukin-1, (4) if sufficient interleukin-1 is
available, activation is enhanced in microgravity, and ( 5 ) there appears to be a
selective effect of microgravity on the secretion of interleukin-1 and tumor necrosis
factor by monocytes and on the production of interleukin-2, interleukin-2 receptors
and interferon-gamma by T-lymphocytes.
Figure 4. Lymphocytes, monocytes, and blood platelets attached to rnicrocarrier

beads in microgravity. After incubation, the cells were treated inflight with 10%
dimethylsulfoxide and stored at -2OOC. Left: Scanning electronmicrograph, magnifi-
cation x730. Right: Transmission electronmicrographshowing monocyte, lymphocyte
(below),and platelets, magnification x3400. Electronmicrographby E. Hunzinger and
D. Miiller.23
Some interesting effects were observed by Chapes et al. in cultures of three types
of immune cells in space.45Unfortunately, these cultures were kept in the mid-deck
of the Space Shuttle at ambient temperature throughout the incubation time rather
than in an incubator at 37°C as desirable for mammalian cells. Hence, the results
must be treated with caution. Upon activation with lipopolysaccharide,the anchor-
age-dependent bone marrowderived macrophage cell line B6MP 102 secreted
significantly more interleukin- 1 and interferon-gamma in space than on the ground.
Murine spleen cells, stimulated with polyinosinic-polycytidylic acid, released
significantly more interferon-alpha in space than on earth. Human peripheral blood
lymphocytes as well as murine lymph node T-cells, activated with concanavalin A,
also produced significantly more interferon-gamma in space than on Earth.
Another important investigation was carried out on the Soviet biosatellite Cos-
mos 2044 with Jurkat cells, a cell line derived from T-lymphocytes, and with THP-I
m ~ n o c y t e sCells
. ~ ~ were incubated at 3 7 T at a low starting concentration (2 x lo5
cells per ml) in plastic bags for 5 days before launch (due to operational constraints).
Activation occurred 6 hours after launch. Jurkat cells were activated to produce
interleukin-2, either by monocyte-derived THP-1cells plus monoclonal antibodies
AUGUST0 COGOLI and MARIANNE COGOLI-GREUTER
1200
1000
800
600
400
200
0
+MC -MC +MC -MC +MC -MC
og 09 19 lg lg lg
SPACE SPACE SPACE SPACE CROUND CROUNO
figure 5. Interleukin-1 secretion by monocytes in microgravity. Data are from the

experiments shown in Fig. 1. The concentration of IL-1 in the supernatants-after 2
days of incubation with concanavalin A-is expressed in pg/ml. In samples with cells
attached to microcarrier beads (+MC) and processed 3 days after addition of conca-
navalin A, interleukin-1 levels are considerably decreased compared to the 2-day
sample. This is probably due to its removal from the medium through binding to
lymphocytes. (Redrawnfrom Ref. 23.)
against CD3R cell receptor, or by A 23 187 plus phorbol myristate acetate. THP- 1
monocytes were activated to produce interleukin-1, either by Jurkat cells plus
anti-CD3 monoclonal antibodies, or by phorbol myristate acetate. Although in the
1-G controls interleukin-2 production under the experimental conditions was only
10% of that under normal culture conditions, it was still well above the detection
limit of the test. Interleukin-2 production in the presence of THP-1 cells plus
monoclonal antibodies against CD3R was not different from that in 1-G ground
controls, but it was fully inhibited by A 23187 plus phorbol myristate acetate.
Similarly, interleukin-1 production in the presence of Jurkat cells plus anti-CD3
monoclonal antibodies was not different from the 1-G ground controls, but it was
85% inhibited by phorbol myristate acetate alone. Glucoseconsumption was nearly
identical (85%) in all cultures, indicating that cell growth and metabolism were not
affected. A control experiment conducted on the ground showed that 85% of the
cells were still viable after 5 days in culture. These results show: (1) cellcell
contacts (JurkabTI-IP-1) are not affected by microgravity, (2) binding of mono-
og 09 19 lg lg lg
SPACE SPACE SPACE SPACE GROUND GROUND
figure 6. Secretion of interferon-gamma by T-lymphocytes in microgravity. Data are

.
from theexperiments shown in Fig. 1 Interferon-gammaconcentration in supernatants
is expressed in IU/ml.(Redrawn from Ref. 23.)
50
40
-
A
E
2a 50
v
0
L
-
a
p 20
LL
z
I-
1c
C
og 09 19 19 'g 19
SPACE SPACE SPACE SPACE GROUND GROUND
Figure 7. Production of tumor necrosis factor by monocytes in microgravity. Data

are from theexperiments shown in Fig. 1. Concentration of tumor necrosis factor alpha
i s given in pg/ml. (Redrawn from Ref. 23.)
55
56 AUGUST0 COCOLI and MARIANNE COGOLI-GREUTER
clonal antibodies to the T-receptor proceeds normally, (3) cosmic radiation effects
are unlikely to be involved, and (4) phorbol myristate acetate, an activator of
phosphokinase C, binds to cells in microgravity, as has been shown in parabolic
flights. Although anti-CD3 monoclonal antibodies are also potent activators of
phosphokinase C in T-lymphocytes, data in the literature do not suggest a phos-
phokinase C-dependence of interleukin-2 production by anti-CD3 monoclonal
antibodies. In comparing these data to those of de Groot et al.,32-34the authors
conclude that a direct action of microgravity are the likely cause of the
The metabolic data of the experiment with Hybridoma 7E3-N cells in Spacelab
IML- 1, described in section I1 C, reveal another interesting pattern of behavior: the
production of monoclonal antibodies, the consumption of glucose and glutmine,
as well as the secretion of waste products like lactate and ammonia, all per cell,
were lower at 0 G than at 1 G.25In fact, the absence of significant differences of
metabolite concentrationsin the supernatantsat 0 G and 1 G is only apparent, since
approximately 40% more cells were present in the cultures at 0 G than in those at
1 G. Although there is not yet an explanation, these data show that gravitational
unloading had a significant effect on the metabolism of hybridoma cells. It appears
that the transition from a two-dimensional configuration of cells sedimented on the
flat bottom of the culture flask at 1 G to a three-dimensional configuration of free
floating cells at 0 G increases cell proliferation despite a lower metabolic turnover.
It also appears that the biosynthesis of a specific cell product is coupled to the
consumption of glucose and glutamineand to the secretion of lactate and ammonia,
rather than to the proliferation rate.
Preliminary data from an experiment with EVI-HI hybridoma cells cultured for
eight days in the Chinese satellite 90 105 revealed an increase of the monoclonal
antibody titer, accompanied by an alteration of the “biosynthesis and secretion of
products” as indicated by the absorption spectra of the supernatants between 200
and 600 nm.26Similar changes in the metabolism of genetic engineered C-4 cells,
producing human growth hormone, and in human adenocarcinoma cells were seen
by the same authors.
Friend Cells
In the experiment with Friend cells in Spacelab IML-1, mentioned in section II

C, the amount of hemoglobin produced upon induction with dimethyl sulfoxide
was the same in the 0-G flight samples and the I-G ground samples.” Counts of
hemoglobin-positive cells show that 60 to 70% of the cells were induced to express
hemoglobin upon exposure to dimethyl sulfoxide. Again, there were no significant
differences between cultures at 1 G and 0 G. Analysis of glucose and glutamine
consumption and lactate and ammonia production clearly showed that Friend cells
do not change their behavior in microgravity. This conclusion is supported also by
centrifuge experiments on the ground, showing that under mild hypergravity
Activation and Proliferationo f Lymphocytes 57
Figure 8. Morphology of Friend cells cultured in rnicrogravity. (A) Friend cells fixed
at 0 C. Scanning electronmicrograph. Bar, 10 pm. (B) Friend cells fixed at 0 G .
Transmission electronmicrograph. The lower cell is undergoing mitosis, the upper cell
shows virus particles in cytoplasmic vacuoles. Bar, 2 p n . (C) Friend cell fixed at 1 G
in the inflight control centrifuge, showing abundant free ribosomes and some virus
particles. Bar, 1 pm. Electronmicrographsby E. Hunzinger and D. Muller.25
5a AUGUST0 COGOLI and MARIANNE COGOLI-GREUTER
conditions no changes appeared. Electronmicrographs of the Friend cells flown in

Spacelab IML-I are shown in Figure 8.
Hamster Kidney Cells

The metabolic behavior of hamster kidney cells and their production of tissue
plasminogen activator was studied in an experiment performed in Spacelab IML-2
(see section I1 C).29In none of the measured parameters, pH, glucose, lactate,
ammonia and glutamine, were any differences found between cell cultures grown
in microgravity, at 1 G in space or on the ground, respectively. The same is true for
the production of tissue plasminogen activator. Therefore, microgravity has nu
influence on the metabolism and production of these cells.
Mouse LM929 Cells from Connective Tissue
This cell line was flown on three different Space Shuttle missions. In the first
experiment the killing of the LM929 cells mediated by tumor necrosis factor was
significantly inhibited compared to ground controls!’ Ground-based studies re-
vealed that activation ofprotein kinaseC by phorbol myristateacetatealso inhibited
tumor necrosis factor mediated killing of LM929 cells. Therefore, inhibitors of
protein kinase C were added to investigateits involvement in the effect detected in
space. Indeed, the presence of inhibitors of protein kinase C restored the tumor
necrosis factor mediated cytotoxicity in microgravity to levels observed in ground
controls!’ Killing of LM929 cells was restored in a dose-dependent manner.
Although these results are very interesting, they need to be interpreted with caution,
as the experiments were performed in the shuttle mid-deck without accurate
temperature control, some at room temperature and others at a temperature defined
by the authors to be “approximately 37OC.”
F. Conclusions
The data on signal transduction, genetic expression and metabolism obtained tiom
experiments in centrifbges, clinostats and in microgravity are summarizedin Table 2.
The studies on lymphocytes in space have shown that microgravity may affect
T-cell activation. In a specific case microgravity was a useful tool to clarify certain
aspects of T-cell activation.23
Of great interest also are the data obtained in the centrifuge, in the clinostat and
in sounding rockets with epidermoid A43 1 cells which show that gravity has an
important and direct impact on signal transd~ction.~~-~’
The work on HeLa cells showed that the duration of the G 1 phase, but not that
of the S, G2, and M phases of the cell cycle are altered in hypergra~ity.’~ Another
study indicates that inositol triphosphateand cyclic adenosine monophosphate may
act as a transducer of a hypergravitational signal and that phosphorylation of
proteins immunoprecipitatedby antibodies against microtubule-associatedproteins
Table 2. Signal Transduction, Genetic Expression, and Metabolism
Cell TYKE Effect Remarks Refs.
Hypergravity
HeLa cells (human) Elevated levels of c-myc mRNA at 18 G, 35 G and 70 G; 13
effect most evident at 35 G: levels of c-myc 3 - 3 . 8 ~
higher than control. Increase of inositol 1,4,5-
~
triphosphate at 35 G: 1 . 5 after 2 min, 2 . 1 after
~ 5
min. DecreaseofcAMPat 35 C: 11%after 10 min. and
16% after 2 0 min.
K-562 cells (human) Exposed to hemin at 10 G: no effect on glucose 31
consumption and proportion of hemoglobin-producing
cells; depression of hemoglobin production.
Friend leukemia-virus Exposed to dimethylsulfoxideat 10 G : reduced glucose 10
transformed (murine) consumption, hemoglobin production unchanged.
A 431 human 18% increase of growth factor induced c-fos expression at 32,33
epidermoid cells 10 G; constitutiveexpression unchanged.
Hypogravity
K-562 (human) Exposed to hemin: 10% decrease in glucose 18
consumption, 50% decrease in hemoglobin
production; number of hemoglobin-producingcells
unchanged.
A 431 human 20-25% decrease of epidermal growth factor-induced c- 32-35 34,35
epidermoid cells fos expression; constitutivec-fos mRNA level
unchanged; phorbolester induced c-fos expression
reduced by 3070, no effect on A231 87 or iorskolin
induced c-fos expression
(continued)
Table 2. (Continued)
~~~ ~ ~~ ~~
Cell Type EffeCl Remarks Refs.

Microgravity
Human lymphocytes 500% increase of alpha-interferon secretion induced by Salyut-6, incubator off during crew sleep 44
various agents
Activation of cells attached to microbeads with Con-A: Spacelab, inflight 1-Gcontrol 22,23
m Interferon-y production 2.5 x increased, interleukin-2
0
production 2 x increased
Binding of Con-A to membrane unaffected; patching Sounding rocket experiments (2) 43
slightly delayed
Human monocytes in Activation by concanavalin A: nearly no interleukin-1 Spacelab, inflight 1-G control 22,23
lymphocyte cultures production by resuspended cells.
Jurkatcells (human Interleukin-2production: unchanged after induction with Satellite 46
T-cell line) anti-CD3 monoclonal antibodies in presence of THP-1
cells; 1 0 0 % inhibited after induction by Ca-ionophore
and phorbolester.
THP-1, myelomono- Interleukin-1 p production: unchanged after induction Satellite 46
cytic cell line with anti-CD3 monoclonal antibodies in presence of
Jurkatcells; 85% inhibited after induction by phorbol
7E3-N hybridoma cells Production of monoclonal antibodies, consumption of Spacelab, inflight 1 -G control 25
glucose and glutamin, and secretion of lactate and
ammonia decreased per cell.
Spleen cells (murine) Increased secretion of interferon* after polyinosinic- Shuttle middeck, ambient temp. 45
polycytidylic acid stimuln.
B6MP102 macrophage Lipopolysaccharide induced interleukin-1 and interferon-y Shuttle middeck, ambient temp. 45
Iine secretion increased
Friend leukemia-virus No changes in glucose and glutamin consumption, Spacelab, inflight 1-G control 25
transformed (murine) production of lactate and ammonia, dimethylsulfoxide
induced hemoglobin production
A 431 epidermoid cells Epidermal growth factor induced expression of c-fos and Sounding rocket expts. (2) 33,34
c-jun 50% decreased; Induction with phorbolester:
26% decrease of c-fos exprn., 51 YOof c-jun exprn.
Hamster kidney cells No effect on metabolism and production of tissue Spacelab, inflight 1-G control 29
Peritoneal neutrophils plasminogen activator Parabolic flight 36
(murine) Phorbol ester induced production of superoxide anion 4 x
increased
LM929 connective Inhibition of tumor necrosis factor mediated cell killing Shuttle middeck, ambient temp. 47
tissue cells (murine)
LM929 connective Protein kinase C inhibitors restored tumor necrosis factor Shuttle middeck, cultures at 37°C 48
' tissue cells (murine) mediated cytotoxicity in dose-dependent manner
62 AUGUST0 COGOLI and MARIANNE COGOLI-CREUTER
suggests that cytoskeletal structures may be influenced by gravity.” Similar data

are not available for microgravity effects.
Data from different and independent experiments show that cell membrane
function is not affected in m i c r ~ g r a v i t y . ~The
~ ~ same
~ ’ * ~is~ also true for antigen-
antibody binding.42
IV. MORPHOLOGY AND MOTILITY

Two questions have always intrigued investigators in gravitational biology: first.
do shape and morphology ofa cell change in microgravity?Second, are free floating
cells, which are not provided with locomotive organelles like cilia, capable of
autonomous movements in microgravity?
In answer to the first question, biophysical calculation of the forces necessary to
alter the shape of a cell show that gravity is not strong enough to influence cell
shape. This means that the flattening of a cell sedimented to the bottom of a cell
culture flask at 1 G is not due to gravitational forces but rather to the spreading of
the cell on the surface. Nevertheless, changes in shape and morphology could
be the result of several small alterations due to the pressure exerted by single
organelles with higher density than that of the cytosol on the cytoskeleton. In
this case, changes in shape and morphology may occur when the gravitational
environment is altered.
The answer to the second question is: yes, in microgravity cells may display
autonomous movements generated either by Brownian motion or by Marangoni
While Brownian motion is difficult to observe, due to the large size
of the cells compared to the length of each impulse, Marangoni convection is
generated by temperature or concentration gradients in a fluid and causes macro-
scopic displacements of the cell. It is conceivable that the microenvironment of a
cell in a culture medium changes continuously due to the metabolism of the cell
with consequent generation of concentration gradients of nutrients and waste
products.
Shape changes and motion may cause considerable changes in important cell
functions, including cell-cell interactions and processes related to cell structures,
like the formation of the mitotic spindle.
Of great importance for morphological and dynamic studies is the advent of
NIZEMI, the low-speed centrifuge microscope which has been used for the fmt
time in the Spacelab IML-2 mission in July 1994.
He1a Cells
Movements ofthese cells on a substratum coated with colloidal gold were tracked
for 48 hours in dark-field illumination at 1 G and 10 G.’ While at 1 G the cells
Activation and Proliferation of lymphocytes 63
showed normal patterns of migration, at 10 G the cells did not change their position.
In addition, whilst after mitosis the daughter cells go in opposite directions
following a symmetrical pattern at 1 G, at 10G the cells remained almost motionless
forming aggregates by successive divisions. The focal contacts between cell and
substratum, however, did not show differences between cells cultured at I G and
I0 G. Neither were shape differences observed by light microscopy. Absence of
shape changes was also reported by Kumei, et a1.12 after 4 days at 35 G.
V79 and JTC-12Cells
A lack of shape changes in hypergravity was confirmed also in V79 Chinese

hamster lung cells (3 days at 18 G) and in JTC-12 monkey kidney cells (4 days at
35 G)."
Cells of the lmmune System
Human peripheral blood lymphocytes were exposed to concanavalin A in the fast

rotating clinostat.I7 Electron transmission micrographs of cells cultured in the
clinostat were compared with those of control cells after fixation with glutaralde-
hyde. Cells activated for 3 days at 1 G were characterized by the presence of
widespread vacuoles in the cytoplasm, by the formation of several pseudopodia,
and by rather low numbers of mitochondria. The formation of vacuoles was more
marked on day 4 of culture, which can be interpreted as an indicator of cell aging.
Cells tended to swell up to a diameter of 1&15 pm. Cells grown in the clinostat
appeared to be equally distributed in two populations.The first population was very
similar to the control cells. The second population appeared to be remarkably
different and, after 3 days, it was characterized by the presence of a high number
of tightly packed and well-developed mitochondria. Cinematographicrecording of
the clinostated cells showed ameboid movements as well as cytoplasmic streaming
in the cells.
A43 1 Epidermoid Cells
It is known that epidermal growth factor causes rapid rounding of A43 1 human
epidermoid carcinoma cells. The effect is dependent upon the temperature.At 37°C
lower concentrations of epidermal growth factor are required for 80% rounding
than at 20°C. A43 1 cells were exposed to epidermal growth factor in the clinostat
and in the centrifuge at 10 G at 20°C and 37°C. In the clinostat rounding of the cells
was significantly increased to 85%, as compared to 72% in stationary cultures. A
slight, non-significant depression of rounding was seen after centrifugation at
10 G."
C. Experiments in Parabolic Flight
Blood Platelets
It is known that stimulation with thrombin and adenosin diphosphate causes
shape changes within 15 seconds. The platelets are transformed to spherocytes with
the appearance of pseudopodia via a calmodulin-dependent mechanism. The effect
of microgravity on shape and aggregation of these cells has been studied recently.39
The activators were injected into the cell suspensions after 5 seconds in micro-
gravity, and 15 seconds later the cells were fixed. Scanning electronmicrographsof
platelets activated by phorbol ester phorbol mynstate acetate, adenosin diphosphate
or thrombin showed no difference between 1 -G controls and microgravity samples.
Transmission electronmicrographs of platelets exposed to thrombin did not reveal
differences.
D. Experiments in Sounding Rockets

Interesting results were obtained recently in two experiments conducted on the
sounding rocket MAXUS In the first experiment, movements ofpurified resting
human lymphocytes kept at 37°C were recorded during 12 minutes ofmicrogravity
by use of a microscope telemanipulated from the ground station. Focus regulation
and object selection could be operated manually during flight. Images were re-
corded by an onboard camera. The recorded images clearly show that the free
floating cells were able to display autonomous motion in random directions. Amore
accurate analysis (one image every 13 seconds) shows that the movements were
much more complex. The cells often changed direction, moved back and forth, and
sometimes crossed the same point several times. The average velocity, calculated
from the displacement in the 13-second increment, was 0.14 f 0.02 pm seconds-',
with a range of (M.49 pm seconds-'. It is also of interest to note that not all cells
in microgravity had a round shape, Very often they exhibited longitudinal forms,
rotated around their axis, and showed contraction waves similar to those described
in the literature for lymphocytes moving under 1 -G conditions. This result is of
primary importance for an interpretation of the behavior of T-lymphocytes in
microgravity. This movement can be attributed to two major causes: First, as
discussed below, the changes of the cytoskeleton may determine at least in part the
random displacements observed. Second, Marangoniconvection due to differences
in the concentration of components dissolved in the medium may generate the
movement of resuspended cells in microgravity. Concentration gradients are gen-
erated by the metabolism of the cells which are consuming nutrients, mainly
glucose and glutamine, and producing waste materials like lactate and ammonia.
In the second experiment, the structure of the microfilaments of vimentin and of
the microtubule network was studiedin Jurkat cells-a human Tcell line-exposed
to microgravity. The cells were chemically fixed in flight at preselected times and
labeled after recovery with fluorescein isothiocyanate-labeledmonoclonal antibod-
ies. Analysis of the cytoskeleton of the Jurkat cells revealed the formation of large
and compact bundles of intermediate filaments of vimentin. Similar structures
appeared in the 1-G controls, but in much fewer number. The changes occurred
after 30 seconds exposure to microgravity, and remained stable throughout the
flight. Similar but less evident changes occurred also in tubulin. These data are in
favor of direct effects of gravity on the cell.
The fhion of the antibody-producing hybridoma cell line G8 with the hypoxan-
tine-aminopterine-thymidinesensitive SP2/O-UZ cell line was attempted in micro-
gravity on a TEXUS sounding rocket flight.53The fusion rate was substantially
increased and the yield of viable cell hybrids was 2-fold enhanced compared to the
ground control experiment. The higher yield is related to a better alignment of the
parent cells resuspended in the electric field in weightlessness, as compared to the
system at I G in which cells rapidly sediment to the bottom of the fusion chamber.
A43 1 -Epidermoid Cells
The clustering of epidermal growth factor receptors was used as a marker to

investigate whether the effects detected on the epidermal growth factor-induced
c-fos and c-jun expression in microgravity are due to the inhibition of processes
occurring at the beginning of the epidermal growth factor-induced signal transduc-
tion. The clusters were visualized in the electronmicroscope by immunogold
labeling with monoclonal antibodies of samples chemically fixed in a sounding
rocket flight at the beginning of microgravity and after 5 minutes exposure to
mi~rogravity,~~ No difference was detected between the flight and ground control
samples. This suggests that the effect of microgravity on epidermal growth factor
signal transduction occurs downstream of growth factor binding and receptor
redistribution, but upstream of early gene expression.
E. Experiments in Orbit
Samples of lymphocytes activated for 3 days with concanavalin A and frozen
inflight in Spacelab 1 with hydroxyethylstarch showed the formation of cell
aggregates in microgravity.]Transmission electronmicroscopic analysis of samples
activated with concanavalin A and fixed with glutaraldehyde inflight (Spacelab
D- 1) confirmed the formation of aggregates. Lymphocytes, however, appeared to
‘suffer’ under microgravity conditions as shown by the appearance of a large
number of vacuoles. There are strong indications that apoptosis (programmed cell
death) is enhanced at 0 G. This is in agreement with the data on DNA synthesis.
The structure of the monocytes was quite different. Their ultrastructure appeared
intact while a stronger membrane ‘activity’, i.e., a display of a large number of
66 AUGUST0 COGOLl and MARIANNE COGOLI-CREUTER
pseudopodia, was observed in the 0-G sample^.^'.^^ Transmission and scanning

electronmicrographsof cultures of cells attached to microcarrier beads, flown in
SpacelabSLS-1, revealed that (1) cells had a strong interaction with the substratum;
(2) attached cells did not suffer in microgravity as free-floating cells did.2223
Ultrastructural analysis of an experiment with hybridoma cells conducted in
Spacelab D- 1 did not yield conclusive results.24
Human Embryonic Kidney Cells
The attachments to a substratum of adhesion-dependent cells was tested in

microgravity in an experiment carried out in an incubator installed in the flight deck
of Space Shuttle flight STS-8.55Microcarriers were added in flight to the cells that
were cultured at 37°C. Scanning electronmicroscopy showed that cell attachment
took place qualitatively and quantitatively as in the ground controls, thus confirm-
ing that the related membrane functions are not altered at 0 G.
Friend Cells
In an experiment conducted in Biorack in Spacelab IML-1, extensive analysis

(scanning and transmission electronmicroscopy, volume measurements) of the
ultrastructure of cells cultured for 6 days in the presence of dimethyl sulfoxide did
not reveal differences between cells cultured at 0 G and in the 1-G control
centrif~ge.’~
W138 Human Embryonic Lung Cells
In an experiment carried out in Skylab and described in section I1 C, cinema-

tographic recording, phase, electron and scanningmicroscopy produced no observ-
able differences in ultrastructure and in cell migration between flight and ground
controls.27
Human lung Adenocarcinoma Cells
Interesting changes were reported in a preliminary study on cells cultured for

eight days in the Chinese satellite 90105.26The flight cells showed poly-polariza-
tion, an increased number of granules, degradation of cytoskeletal structures
resulting inrounding up ofthe cell shape, and the appearance of ‘bubbles’.Although
these data, together with those reported on other cell lines in sections II C and III
E, are interesting, they lack proper controls and adequate statistical analysis, and
thus must still be considered preliminary.
Erythrocytes
The peripheral blood from several donors (either healthy, or with a history of
various diseases) was diluted with autologous plasma. Storage at ambient tempera-
Table 3. Morphology and Motility
Hypergravity
HeLa cells (human) No migration and shape changes af 10 G and 35 G 7,12
V-79 hamster lung cells No shape changes after 3 days at 10 G 12
JTC-12 renal tubules No shape changes after 4 days at 35 G 12
(monkey)
Hypogravity
Human lymphocytes 3-d Con-A exposure: many tightly packed mitochondria 17
in 50% of cells; ameboid movements of cells
A 431 epidermoid cells Increase of epidermal growth factor induced cell rounding 51
(human)
Microgravity
Human lymphocytes Con-A induced cell aggregation; suspended cells 'suffer', Spacelab, inflight 1 G 1,21,23
microbead-attached cells normal control
Human lymphocytes Resting, suspended cells display autonomous random Sounding rocket; on-board 52
motion. Cells exhibit elongated shape and contraction microscope and video
waves camera
lurkat cells &cell line) Cytoskeletal changes after 30 seconds: formation of large Sounding rocket 52
bundles of vimentin
(continued)
Table 3. (Continued)
M krogravity
A 431 epidermoid cells No changes in clustering of epidermal growth factor Sounding rocket 54
receptors
Embryonic kidney cells Normal attachment of cells to microcarrier beads Spaceshuttle 55
Friend leukemia-virus trans- No changes in the ultrastructureof the cells Spacelab, inflight 1 C 25
formed (murine) control
WI 38 embryonic lung cells No ultrastructural changes, no effect on cell migration Skylab, on-board time 27
(human) lapse cine camera
Lung adenocarcinoma cells Cells show polypolarization, increased number of Satellite, no statistical 26
(human) granules and cytoskeletal degradation analysis, no proper
controls
Erythrocytes (human) Dramatic decrease of cell aggregation Space Shuttle experiments 56,57
(2)
Blood platelets (human) No shape changes after 15 sec exposure to phorbol ester, Parabolic flight 39
QI ADP or thrombin
03
L8 rat myoblast cells Flight cells fail to fuse and differentiate into myotubes Space Shuttle, postflight 30
when cultivated at 1 C; show atypical morphology ground
ture on two Space Shuttle flights showed a dramatic decrease of red blood cell
aggregation compared to the ground controls. Without specifying the nature of the
effect, the authors concluded that membrane function appears to be altered in
mi~rogravity.’~.’’
L8 Rat Myoblast Cells

Cells, grown for 9 days under microgravity conditions (see section I1 C) and
cultivated after recovery under 1-G conditionson the ground, showed an interesting
and unexpected beha~ior.~’ The flight cells failed to fuse and differentiate into
myotubes when placed under in vitro fusing conditions, while the ground control
cells exhibited normal fusion behavior under these conditions. The inability of the
flight cells to fuse was found to be a permanent phenotypic alteration. Scanning
electronmicroscopy of the flown cells, grown under 1-G conditions, showed an
atypical morphology as compared to control cells. The cells also piled up on top of
each other.
F. Conclusions
The data on morphology and motility obtained from experiments in centrifuges,
clinostats and in microgravity are summarized in Table 3.
1. Gravitational unloading does not cause changes of the cell shape. However,
changes of the cytoskeleton, probably due to the change in pressure of dense
organelles, may
2. Data on A431 epidermoid cells confirm that binding of an inducer and
clustering of signal receptors are not affected in mi~rogravity.’~
3. Free-floating cells are capable of autonomous movements 52 and of aggre-
gation in microgravity.’ Therefore, cell-cell contacts occur, but the number
of aggregates is reduced at 0 G.
4. Ultrastructural changes observed in lymphocytesmay be related to increased
apoptosis in microgravity.2’z3
5. The fast rotating clinostat is a valid instrument to assess, at least qualitatively,
gravitational effects on single cells in microgravity.
V. THEORIES AND MODELS

A. Biophysical and Thermodynamic Theories
Based either on experimental results or on theoretical considerations, several

authors have discussed gravity effects at the cellular level. The observed effects in
microgravity may be due to important changes in metabolism andor molecular
organization occurringwithin the cell, which allow it to adapt to a new gravitational
environment. Other environmental factors such as radiation may phy a role as well.
This idea of adaptation is not peculiar to the space environment, as many other
changes in the environment. such as temperature, solute concentration, pH or
pressure, are also followed by alterations in cell behavior reflecting a process of
adaptation. Therefore, it is not surprising that single cells adapt to altered gravita-
tional conditions.
The following discussion is an updated continuation and in some aspects a
repetition of that presented in a previous paper.5 Essentially, three theoretical
approaches have been proposed, representing three different types of gravitational
effect on cells:
(1) A direct effect: the direct interaction of gravity with one or more cellular
organelles of a density, different from that of the cytoplasma, generates a pressure
on neighboring structures (e.g., the cytoskeleton) and consequently a signal that is
transduced into a biological event, (2) a non-equilibrium thermodynamic effect: the
interaction of gravity with a few organelles is not sufficient to trigger one event,
but a series of small changes is amplified to generate an important effect, and (3)
an indirect effect: alterations of the gravitational environment cause important
effects on the microenvironmentofthe cell with consequencesfor their metabolism.
B. Direct Effect: Gravity Receptors

P ~ l l a r dassumed
~ ~ * ~ that
~ diffusion and sedimentation inside a single cell with a
diameter larger than 10 pm might be affected by gravity. Calculations show that
the process of diffusion, which is often referred to as Brownian movement, is
significant enough to counterbalance gravity effects in cells of roughly spherical
shape with a diameter below 10 pm. However,in cells exceeding 10 pm in diameter,
sedimentation processes involving organelles may occur.
This approach to explain gravity effects by sedimentation and diffusion has come
into difficulty on two scores: the limited fraction of free water, and the presence of
the cytoskeleton in eukariotic cells. It is now understood that the cytoplasm is not
a solution of proteins in a water-like liquid nor a suspension of organelles in the
cytosol. Rather, the fraction of free water in the cytoplasm is small, most of the
water being adsorbed to proteins with little bulk water left. In eukaryotic cells, the
complex latticework of the cytoskeleton fkther reduces the mobility of organelles.
Nace6' proposed that cells may sense gravity by means of the cytoskeleton.
Calculations show that gravity exerts a considerable torque on the cytoskeleton.
The torque imparted by starch granules and oil vacuoles on a cell with a diameter
of -6 pm amounts to 2.5 x dyne cm. The force developed by a bundle of 6
microtubules was found to be -lo4 to lK5 dyne. If this force is applied to a lever
arm of 6 pm, this yields a torque of -5 x 10-9 dyne cm. Neither the actual length
ofthe lever arm ofthe microtubules(presumably smallerthan 6 pm) nor the number
of microtubules acting on the organelles are known. Although the torque imparted
by starch granules and oil vacuoles appears to be small compared to that produced
by microtubules, energy is needed to maintain positional homoeostasis under
gravity conditions. In microgravity, this requirement for energy expenditureis zero.

Thus, cells in space may be expected to use less energy than do cells on Earth and
this might be accompanied by structural or biochemical changes.
C. Non-Equilibrium Thermodynamics: Bifurcation Theory
An interestingview of a direct action ofgravity on single cells has been proposed

by Mesland.61This endeavor is a completely new way to explain gravity effects in
living organisms. Based on the work of Prigogine et al.62v63 Mesland applies
non-linear non-equilibrium thermodynamics to living cells under changing gravity
conditions. Prigogine and coworkers predicted gravitational effects in chemical
reactions far from equilibrium. Under the conditions of chemical non-equilibrium
the three laws of thermodynamics do not apply, and chemical reactions do not
proceed linearly. In thermodynamic terms, this is precisely the principal charac-
teristic of biological systems. Biochemical reaction chains catalyzed by enzymes
and controlled by complex feedback mechanisms are nonlinear and far from
equilibrium. Under these conditionsa cell may within a given latitude display quite
unexpected behavior, which is often referred to as chaotic. Theoretically, there are
three possibilities: the constituents of a reaction may remain constant (which is,
however, very unlikely); they may oscillate with a known phase, frequency and
amplitude; or they may fluctuate chaotically. At this crossroad, the decision in
which way the reaction develops depends on minuscule differences in the reaction
conditions,such as concentration of substrates,products, catalyst, temperature, and
pressure. At the crossroad, or point of bifurcation, the system is extremely sensitive
to changes in the environmental conditions. In fact, the presence of gravity alone
can force the reaction to take one direction or the other. Mesland suggested that a
lack of gravity could cause a cell to behave differently from its behavior under
normal gravity conditions. This hypothesis implies that for each cell there must be
a threshold gravity force at which the system switches from gravity to microgravity
behavior, or vice versa.
In the case of lymphocytes, the space experiments are not in conflict with the
bifurcation theory. In microgravity, lymphocyte responsiveness is virtually nil
compared with normal gravity. In a lymphocyte proliferation experiment the
individual cell has two possibilities: Upon the addition of the mitogen it can remain
dormant in the resting phase of the cell cycle or it can enter the mitotic phase and
start to proliferate, With respect to an individual cell, there are no intermediate
reactions. Because the lymphocytepopulation used in space studies is not uniform,
but comprises a great number of subpopulations and clones, the sum of all
individual cell reactions may result in a blurred transition from microgravity to
gravity behavior. The use of transformed lymphocyte cell lines, deriving from a
single clone, could help to elucidate this interesting question.
In a recent article, TabonyMdiscussed the role of gravity in pattern formation of
microtubules in solutions of purified tubulin. The patterns were different, depend-
ing on whether the reaction containers were in the upright or in the horizontal
position. This phenomenon is considered in relationship with the bihrcations
occurring in non-linear, out-of-equilibrium states. The data on cytoskeletal changes
in lymphocyte^,'^ described in section IV D, together with the previous findings
on the activation of T-cells in space strongly support the bifurcation theory and the
control of pattern formation of cytoskeletal structures by gravity.
D. Indirect Effect: Physicochemical Effects
With respect to changes in the physicochemical environment. lack of sedimen-

tation and thermal convection in microgravity may result in gradients of nutrient,
oxygen and waste products. Schatz and Linke-Hommes6’ pointed out that electric
potential and solute variations may occur at the cell-solution interface. Micro-
gravity conditions favor the formation of stationary films (boundary layers) around
the cells. In cells in which the uptake rate of oxygen and nutrients exceeds the
diffusion rate, the cell metabolism may be markedly affected. Schatz, Linke-
calculated that in 1-G the density convection may be sumcient to
partially counterbalance this effect. As a model they used a phospholipid membrane
with a surface charge density of 9,= - 4.824 x 10-6 As. cm-*, in contact with an
electrolyte. In this case the positive ions, such as sodium, magnesium, and calcium.
accumulate near the membrane surface, while the negative ions are excluded in
response to the electrostatic repulsion of the negatively charged phospholipid
groups. In other words, a concentration gradient is generated by the membrane
potential leading to maximal density variations of -4 x lop2g. cm-3 over a range
of 1 to 6 nm. In gravity, two configurations are considered, (1) the membrane
surface is horizontally oriented, and (2) the membrane surface is perpendicular. In
the first case the convection in the boundary layer can take place along the
membrane surface. The movement of ions is then not affected by the electric field,
because the charges move perpendicular to the field lines. In the second case,
however, the action of gravity may result in a displacement of the surface layer
away from the membrane surface resulting in a change of the surface potential. In
gravity, convection continuously supports the supply of fresh electrolytes. In
microgravity, however, the supply of these ions is due to diffusion alone. Likewise,
a concentration gradient may develop in the close vicinity of the cell surface when
the cells are rapidly consuming substratessuch as glucose and oxygen. A boundary
layer impoverished in glucose and oxygen will then develop (see also ref. 66).
On the other hand, a ‘solutal’ convection may be generated by concentration
gradients in cell cultures,which favorsthe movements ofcells in the m e d i ~ m . ~ ~ . ’ ~ . ~ ’
This is the Marangoni convection, which is not detectable at 1 G, but may, at least
in part, compensate for the lack of sedimentation and thermal convection in
microgravity. The movements of free-floating cells, which have been clearly
observed in rnicrogravity,may thus be due to the Marangoni convection.
Based on Pollard’s considerations (see section V B), direct gravitational effects

in bacteria because of their small size were ruled out also by Kondo.68However,
the possibility of indirect effects due to intercellular interactions has been recog-
nized. Further detailed analyses of gravity sensing in single cells have been
presented in special reports by Albre~ht-Buehler~~ and by
E. Experimental Models
As described in the previous sections, only few single cell systems showed
significant changes in space. In this section, the characteristics of systems which
are recommended for future activities for the study of gravitational effects are
described.
Several investigators in different laboratories have used either peripheral blood

lymphocytes or derived cell lines for experiments in gravitational biology. Most
frequently the lymphocyte-monocyte system has been used.”8’19‘44372*73 Lympho-
cytes are easily separated as resting cells from peripheral blood of healthy donors.
The separation from fresh blood is based on centrifugation on FicolVmetrizoate
gradients, yielding a cell population consisting of 90430% lymphocytes (T- and
B-cells), 10-1 5% monocytes and 5% granulocytes. By exposure to mitogens the
lymphocytes can be activated polyclonally, leading to proliferation and in the case
of T-cells to the production of a number of lymphokines. Alternatively, activation
can be triggered in samples of whole blood diluted with culture medium. In some
cases, lymphocytes can be isolated from the spleen of mice or rats. The terms
activation, stimulation or proliferation refer to the measurement of the amount of
3H-thymidineincorporated into DNA (precipitated with cold trichloroacetic acid)
after a 2-5 hr pulse.
The mitogenic activation of lymphocytesin v i m constitutes a system of interest,
since the process mimics the events occurring in vivo during antigenic challenge.
The mitogenic activation assay is also used as a diagnostic tool to assess the
efficiency of the immune system of an individual. The test has been employed to
determine the effects of spaceflight on the immune system of astronauts for over
two It could be used to detect alterations of the immune system on
future long-duration space missions and to investigatein v i m neuroimmunological
interactions under the physical and psychological stress of spaceflight.
The transition from the resting cell to the activated cell can be considered an
example of cell differentiation that may be controlled in vitro by addition of the
mitogen. T-lymphocytesare activated by a number of different mitogens. The most
frequently used mitogen is the lectin concanavalin A from lentil seeds. The
mechanism of activation is very complex, and is not yet completely understood.
Studies in microgravity may contribute to the understanding of the basic mecha-
nism underlying lymphocytedifferentiation.In fact, certain steps of activation,such
as the production of interleukin- 1 (the second signal required for T-cell activation)
may be switched off at 0 G. This provides the opportunity to dissect the activation
process into separate phases which can then be analyzed in detail.23This approach
is similar to that in which specific inhibitors of metabolic processes are used to
study sequential reaction steps.
An alternative to the lymphocyte/monocyte system from peripheral blood lym-
phocytes is provided by cell lines, such as the Jurkat (T-cells)or THP-1 (monocytes)
lines. Cell lines have the advantage of consisting of homogeneous and well
characterized cell population^.^^.^^ Peripheral blood lymphocytes, in contrast,
comprise several T- and B-cell populations. Nevertheless, while the possibility of
regulating in vitro the b c t i o n of derived cell lines is rather limited, the peripheral
blood lymphocytes system is more flexible to mitogenic activation and certainly
much closer to the in vivo tinction of the cells of the immune system.
Less interesting for experiments in microgravity and possible bioprocessing
applications (in terms of proliferation rate and antibody production) appear to be
hybridoma cells. This is not surprising, since hybridoma cells are ultimately
committed to divide and to secrete antibodies with little or no possibility of
regulation by external Conversely, the electrofusion of immune cells to
produce antibodies gave promising results in mi~rogravity.’~
A43 1-Epidermoid Cells
The use of these carcinoma cells in combination with epidermal growth factor
has proved to be a very useful model for the study of gravitational
In particular, important data were gathered, first, on the intracellular signal
transduction cascade involving either protein kinase A or protein kinase C; second,
on the binding of epidermal growth factor to the cell membrane; and, third, on the
early expression of oncogenes. Similar studies are certainly needed also on the
T-lymphocyte/monocytesystem.
He1a Cells
These cells can be used in similar fashion as the A43 1-epidermoid cells, as shown
’
by the work of Kumei et al. 12*13,3on oncogene expression and cell cycle. However,
HeLa cells are not subject to regulation by mitogens or growth factors as peripheral
blood lymphocytes and A43 1 cells are.
VI. CONCLUSIONS AND SUMMARY

The experimental findings reviewed in this chapter support the following conclu-
sions:
Proliferation. Human T-lymphocytes, associated with monocytes as accessory
cells, show dramatic changes in the centrifuge, in the clinostat and in space. In
free-floating cells the mitogenic response is depressed by 90% in microgravity,
Activation and Proliferationo f Lymphocytes 75
whereas in cells attached to a substratum activation is enhanced by 100%compared

to 1-Gground and inflight controls.
The duration of phase G1 of the mitotic cycle of HeLa cells is reduced in
hypergravity, resulting in an increased proliferation rate. Other systems like Friend
cells and W138 human embryonic lung cells do not show significant changes.
Genetic expression and signal transduction. Human T-lymphocytes and mono-
cytes show important changes in the expression of cytokines like interleukin-1,
interleukin-2, interferon-gamma and tumor necrosis factor. The data from space
experiments in Spacelab, Space Shuttle mid-deck, and Biokosmos have helped to
clarify certain aspects of the mechanism of T-cell activation.
Epidermoid A43 1 cells show changes in the genetic expression of the proto-on-
cogenes cfos and cjun in the clinostat and in sounding rockets.
Membrane function, in particular the binding of ligates as first messengers of a
signal, is not changed in most of the cell systems in microgravity.
Morphology and Motility. Free cells, lymphocytes in particular, are able to move
and form aggregates in microgravity, indicating that cell-cell contacts and cell
communications do take place in microgravity. Dramatic morphological and ultras-
tructural changes are not detected in cells cultured in microgravity.
Important experiments with single mammalian cells, including immune cells,
were carried out recently in three Spacelab flights (SL-J, D-2, and IML-2 in 1992,
1993, and 1994, respectively). The results of the D-2 mission have been published
in ref. 75; those of the IML-2 mission in ref. 76.
Finally, many cell biology experiments in space have suffered in the past from a
lack of adequate controls (like 1-G centrifuges) and of proper experimental condi-
tions (like well-controlled temperature). In this respect the availability of Biorack,
outfitted with proper incubators with 1-G control centrifuge as well as a glovebox
with a microscope, is a great advantage. It is also desirable that cell biology
experiments in space are accompanied or even preceded by a program of ground-
based investigations in the fast rotating clinostat and in the centrifuge, and that
preparatory experiments be done in parabolic flights and sounding rockets, when-
ever possible.
Proper publication of the results of space experiments is another important need.
A great number of data have been published in proceedings and reports that are not
available to the broad scientific community. To guarantee the credibility and the
international recognition of space biology it is important that the results be publish-
ed in international, peer reviewed journals.
ACKNOWLEDGMENTS
The work of the authors has been supported by the ETH Zurich, the Swiss National Research
Foundation, the Italian Space Agency (ASI), the National Aeronautics and Space Admini-
stration (NASA), the European Space Agency (ESA), and Oerlikon Contraves AG. They
wish to acknowledge the work of all present and former members of the Space Biology
76 AUGUST0 COGOLI and MARIANNE COGOLI-CREUTER
Group at the ETH Ziirich and, in particular, Birgitt Bechler, Felix K. Grniinder, Juliet Lee,
Giovanna Lorenzi, Alex Tschopp, Pia Fuchs-Bislin, Myriam Valluchi-Morf and Isabelle
Walther. Finally. w e would like to thank Sue B. Criswell. Elisabeth Hunzinger, Helen Joller,
Peter Joller. Maria Antonietta Meloni, Otfkied Muller, Proto Pippia, Luigi Sciola a n d
Alessandra Spano for their fruitful collaboration.
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Chapter 3
PSYCHOSOCIAL VALUE OF SPACE

SIMULATION FOR EXTENDED
SPACEF L I G HT
Nick Kanas
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
I1. Past Simulation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
In . Psychosocial Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
A . Psychological Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
B . PsychiatricIssues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
C . Interpersonal Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
IV. Future Simulation Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
A . Social and Cultural Factors . . . . . . . . . . . . . . . . . . . . . . . . . . 86
B . Career Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
C . Monotony and Reduced Activity . . . . . . . . . . . . . . . . . . . . . . . 87
D . Leadership and Authority . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
E. Relation between Crew and Ground Personnel . . . . . . . . . . . . . . . 88
V. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Copyright 0 1997 by JAI Press Inc .
ISBN:0-7623-0147-3
81
82 NICK KANAS
1. INTRODUCTION
During future space missions involving a space station, a lunar base, or a trip to
Mars, international crews of men and women will engage in complicated tasks for
long periods of time. Under these conditions, psychological and social factors will
play an important role in influencing crew morale and performance. In order to
prepare for such missions, it is important to understand the impact of these
psychosocial factors, particularly those that may have negative consequences. In
this way, crews can be trained to recognize and deal with these factors before they
become problematic. Although there have been a number of anecdotal reports and
diary entries from space travelers participating in American and Russian long-term
missions, the role of psychosocial issues has not been examined in space under
controlled conditions. Space simulations on Earth offer the possibility for such
study.
II. PAST SIMULATION STUDIES

There have been well over 60 studies on Earth which have involved the exposure
of humans to conditions of monotony, isolation, danger and confinement.'"
Because these activities have characteristics in common with manned spaceflight,
conclusions have been drawn that have ramifications for the training and planning
of space mission^.^.' These space analogs have been conducted for decades, and
they have been supponed by the American, Russian and European space programs.
Examples of simulation environments are shown in Table 1.
Despite offering some similarities to spaceflight, however, none of these simu-
lations can be considered as a perfect analog of space conditions. For example, all
simulation studies have taken place on Earth where conditions of microgravity and
high radiation do not exist. In many cases the duration of isolation was only a few
weeks, and thus the psychosocial stresses that are related to longer periods of time
have not been addressed. Finally, a number of these simulation activities have dealt
with relatively homogeneous crews comprised of males from a single country of
Table 1. Space Simulation Environments

Expeditions (e.g., Antarctic, Arctic)
Submarines
Submersibles (e.g., Sealab, Ben Franklin, Tektite, La Chalupa)
Land-based simulators, e.g.:
U.S. Air Force School of Aviation Medicine simulator,
Douglas and McDonnell Douglas capsules,
Norwegian Underwater Technology Center "N UTEC" hyperbaric chamber,
Russian Mir simulator
Hypodynamia (bed rest) environments
Psychosocial Value of Space Simulation 83
origin, thus failing to account for issues related to gender and the ethnic and cultural
diversity that will be present on future long-duration international space missions.
Sells8 has studied eleven social systems that he regarded as pertinent to long-
duration space flight. After first developing 56 characteristics of space missions,
he rated the social systems on each characteristicusing a 3-point Likert scale from
0 (dissimilar) to 2 (highly similar). He found that submarines and exploration
missions had the highest similarity scores, whereas industrial work groups and
shipwrecks/disastersrated lowest. Sells’ study did not include more recent land-
based systems (e.g., NUTEC chamber, Mir simulator) or non-submarine submers-
ible habitats (e.g., La Chalupa).Nevertheless, his study reminds us that the validity
of Earth simulations varies, with some analogs being more characteristic of space
travel than others. Although much information has been obtained from these
simulations,a concern arises as to how applicablethis information really is to space
missions.
111. PSYCHOSOCIAL FACTORS

A number of psychosocial factors have emerged from the simulation literature that
relate remarkable well to important issues that have been reported anecdotallyfrom
space These are summarized in Table 2 and will be described briefly
in terms of their relevance for manned spaceflight.
Table 2. Psychosocial Factors

Psychological
Sleep problems
Time sense alterations
Transcendent experiences
Demographic issues
Career motivation
Homesickness
Perceptual changes
Psychiatric
Anxiety, depression, psychosis
Psychosomatic symptoms
Stage-dependent emotional problems
Asthenia
Post-flight personality and marital problems
Interpersonal
Tension due to crew heterogeneity
Decreased cohesion over time
Need for privacy
Leadership roles and lines of authority
a4 NICK KANAS
A. Psychological Issues
Psychological issues pertain to the normal responses of people to the conditions

of simulated or actual spaceflight. Sleep problems, such as insomnia and changes
in the characteristics of the sleep cycle, often accompany the excitement taking
place at the beginning or end of a space mission. Disruptions in sleep may also be
related to the depression and asthenia that sometimes occurs during the middle
phase of long-duration missions.’ Alterations in time sense also have been reported,
where short time intervalsare over-estimated and people are unable to perform their
duties in the time that is allotted.” Several astronauts and cosmonauts have had
transcendent experiences(e.g.. religious conversions, derealization)in space,” and
these are reminiscent of the break-off phenomenon experienced by jet pilots flying
at high altitudes.l 2 Some submersible simulations have suggested that first-born
individuals and those growing up in large cities may be less adaptable to stressfid
conditions than later-born people and those from small towns.13 although these
demographicissues have not yet been reported under spaceflightconditions.Career
motivation has been shown to be an important factor during Antarctic expeditions,
where people who are able to use unstructured time to do work-related projects
experience fewer psychological problems during the long wintering over period
than those whose jobs are more action-oriented.14As will be discussed later, this
has important ramifications for long-duration space missions. Homesickness and
lonelinessoccur in space as well as in Earth-bound simulations.” Finally, in a study
of 54 astronauts and cosmonautsAlan Kelly and 1 found evidence that auditory and
visual perceptual sensitivities are increased in the space environment,” thus
supporting reports of increased sensitivity to loud sounds during the Salyut-6 and
~alyut-7mission^.'^
B. Psychiatric Issues
In contrast to psychological factors, psychiatric issues pertain to abnormal

responses of people to simulation and off-Earth environments. Although severe
anxiety, depression and psychosis have not been reported in space, they have
occurred during Antarctic and submarine mission^.^ In addition, irritability, mood
instability and hypoactivity are components ofthe asthenic reactions that have been
observed during the monotonous phase of long-term space missions.’ Psychoso-
matic symptoms also have been reported during space activities.I8
Emotional problems may be related to the stage of the mission. On the basis of
an extensive examination of Antarctic and submarine missions, Rohrer” described
three stages of the reaction to isolation and confinement:
1. The first stage occurs early and is characterized by heightened anxiety due
to the novelty of the situation;
2. The second stage takes place during the long monotonous middle part of the
mission and is characterized by depression and homesickness;
3 . The last stage occurs just before the end of the mission and is characterized
by anticipation and juvenile behavior.
Elements of these three stages have been described for space missions.’520
During the second stage asthenia is most likely to occur, which is characterized by
hypersensitivity, emotional lability, irritability, hypoactivity,psychosomatic symp-
toms, sleep problems, and poor appetite.’ Finally, postflight personality changes
can occur as astronauts and cosmonauts readjust to life on Earth.2’ In addition. their
spouses sometimes react with tension and depression, as they too must adapt to the
presence of their long-absent mate. This phenomenon has been called “the subma-
riners’ wives syndrome.”22
C. Interpersonal Issues
Interpersonal issues relate to the problems that groups of confined or isolated

people have in interacting with one another or with people on the outside who are
monitoring their activities. In space as well as in ground-based simulations,
interpersonal tension is enhanced when crews are heterogeneous or composed of
people with conflicting work tasks. This can result in a number of intra-crew
problems as well as the displacement of tension to people on the outside (e.g.,
mission control), resulting in anger, blame and difficulties in crew-to-ground
communication.‘5s2s26Groups of people confined in space or on the ground for
long periods of time also show decreased cohesion as time goes on, manifested by
subgrouping, scapegoating and territorial Individuals, isolated
together in a small habitat, experience a great need for privacy, and inadequate
attention to this issue can result in increased interpersonal tension in space.28
Finally, during short-term simulations and space missions, the leadership structure
is clear and often revolves around task-oriented lines of authority. However, during
long-term missions, there is a tendency for status leveling to occur. and the most
valued leaders are those who are able to relate to the emotional needs of the crew
and to carry out the tasks of the mission in a more democratic manner.15.27Thus,
both task and emotional leadership skills are necessary.
IV. FUTURE SIMULATION OBJECTIVES

Although the psychosocial factors mentioned above have been studied on Earth
and reported anecdotally from space, more simulation work still needs to be
accomplished. Many of the ground-based studies conducted in the past were
relatively short, dealt with crews that were homogeneous in terms of gender and
cultural background, and used scientific methods that are not up to modern
standards. Since future space missions will involve heterogeneous crews working
86 NICK KANAS
Table 3. Areas of Interest for Future Simulation Studies

Social and cultural factors
Career motivation
Monotony and reduced activity
Leadership and authority
Relation between crew and ground personnel
on complex objectives over long periods of time, hture simulation studies should
take these features into account. Several areas of interest that still need to be
evaluated are shown in Table 3.
A. Social and Cultural Factors
The first area of interest relates to the impact of social and cultural factors on
space missions. Examples include language and dialect, cultural differences, and
gender biases. In our study of 54 astronauts and cosmonauts,16 we found that
although space travelers believe that space crews should be fluent in one common
language, “international” astronauts are significantly more tolerant of dialect
differences than their American or Russian counterparts. The Czechoslovakian
cosmonaut Vladimir Remek, who visited the Salyut-6 space station, felt like he was
being treated as an outsider when he tried to interact with his Russian counterparts
in space.29 Similarly, Russian cosmonauts have reported feeling uncomfortable
when non-Russian visitors boarded the Salyut space station.” Evidence of sexual
stereotyping was reported during one Salyut-7 mission when upon her arrival, a
female cosmonaut visitor was greeted with flowers and an apron and was asked to
prepare the meals.” There is evidence that social and cultural factors such as these
play an important role in crew cohesion and on-board tension. Since hture
long-duration space missions are likely to involve crews of men and women from
different cultures with different native languages, it is necessary to examine the
impact of these issues in controlled simulation studies on Earth. For example, crews
of different gender and cultural backgrounds can be studied, and confined groups
with one common native language versus more than one can be evaluated in terms
of performance and efficiency. This has been done in the European ISEMSI and
EXEMSI projects.4,’
B. Career Motivation
A second important factor to study in simulations has to do with career motiva-
tion. Future space travelers will be highly specialized and will differ from one
another in terms of training and career goals. For example, career pilot astronauts
and payload specialists will be expected to work together on a space station even
though each group has different motivations for being in space. In a study of five
U.S. Antarctic stations, military personnel experienced more insomnia, depression

and hostility than civilian scientists during the long wintering over period.14 The
explanation for this is that the latter were involved with activities congruent with
their training and career goals (e.g., conducting experiments, writing scientific
reports), whereas the former had little to do and reacted with boredom and
interpersonal tension. Conflicts between scientists and seamen also have been
reported, which have resulted in the loss ofdata and overt hostilitieson ocean-going
research vessels.30Alan Kelly and I did not find major differences in the ways that
researchers and pilots/commanders who had flown in space viewed various aspects
of intra-crew and crew-to-ground c ~ m m u n i c a t i o n . However,
' ~ ~ ~ ~ this is not to say
that in future long-term space missions tensions won't arise between these scientific
and operational groups, particularly in a space station where non-astronaut scien-
tists regularly will be shuttled up from Earth to perform tasks that they feel are
critical for their work. The ways in which these and other task-motivated groups
(e.g., engineers,physicians, politicians,journalists, businessmen) will interact over
long periods of time in space need to be studied in future simulations in terms of
crew cohesion, subgrouping, scapegoating and territorial behavior. For example,
these factors could be measured in different space simulation crews with members
that are varied in terms of operational, scientific,medical, political, journalistic and
economic backgrounds.
C. Monotony and Reduced Activity
The effects of monotony and periods of low activity also need to be studied
further. Many simulations in the past have been relatively short-term, and tasks
have been quite active and stimulating for the crews. However, many ofthe negative
psychosocial effects reported from space have occurred during the long middle
stage of a mission, where activities have become routine and where the crew
members have periods of free time. Asthenia, crew member withdrawal and
territorial behavior we most likely to occur during this stage.'.'' In addition, issues
arise about the best ways to occupy leisure time. From our analysis of leisure time
activities in space, Alan Kelly and I concluded that cosmonauts and long-duration
space travelers were more sensitiveto the absence of various media that would help
them fill leisure time (e.g., movies, letters, reading material) than astronauts and
those who had been in space for shorter periods of time.32Furthermore, although
international events, national events, and historical subjects were of most interest
to our respondents, a number of other topics also were highly rated. Thus, variety
in leisure media and topics needs to be planned in future long-term space missions,
since individuals differ greatly in their preferences for free time activities. Some of
the different ways of using leisure time could be examined in fbture Earth-bound
space analogs. For example, periods of free time could be built into the mission
plan, and different types of activities that are of potential use to the crews in
occupying this time productively could be examined.
aa NICK KANAS
D. leadership and Authority
As a fourth issue, leadership and lines of authority need to be studied further. In

short-term spaceflight,where the identified leader is the mission commander, lines
of authority are clear and related to specific tasks. However, during long duration
missions status leveling tends to ~ c c u r . ~Sometimes
~,~’ the mission requirements
affect the leadership structure. For example, on one Salyut-6 mission, the com-
mander had less experience in space than his partner, who was an older veteran with
specific skills that were needed to repair a problem in the space station. The two
men agreed to share decision-making responsibilities through mutual discussion,
and this resulted in the successful completion of the mission goals2’
The leader not only has to be aware of keeping the crew members on task, but
he or she also must be sensitive to their emotional needs as well. Over time. the
role of the leader may become diffuse and unclear. This was experienced during
the European Space Agency’s EXEMSI simulation, in which a crew of three men
and one woman were isolated for 60 days in a hyperbaric chamber. The crew went
through several distinct stages in terms of its cohesion and the ability of the
members to relate to one another. Although the designated leader generally kept to
his role throughout the mission, there was some competition for his position, and
there was evidence of status leveling and leadership role confusion.’ Thus, issues
involving proper leadership role, competition for leadership, and status leveling
need to be examined further in future space simulations.
E. Relation between Crew and Ground Personnel
Finally, the relationship between confined crews and the personnel monitoring
their activities needs to be characterized in order to minimize the displacement of
tension to the outside. In-group/out-group problems have occurred during space
mission^^^**"^ as well as during space sir nu la ti on^.^*^^^^ For example, during the
EXEMSI simulation, territorial behavior, subgrouping and scapegoating took
place, and there was evidence of covert intra-crew tension. There was a tendency
for the crew to displace this tension to the people outside who were monitoring
their performance, particularly during the middle three to six weeks of the isolation
peri~d.~
Since inter-group tension can lead to miscommunications that can threaten a
space mission, it is important to study the causes and effects of this problem, along
with ways to deal with it. For example, there is evidence that private audio-visual
contact with loved ones on Earth can be of benefit to long-term space travelers9’15
We also found that astronauts and cosmonauts significantlyendorsed the value of
contact with loved ones on Earth as having a positive influence on mission
performance, especially during long-duration space mission^.^' During future
simulations,the relationship between the crew members and people on the outside
can be observed and measured in a naturalistic way in order to look for displacement
effects. Alternatively, conflictual situations could be built into the simulation plan
in order to test the effects ofpre-training awareness and mission support in resolving
any in-grouplout-groupdifferences that result.

There have been over 60 studies of Earth-bound activities that can be viewed as
simulations of manned spaceflight. These analogs have involved Antarctic and
Arctic expeditions, submarines and submersiblesimulators, land-based simulators,
and hypodynamia environments. None of these analogs has accounted for all of the
variables related to extended spaceflight (e.g., microgravity, long-duration,hetero-
geneous crews), and some of the simulation conditionshave been found to be more
representative of space conditions than others.
A number of psychosocial factors have emerged from the simulation literature
that correspond to important issues that have been reported from space. Psycho-
logical factors include sleep problems, alterations in time sense, transcendent
experiences, demographic issues, career motivation, homesickness, and increased
perceptual sensitivities.Psychiatricfactors include anxiety, depression, psychosis,
psychosomatic symptoms, emotional reactions related to mission stage, asthenia,
and postflight personality, and marital problems. Finally, interpersonal factors
include tension resulting from crew heterogeneity, decreased cohesion over time,
need for privacy, and issues involving leadership roles and lines of authority.
Since future space missions will usually involve heterogeneous crews working
on complicated objectives over long periods of time, these features require further
study. Socio-cultural factors affecting confined crews (e.g., language and dialect,
cultural differences,gender biases) should be explored in order to minimize tension
and sustain performance. Career motivation also needs to be examined for the
purpose of improving crew cohesion and preventing subgrouping, scapegoating,
and territorial behavior. Periods of monotony and reduced activity should be
addressed in order to maintain morale, provide meaningful use of leisure time, and
prevent negative consequences of low stimulation, such as asthenia and crew
member withdrawal. Leadership roles and lines of authority need to be studied
further to understand the factors leading to status leveling, leadership competition,
and role confusion. Finally, the relationship between crews and ground personnel
should be characterizedin order to minimize the displacement of anger and tension
to the outside, to counter -the effects of inter-group miscommunications, and to
develop support strategies that can help to counter in-group/out-group conflicts.
Ground-based space simulationsstill have a role to play in terms of understanding
the impact of these factors and ways of dealing with them. In particular, issues
involving language, cultural differences, gender biases, career motivation,monoto-
nous conditions,use of free time, leadership, lines of authority,and the relationship
between crews and outside monitoring personnel need to be further characterized
and examined under controlled conditions. Until such time as these factors can be
90 NICK KANAS
studied directly in space, simulations provide an opportunity to learn more about

these psychosocial issues and to plan ways of minimizing their negative conse-
quences during actual space missions.
ACKNOWLEDGMENT
Parts of this paper were presented at the Symposium on Human Behaviour in Space
Simulation Studies, ESA, Paris, France, December 1-2, 1993.
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cosmonauts. Aviation. Space and Environmental Medicine, 63:72 1-726, 1992.
17. Grigoriev, AJ., Kozerenko, O.P., Myasnikov, V.I., Egorov, A.D. Ethical Problems of Interaction
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18. Chaikin. A. The loneliness ofthe long-distance astronaut. Discover, February 1985, pp. 2&31.
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20. Grigoriev, A.I., Kozerenko, O.P., Myasnikov, V.I. Selected Problems of Psychological Support of
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23. Cooper, H.S.F. Jr., A House in Space. Holt, Rhinehart & Winston, New York, 1976.
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25. Bluth, B.J. Soviet space stress. Science, 81:3&35, 1981.
26. “More back talk from space”, Sun Francisco Chronicle, December 8, 1983.
27. Oberg, J.E., Red Star in Orbit. Random House, New York, 1981.
28. Bell, L. Designing a village in space. Futurist, October 1981, pp. 3 W 6 .
29. Bluth. B.J. The benefits and dilemmas of an international space station. Acta Astronautica,
11:149-153, 1984.
30. Finney. B. Scientists and seamen. In: From Antarctica to Outer Space (A.A. Harrison, Y.A.
Clearwater, C.P. McKay, Eds.), pp. 89-101. Springer-Verlag, New York, Berlin, 1991.
3 1. Kelly, A.D., Kanas. N. Communication between space crews and ground personnel: A survey of
astronauts and cosmonauts. Aviation. Space and Environmental Medicine, 64:795-800, 1993.
32. Kelly, A.D., Kanas, N. Leisure time activities in space: A survey of astronauts and cosmonauts.
Acta Astronautica, 3245 1-457, 1994.
33. Flinn, D.E., Monroe, J.T. Cramer, E.H.Observations in the SAM two-man space cabin simulator.
Behavioral factors in selection and performance. Aerospace Medicine, 32:610415, 1961.
34. Dunlap, R.D. (Ed.), The Selection and Training of Crewmenfor an Isolation and Confinement
Study in the Douglas Space Cabin Simulator. No. 3446, Douglas Aircrai? Company, Santa Monica,
California. 1965.
35. Rodgin, D.W., Hartman, B.O. Study of man during a 56-day exposure to an oxygen-helium
atmosphere at 258 mm Hg total pressure. XIII. Behavior factors. Aerospace Medicine, 37:605-
608. 1966.
36. McDonnell Douglas Astronautics Company, 60-Day Manned Test of a Regenerative Life Support
System with Oxygen and Water Recovev. Part II: Aerospace Medicine and Man-Machine Test
Results. NASACR-98501, McDonnell Douglas Astronautics Company, Santa Monica, California,
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37. Ferguson, M.J. (Ed.), Use of the Ben Franklin Submersible as a Space Station Analog. Vol.
II-Psychology and Physiology. OSR-7&5, Grumman Aerospace Corporation, Bethpage, New
York, 1970.
38. Jackson, J.K., Wamsley, J.R., Bonura, M.S.,Seeman, J.S. (Eds.), Program Operation Summay:
Operationa190-DayManned Test ofa Regenerative LifeSupport System. NASACR- 1835, NASA,
Washington, DC, 1972.
Chapter 4
PHARMACOLOGY IN SPACE:
PHARMACOTH ERAPY
A. Pavy-Le Traon, S. Saivin, C. Soulez-LaRivi&re,

M. Pujos, A. Guell, and G. Houin
I. Introduction . . . . . . . . . . . . . . . . . . . . . , . . . . . . , . . . . . . . 94
11. Development of Medical Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
A. Medical Kits on Early American Flights . . . . . . . . . . . . . . . . . . . 94
B. Medical Kits on Space Shuttle Flights . . . . . . . . . . . . . . . . . . . . 95
C. Medical Kits on Russian Flights . . . . . . . . . . . . . . . . . . . . . . . 99
111. Main Drugs used during Spaceflight . . . . . . . . . . . . . . . . . . . . . . 100
IV. Pharmacological Countermeasures . . . . . . . . . . . . . . . . . . . . . . . 101
V. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . , . 103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

ISBN: 0-7623-0147-3
93
94 PAVY-LE TRAON et al.
1. INTRODUCTION
Drugs are necessary in space to treat ailments that are common to humans on Earth
as well as disturbances caused by the space environment.
Since the beginning of manned spaceflight, medical kits have been carried
onboard the space vehicles to cope with illnesses and injuries that may occur during
such missions. The choice of drugs for the medical kits has mainly been determined
by the duration of a flight, but other factors such as the nature of the mission and
the presence or absence of a physician among the crew members have also played
a role. With increasing experience from previous manned missions and the increase
in flight duration, there has been a steady growth in the number of drugs contained
in these kits. The choice of drugs used in space and their indications are discussed
in this chapter.
On the other hand, the disturbances due to the space environment, particularly
weightlessness, can influence drug disposition in the human system. The possible
effects of the space environment on the pharmacokinetic behavior of drugs admin-
istered in space open up an entirely new field of pharmacological research.
II. DEVELOPMENT OF MEDICAL KITS

A. Medical Kits on Early American Flights
Initially, during the American Mercury program, the basic concept regarding
drugs carried into space was that they would be made available for emergency use
only. Autonomic injections made it possible for the astronaut to self-administer
drugs through the pressure suit.' For the first missions, these drugs included an
anodyne, an anti-motion-sickness drug, a stimulant, and a vasoconstrictor for
treatment of shock. In later missions, these were reduced to the anti-motion-sick-
ness drug and an anodyne. For the last Mercury flight, it was decided to make tablets
of the stimulant dextroamphetamine sulfate available both in the pressure suit and
in the survival kit.'
As project Mercury evolved into project Gemini, additional drugs were included
in the medical kit and a first-aid kit was also carried onboard. The various drugs,
their forms and uses are summarized in Table 1.2 They include drugs for the
treatment of motion sickness, pain, fever, inflammation of the respiratory tract,
diarrhea, and infections.
The content of the Apollo medical kit was based on experience gained during
these earlier missions. The drugs for medical emergencies were supplemented with
those for treating the contingency situations most likely to arise.3Preflight drug
sensitivity testing was also performed to determine the response of flight crew
members to each item in the kit and thus to preclude allergic reactions and other
undesirable side-effects during flight.3
Pharmacology in Space 95
Table 7. Gemini-7 Inflight Medical and Accessory Kits

(2 astronauts, 14 days)
~~
Medication Dose and Form indication Quantity

Cyclizine 50 rng tablets Motion sickness 8
d-Amphetamine sulfate 5 rng tablets Stimulant 8
APC (aspirin, phenacetin tablets Analgesic 16
and caffeine)
Meperidine 100 rng tablets Pain (narcotidanalgesic) 4
Triprolidine 2-5 rng tablets Decongestant 16
Pseudoephedrine 60 rng tablets Decongestant 16
Diphenoxylane 25 rng Diarrhea 16
tablets
Tetracycline 250 rng film-coated tablets Antibiotic 16
Methyl Cellulose soln. 15 cc in squeeze-dropper Eye drops 1
bottle
Parenteral Cyclizine 45 rng (0.9 rnl in injector) Motion sickness 2
Parenteral Meperidine 90 mg (0.9 rnl in injector) Pain (narcotidanalgesic)
Source: From Berry (1975).
The contents of the Apollo Command Module medical kit are listed in Table 2 .
A reduced version of this kit was carried in the Lunar Module. Topical drugs were
more numerous. A short-acting barbiturate, secobarbital, was added after reports of
sleeping difficulties by the Apollo-7 crew, as well as medications against cardiac
arrhythmia (procainamide,lidocaine). The adequacy of the kits was reviewed after
each flight and appropriate modifications were made for the next flight. Thus, there
was no standard kit, although the basic contents of the kits remained the same.3
Onboard medical supplies for Skylab were more elaborate in view of the longer
duration of the missions (28 to 84 days). The inflight medical support system was
developed to provide the onboard crew physician or other designated crew member
with adequate information to make a diagnostic assessment of the injuries or
illnesses most likely to occur in the Skylab en~ironment.~ These pharmacological
kits were more elaborate, although the 62 medications for the three missions did
not differ greatly from those for the Apollo mission^.^
B. Medical Kits on Space Shuttle Flights

The U.S. Space Shuttle carries a specially designed medical system that allows
the treatment of simple illnesses and injuries that could occur during flight, as well
as the stabilization of a severely ill or injured crew member until return to Earth is
possible.6This package, known as the Shuttle Orbiter Medical System (SOMS),
comes in three versions-SOMS-A, SOMS-B and SOMS-C4epending on the
flight duration. Only the SOMS-A package has been used to date. The items in the
Table 2. Command Module Medical Kit during Apollo Missions
7 8 9 10 11 12 13 14 15 16 17
Methylcellulose eye drops (1/4%) 2/1 2/2 210 2/0 210 2/0 210 1/o 10/2 1/o
Tetrahydrozoline (Visine) - - - - - - - - - 111
Compress - bandage 210 2/0 2/0 210 210 2/0 210 210 210 210
Bandaids 1 212 1 210 1210 1210 1210 1210 1 210 1210 1210 1210
Antibiotic ointment 1 I1 1/o 1/o 1/o 2/0 2/0 2/0 210 2/1 2/1
Skin cream 1I0 111 111 1 I0 1I0 1I0 1I0 1I0 1I1 1I0
Meperine (Demerol)@injectors, 90 mg 310 310 310 310 310 310 310 310
Cycl izine (Marezine)@injectors 310 310 310 310 310 310 310 3/0
Cyclizine (Marezine)@ tablets, 50 mg 24/3 24/1 2414 1 2/0 - - - - - -
Dextroamphetamine (Dexedrine)0
5 mg tablets 1211 1210 1210 1210 1210 1211 1 210 12/0 1210 1210
@
Propoxyphene(Darvon) compd.,
60 mg capsules 1 212 1 8/0 1 8/0 1 8/0 1 8/0 1 8/0 1 8/0 1 8/0 1 8/0 1 810
Actifed 6010 6011 2 6012 60/0 60/0 6010 6010 6010 6010 60/1
Diphenoxylate (Lomotil)@tablets 2418 2413 2411 2411 3 2410 2 411 2410 2410 24/0 4815
Nasal emollient 1I0 211 2/1 1I0 1I0 1I0 1I0 1/o 1I0 1I0
Aspirin, 5 g tablets 72/48 7218 7212 7211 6 72/6 72/30 7210 72/0 7210 72/0
Tetracycline, 250 mg 2410 2410 24/0 1510 - - - 6010 60/0 60/0
Ampicillin - 60/0 60/0 4510 60/0 6010 60/0 6010 6010 6010
Secobarbital (Seconal)@
100 mg capsules
50 mg capsules
@
Oxymetazoline (Afrin) nose drops
Diphenhydramine (Benadrylf 50 mg
Acetaminophen (Tylenol)? 325 rng
Bacitracin eye ointment
Scopolamine, 0.3 mg
@
Dextroamphetamine (Dexedrine) ,
5 mg capsules
c9 - 4010 4010 4010 4010 4010 4010 4010
Simethicone (My1icon) tablets
Opthalne - - - 110 110 110 110 110
Multi-vitamins - - - - 2010 - - -
Notes: Auxillary Drugs for Apollo 16 & 17: Proca'inamide(Pronestyl)"80/0, Lidocaine 1YO, Atropine 1YO, Meperidine (Demerol)"6/0. * unknown.
v
Source: From: Biomedical Results of Apollo, Hawkins and Zieglscmid, 1975.
packages are reviewed periodically in the light of growing flight experience, and
the contents are revised as appropriate.
The Shuttle SOMS medications (Table 3) comprise primarily:
0 antibiotics for general and local use;

0 analgesics, comprising often used minor substances like aspirin, but also
narcotics like morphine;
antihistamines;
medications for the digestive tract, e.g., against diarrhea, constipation, and
flatulence;
0 drugs to counter motion sickness,
Table 3. Medications Flown on Space Shuttle

ANTACl DS HEMORRHOIDS
Aluminum hydroxide (Mylanta@) ZindBismuth oxyde (AnusoI@ointment)
ANTIBIOTICS MUSCLE RELAXANTS
Am ikacin Diazepam (Valium@)(PO and IM)
Amoxicillin PAIN
Sulfamethoxazole+trimethoprime (Bactrim@) Aspirin
Cefadioxil (Duricep) Codeine
Erythromycin Meperidine (Demerol@)
8
Metronidazole (Flagyl ) Morphine
CONSTIPATION Ibuprofen (Motrin@)
Bisacodyl (Dulcolax@) Phenazopyridine(Pyridium?
Acetaminophen (Tylenol8)
DECONGESTANTS
Oxymetazoline (Afrin@)nasal spray SEIZURES
Pseudoephedrine (Sudafed@) Diazepam (Valium@)(IM or IV)
DIARRHEA SLEEPING PILLS
@
Pepto-Bismol Flurazepam (Dalmane )
Diphenoxylate (Lomotil@) Temazepam (Restoril@)
HEART MEDICATIONS STIMULANTS
Atropine Dextroamphetamine(Dexedrine@)
Epinephrine OTHERS
Lidocaine Haloperidol (Haldol@)
Verapam il Naloxone
MOTION SICKNESS Norgestrel/Ethinyl Estradiol
Promethazine (Phenergan@)(PO, PR, IM) Diphenhydramine (Benadryl@)
Scopolamine
Dextroamphetamine (Dexedrine@)
Note: @givesthe trade name of the product in the USA
Source: From: Flight Data File Medical Checklist, JSC 17327, 1989.
0 neurotropic and psychotropic agents, including sleeping pills, tranquilizers

and stimulants,
0 cardiovascular medications, including most of the injectable drugs for emer-
gency situations,
0 locally applied medications, often with decongestive or antibioticaction, such
as eye drops, nasal drops, creams, and ointments.
C. Medical Kits on Russian Flights
Few data are available on the medical kits used on the Russian flights. Table 4
lists the drugs used during a 166-day mission on the space station Mir.7 As on the
American flights, the Russian medical kits appear to contain a relatively small
assortment of drugs, primarily consisting of cardiovascular agents, analeptics,
analgesics, psychotropic agents, antibiotics and vitamins. There was, of course, a
need to develop pharmacological countermeasures for the long-duration flights in
the context of the Salyut and Mir space stations.*
Although a detailed list of the drugs carried onboard the Mir Station cannot be
given here, the medical kits (B. Comet & A. Cornac, personal communication)are
believed to contain the following medications:
The main types of drugs are antibiotics, drugs against digestive tract distur-
bances, cardiovascular drugs (diuretics, antiarrhythmics, sympathomimetic
agents), drugs against fever, pain and inflammation,and antiallergics.
Several drugs acting on the central nervous system (hypnotics, tranquilizers,
psychostimulants).
Multivitamins and minerals.
Table 4. Drugs Used on 166-Day MIR Mission

Drug Time Period Purpose
Ribox in Month 3, 20 days Prevents myocardial metabolic

disorders
(9-Ribofuranozyl-Hypoxanthin) increases myofibril potential
Panagin Month 3, 20 days Supplies potassium and other minerals
Riboxin Month 3, 10 days See above
Potassium Orotate Nonsteroidal anabolic drug
Nootropyl-2-0x0-1 Month 4 Positive effect on cerebral blood flow
Pyrrolidinyl-Acetamide
Riboxin Month 5 See above
Panagin See above
Bifidum Bacterine Month 6 Prevents dysbacteriosis
Multivitamins Throughout flight Prevents disruption of vitamin and
mineral metabolism
Source: From: J. Vernikos-Danielis (1991).

Multivitamins and minerals have been deemed important for long-duration

flights. Startingwith the first Vostok flights, spaceflight rations for the cosmonauts
have always included multiple vitamin supplements. On Salyut and Mirythe crew
has taken the Aerovit multivitaminpreparation, which contains 12 vitamins.' Fewer
topical drugs have been made available than on Shuttle flights.
111. MAIN DRUGS USED DURING SPACEFLIGHT

Space motion sickness has proved to be one of the main reasons for administering
pharmaceuticals during spaceflight," along with the management of altered sleep
patterns," especially during Shuttle flights.
Marezine was first used for the treatment of space motion sickness during the
Apollo program. After the Apollo-11 and -14 missions it was replaced by a
combinationof scopolamine and dextroamphetamine,when ground-based tests had
indicated that the latter combination was more effe~tive.~
Aspirin was often used for the relief of headache, pain, and strains. The barbitu-
rate secobarbital was used during several missions for the prevention of sleep
disturbances. Astronauts took diphenoxylate (Lomotil@)as an antidiarrheic sub-
stance for gastro-intestinal disturbances, probably caused by viral infection, and
dimeticone (Simethicone') for the treatment of flatulence. Actifed@,which is a
combination of three agents (antihistaminic, anti-inflammatory, analgesic and
antipyretic), was often used for colds or rhinitis related to the fluid shift caused by
microgravity. Among local medications, decongestant nasal drops were used par-
ticularly during the first days of flight for congestion due to the fluid shift.
During the Apollo-15 mission, one astronaut experienced a cardiac arrhythmia
due to premature ventricular contractions, while another crew member exhibited
premature supranodal contractions. These symptoms were associated with
fluidelectrolyte disorders.To prevent such cardiac rhythm disturbances, astronauts
routinely took potassium salt during the Apollo-16 and - 17 missions. No medically
significant arrhythmia occurred during these flights.
An inflight malhction of the service module caused early termination of the
Apollo- 13 mission. Examination of the crew immediately after the flight confirmed
that one crew member had contracted a severe urinary tract infection inflight, from
which Pseudomonas aerogunisae was isolated. Several antibiotics were used to
treat this condition: tetracyclinewas used inflight, and after return phenazopyridine
(Pyridium@)and nitrohntoin (Furadantin@),all without success. Finally, the
infection was successfully treated with colistin (Colimycin@).12
During the Skylabprogram, the crew surgeon reported that many crew members
took scopolamine/dextroamphetamine for space motion sickness, either as a pre-
ventive measure or when the symptoms of space motion sickness appeared. Aspirin
was prescribed for transient headaches, and hypnotics were used to treat sleep
disturbances. Vitamins were added to the alimentation during Skylab-3 and 4.
Decongestants (topical and systemic) were used both prophylactically during
extravehicularactivities and for specific relief of the feeling of fullness in the head,
nose, and ears. Other local medications prescribed included: ophthalmic antibiotic
ointment for painless sty, topical steroid cream for a skin inflammation,and topical
treatment of a probable skin infection (mycosis).
Turning to the Shuttle Program, the significanceof drug prescription is reflected
by the fact that 83 of 107 (78%) crew members took medi~ati0n.l~ Medications
were taken essentially for: space motion sickness (30%), headache (20%), sleep-
nessless (15%), and back pain (1 0%).
Many drugs (taken orally or as suppositories)and drug combinations have been
tested for effectiveness against space motion sickness. Oral combinations of
scopolamine (0.4 mg) and dextroamphetamine (5 mg) (Scopdex') o r of
promethazine (25 mg) and ephedrine (50 mg) were most frequently used, but they
proved less efficient in flight than on Earth.I4A recent study showed that after oral
administrationof Scopdex@space motion sickness symptoms were delayed in some
crew members, but not in all. Use of these drugs produced side effects, most of
which can be attributed to the scopolamine. Amphetamine, which is also efficient
against space motion sickness, tends to counteract the sedative effects of scopola-
mine. Metoclopramide, which acts on the digestive tract, was not efficient inflight.
Recently, intramuscular injections of promethazine (Phenergan@)have been
given to crew members for space motion sickness. Data on Space Shuttle missions
up to July 1991 indicate that 28 of 29 subjects have been successhlly treated for
space motion sickness by intramuscular Phenergan' administration.l5 It is unclear
whether the increased efficacy of intramuscular Phenergan@is due to the pharma-
cological behavior of the drug itself, or whether the route of administration and the
correspondingbio-availability are responsible. This drug can result in dizziness and
disturbanceof psychomotor performance, but no appreciable signs of sedation were
reported by the astronauts.l 4 There is, however, a need for scientificallycontrolled
studies to evaluate the impact of drugs on psychomotor perf~rmance.'~
The drugs used during a 166-day Mir mission (see Table 4) were mostly
prescribed for prevention of cardiovascular, neurologic and digestive disturbances.
They presented no side effects. No medication was prescribed during this mission
to treat illnesses or space-induced disorders. This approach differs from that for the
short-duration Shuttle flights, where crew members have to cope with operational
constraints throughout the short flight.
IV. PHARMACOLOGICAL COUNTERMEASURES

Drugs can also be used as countermeasures to prevent physiological disturbances
induced by spaceflight and by retum to Earth gravity. After landing re-adaptation
of the cardiovascular system and orthostatic intolerance remain an issue which
needs to be addressed.
Many drugs have been tested for the prevention of the orthostatic arterial
hypotension occurring after ground simulations, particularly bedrest studies.I6
Despite a number of extensive studies, post-bedrest and post-spaceflight hypoten-

sion have not been adequately ~haracterized.~ The problem is complicated by the
fact that several factors contribute to orthostatic intolerance: hypovolemia and
hormonal changes, increased venous compliance favored by muscle atrophy,
baroreflex changes,17and so forth.
Pharmacological agents generally act on only one or two factors, and the role
played by different factors may vary with the individual' and the flight duration."
To counteract hypovolemia, an inflight saline-loading procedure has been devel-
oped. Shortly before landing water and salt tablets equivalent to a liter of 0.9%
saline are ingested. This improves orthostatic tolerance, but the procedure is not
completely suc~essfu1.l~ Solutions of various concentrations of salt and glucose
have also been tested.20Fludrocortisone increases the plasma volume and also the
responsiveness of vascular smooth muscle to n~repinephrine.~ After bedrest experi-
ments, fludrocortisonewas the most effective drug against orthostaticintolerance.21
This medication has recently been used on Space Shuttle flights.
Numerous other drugs have been tested during simulation experiments. Propra-
nolol (Intravenous),a beta blocker, also had beneficial effects after bedrest, but only
a few studies have been made?2 Some authors concluded that atropine improves
orthostatic tolerance by blocking vagal reflexes after immersion experiment^^^ and
bedrest experiment^.^'.^^ Other drugs have no, or only incomplete, beneficial
effects: central nervous system agents like amphetamine and caffeine, calcium
blocking agents like isoptin, and clonidine, which is a partial alpha-2 agonist
generally used as an anti-hypertensive agent.25926 A combination of drugs acting on
the different factors involved in orthostatic intolerance might improve its preven-
tion, but the interactions of such drugs need to be studied.
Changes in bone and Ca/p metabolism also remain an issue for long-duration
flights. These changes are not well-characterized. Various medications have been
tested during prolonged bedrest experiments. Increase of phosphate in the diet (1.3
g/day) prevented the hypercalciuria observed during prolonged head-down tilt, but
the Ca/P balance still remained negative and no effect on the heel bone density was
noted.27Ingestion of phosphate and calcium prevented the hypercalciuria and the
hydroxyprolinuria (an indicator of bone catabolism) observed during head-down
tilt, but the effects on the calcium balance were very limited.**Ingestion of fluoride
had no significant effect on the calcium balance of immobilized subjects.29
Some agents that inhibit bone resorption have been tested. Calcitonin had no
effect on the calcium balance changes induced by head-down tilt.28Diphosphon-
ates, prescribed in osteoporosis, could be used to prevent the bone resorption
induced by spaceflight.
Radiation effects will probably be a limiting factor in long-duration space
missions. Chemical radioprotection is still being studied, but its use should prob-
ably be limited to cases of massive irradiation.
Spaceflight typically takes place in a confined and isolated environment, which

can produce psychological problems. The treatment of such problems should not
differ from that prescribed on Earth.

This chapter summarizes the information available on the pharmacological kits
onboard spacecraft and on the use of drugs in space, while the next chapter is
dedicated to the impacts of weightlessness on drug pharmacokinetics. The need of
a selected group of drugs for the use of astronauts during short-term and long-term
spaceflights has been discussed. Recommendations are made for a Space Pharma-
copoeia as well as for the areas of research needed to adapt medication to the
weightlessness of the space environment.
Although the usefulness of these drugs has been clearly demonstrated, their use
also raises several problems. Physiological changes due to weightlessness may
induce changes in pharmacokinetic behavior of drugs and influence their dosage
regimen. Inflight data obtained by salivary drug monitoring have shown changes
in the distribution of scopolamine and a significant change in the disposition of the
common pain-relief agent acetaminophentaken inflight, in both drug concentration
and time course.3oThe authors of this study emphasize, however, that their data are
preliminary and as yet incomplete. Further simulation studies and, if possible,
inflight experiments are required.
In vitro studies of the antibacterial activity of antibiotics under space conditions
have shown an increased resistance of Escherichiu Coli to colistin and kanamycin,
and a lowered resistance of StuphyfococcusAureus to oxacillin, chloramphenicol,
and The possible consequences of these findings for the treat-
ment of infections contracted by astronauts are yet to be elucidated.
There is still a lack of pharmacological countermeasures, particularly for pre-
venting the progressive bone demineralization occurring in weightlessness. The
treatment of space motion sickness with drugs carries with it the problem of
undesirable side-effects on psychomotor performance.
In order to arrive at the most appropriate medical kit for a particular mission, the
best trade-off of risk versus benefit for the individual and the mission must always
be attempted for any pharmacological agent.
ACKNOWLEDGMENT
This work was supported by a grant (n09611/91/FL) from the European Space Agency.
REFERENCES
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1965.
2. Berry, C.A. Medical care of space crews (medical care, equipment, and prophylaxis). In: Foun-
dations of Space Biology and Medicine. Vol. 111. (M. Calvin, O.G. Gazin. Eds.), NASA SP-374,
U.S. Government Printing Ofice, Washington, D.C., pp. 345-371, 1965.
3. Hawkins, W.R., Zieglschmid, J.F. Clinical aspectsof crew health. In: Biomedical Results ofApollo
(R.S. Johnston, L.F. Dietlein, C.A. Berry, Eds.), NASA, SP-368, pp. 4 M 1 , 1975.
4. Hordinsky, J.R. Skylab crew health - Crew surgeons’ reports. In: Biomedical Results of Skylab
(R.S. Johnston, L.F. Dietlein, Eds.), NASA SP-377, pp. 30-34, 1977.
5. Chassay, C., Rose, S.A. In-flight Medical Support. In: Biomedical Results of Skylab (R.S.
Johnston, L.F. Dietlein, Eds.), NASA SP-377, pp. 463-473, 1977.
6. Nicogossian, A.E., Pool, S.L. Medical care and health maintenance in flight. In: Space Physiology
andMedicine, (A.E.Nicogossian, C. LeachHuntoon,S.L. Pool, Eds.), 2nded., pp. 349-363. 1989.
7. Vernikos, J., Dallman, M.F., Van Loon, G., Keil, L.C. Drug effects on orthostatic intolerance
induced by bedrest. Journal of Clinical Pharmacology, 31:974-984, 1991.
8. Shashkov, V.S., Yegorov, B.B. Problems of Pharmacology in Space Medicine. Farmakologiya i
Toksikologiya, 4:325-329, 1979 (Russian).
9. Popov, 1. Alimentation during long-duration spaceflight. Aviatsiia Kosmonautika. 1:42-43, 1981
(Russian).
10. Lathers, C.M., Charles, J.B., Bungo, M.W. Pharmacology in space. Part 2: Controlling motion
sickness. Trends in Pharmacological Sciences. 10 193-200, 1989.
11. Lathers, C.M., Charles, J.B., Bungo, M.W. Pharmacology in space. Part 1: Influence of adaptative
changes on pharmacokinetics. Trends in Pharmacological Sciences, 10:193-200, 1989.
12. Ferguson, J.K., Taylor, G.R., Mieszkuc, B.J. Microbiological investigations. In: Biomedical
Results of Apollo (R.S. Johnston, L.F. Dietlein, C.A. Berry, Eds.). NASA SP-368, NASA,
Washington, D.C., pp. 83-103, 1975.
13. Santy, P.A., Kapanka, H., Davis, J.R., Stewart, D.F. Analysisof sleep on Shuttlemissions. Aviation
Space and Environmental Medicine, 59: 1094-1097, 1988.
14. Kohl, R.L., MacDonalds. New pharmacological approaches to the prevention of space motion
sickness.Journal of Clinical Pharmacology, 31:934-946, 1991.
15. Bagian, J.P. First intramuscular administration in the US Space Program, Journal of Clinical
Pharmacology, 31:920, 1991.
16. Sandler, H. Cardiovascular effects of inactivity. In: Inactivity Physiological Effects (H. Sandler,
J. Vernikos-Danellis, Eds.), Academic Press, London, pp. 1147, 1986.
17. Eckberg, D., Fritsch, J. Human autonomic responses to actual and simulated weightlessness.
Journal of Clinical Pharmacology, 31:951-955, 1991.
18. Charles, J.B., Lathers, C.M. Cardiovascularadaptation to spaceflight.Journal of Clinical Phar-
macology, 31: 1010-1023, 1991.
19. Bungo, M.V., Charles, J.B., Johnson, P.C. Cardiovasculardeconditioningduring space flight and
the use of saline as a countermeasureto orthostaticintolerance.Aviation Space and Environmental
Medicine, 56:985-40, 1985.
20. Frey, M.A.B., Riddle, J., Charles, J.B. Bungo, M.W. Blood and urine responses of ingesting fluids
of various salt and glucose concentrations. Journal of Clinical Pharmacology,31:88M87, 1991.
21. Vernikos, J. Metabolic and endocrinechanges. In: Inactivity Physiological Effects (H. Sandler ,J.
Vernikos-Danielis. Eds.), pp. 92-121. Academic Press New York, 1986.
22. Sandler, H., Goldwater, D.J., Popp, R.L., Spacavento, L., Harrison, D.C. Beta blockade in the
compensation for bedrest cardiovascular deconditionning: Physiological and pharmacological
observations.American Journal ofcardiology, 55:1 14D-I20D, 1985.
23. Stegemann, J., Framing, H.D., Schiefeling,M. Effects ofmulty hours immersion with intermittent
exercise on urinary excretion and tilt tolerance in athletes and non athletes. Aviation Space and
Environmental Medicine, 46:26-29, 1975.
24. Murray, R.H., Shropshire, S.Effect of atropine on circulatory responses to lower body negative
pressure and vasodepressor syncope. Aerospace Medicine, 41:7 17-722, 1970.
25. Bonde-Petersen, F., Giiell, A., Skalen, K., Henriksen, A. The effects of clonidine on peripheral
vasomotor reactions during simulated zero gravity. The Physiologist, 24 (6):58%590, 1985.
26. Giiell, A., Gharib, CI., Gauquelin, G., Montastruc, P., Bes, A. Clonidine as a countermeasure for
metabolic studies during weightlessness simulation. The Physiologist, 25 (4):69-70, 1982.
27. Hulley, S.B., Vogel, J.M., Donaldson, C.L., Bayers, J.H., Friedman, R.J., Rosen, S.N. Effect of
supplemental oral phosphate on the bone mineral changes during prolonged bed rest. Journal of
Clinical Investigation, 50:2506-25 18, 1975.
28. Schneider, V.S., MacDonald, J. Skeletal calcium homeostatis and countermeasures to prevent
disease osteoporosis. Calcified Essue International, 36:s 151-1 54, 1984.
29. Maheshwari, U.R., Brunetti, A.J.. Leybin, L., Newbrun, E., Hodge, H. Fluoride balance studies
in healthy men during bed rest with and without a fluoride supplement. American Journal of
Clinical Nutrition. 36:211-218, 1982.
30. Cintrom, N.M., Putcha, L., Chen, Y.M., Vanderploeg, J.M. Inflight salivary pharmacokinetics of
scopolamine and dextroamphetamine. In: Results of the life sciences D.S.O.S.conducted aboard
the Space Shuttle, 1981-1986 (NASA Editorial Review Board), NASA TM 58280, NASA,
Washington, D.C., pp. 153-158, 1987.
31. Tixador, R., Richoilley, G., Gasset, G. Preliminary Results of the Cytos-2 Experiment. Proceed-
ings of the 34th Congress of the International Astronautical Federation, Paper IAF-83-192, 1983.
32. Lapchine, L., Moatti, N., Richoilley, G., Templier, J., Gasset, G., Tixador, R.R. Antibacterial
activity of antibiotics in space conditions. In: Scientific Results of the German Spacelab mission
D I , Pmc. Symposium Nordemey, ESA, Paris, pp. 395397, 1986.
Chapter 5
PHARMACOLOGY IN SPACE:
PHARMACOKINETlCS
S . Saivin. A . Pavy-Le Traon. C . Soulez.LaRivi&-e.

A . Guell. and G . Houin
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
I1. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
A . Membrane Passage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
B . Effects of Route of Administration . . . . . . . . . . . . . . . . . . . . . 109
111. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
A . Protein Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
B. BloodFlow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
C . Physical Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
IV. Elimination of Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
A . Hepatic Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
B . Renal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
V. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Volume 6. pages 107-121
All rights of reproduction in any form reserved .
ISBN: 0-7623-0147-3
107
108 S. SAlVlN et at.
1. INTRODUCTION
During spaceflight the human organism undergoes various physiological modifi-

cations due to its adaptation to weightlessness. Some of these modifications last
only a brieftime, while others persist during the entire flight. During this adaptation
process the human organism reaches a new state of homeostasis. The physiologjcal
and biochemical modifications taking place during spaceflight can be chronologi-
cally divided in three phases: the adaptation phase, the equilibrium phase and the
landing phase. Figure 1 illustrates the most important modifications occurring in
microgravity.I
From a pharmacokinetic point of view, similar physiological changes occurring
on Earth are well known to greatly modify drug disposition. To some extent it is
possible to extrapolate from the pharmacokinetic changes observed on Earth to
what may be expected to happen to drug disposition in space. The process of
disposition of a drug in the body can be divided in three parts: absorption at the site
of administration, distribution in the tissues, and elimination by metabolism and
excretion. It is necessary, therefore, to know what will happen in weightlessness to
absorption, distribution and elimination for each route of administration.2
II. ABSORPTION
Absorption is the first step of drug disposition after administration. It corresponds
to the appearance of the drug and in certain cases its metabolites in the circulation
IRREVERSIBLE PROCESSES
I I
FLUIDS AND ELECTROLYTES
REDBLOOOCELLWASD
09
set
Point
1.p
Set
Point
3 4 6 6
POW OF ADUTATIUU
Time suls (months)
figure 1. Time course of physiological shifts during spaceflight according to Nico-

1
gossian.
Pharmacology in Space: Pharmacokinetics 109
from the site of administration. The rate of absorption and the amount absorbed
characterize the mechanisms involved in absorption. These are a function of the
form in which the drug is presented, the membranes through which it passes, and
the site of loss. In pharmacokinetics, the rate of absorption and the absorbed amount
are characterizedby the bioavailability of the drug. It includesthe ‘first pass effect’,
which is defined as any mechanism responsible for a loss of drug between the site
of administration and the circulation. In most cases, the first pass effect occurs in
the liver, but metabolism can occur at other sites.
A. Membrane Passage
Membrane passage is required whenever a drug is administered extravascularly.

This step depends on numerous parameters, such as the physicochemical properties
of the drug and the specificmembrane properties. Specific cases are the blood-brain
barrier and the hemo-placental barrier. Diffusion through membranesoccurs essen-
tially according to three mechanisms: passive diffision according to Fick’s law,
facilitated diffusion, and active transport. Diffusion is the most important one, and
the rate of diffision is proportional to the concentration gradient across the
membrane. For drugs which diffise freely, blood flow becomes the limiting factor.
In weightlessness, the intrinsic ability of a drug to cross a membrane or to be
actively transported is unlikely to be changed, but the blood flow in a specific tissue
may be modified. Membrane permeability may also be reduced by local edema due
to redistribution of fluid.3
B. Effects of Route of Administration
Routes of administration, their specific sites of loss and the different steps
involved may influence the bioavailability of a drug, as illustrated in Figure 2.
Weightlessness may have specific effects for each route of administration.
Intravenous Route
The intravenous route is considered as the reference mode of administration,

since the drug is directly introduced into the circulation and thereupon the entire
administered dose is available to induce its pharmacological effect. An exception
is the ability of the lungs to metabolize drugs such as norhiptyline, d-methadone,
mescaline or ibuter01.~In this case, a first pass will occur before the drug reaches
the pharmacological target organ, and the corresponding effect will be decreased
when compared to arterial administration. Microgravity may influence lung meta-
bolism by a possible increase in the perfusion rate of this
Subcutaneous and Intramuscular Routes
Absorption after subcutaneous or intramuscular injection depends on blood flow

and muscle activity. In microgravity, redistribution of the blood volume may
110 S. SAlVlN et al.
increase the blood flow in the upper part of the body and decrease it in the lower
part. Therefore, bioavailability may vary depending on the site of injection. This
may also affect the amount of drug metabolized before the remainder reaches the
general circulation. In space,muscle atrophy,characterizedby a reduction in muscle
strength, tone and endurance, has been reported.'+ Intramuscular injections of
promethazine have been performed during a US. spaceflight with better efficacy
than on Earth and without any sign of toxicity." However, since bioavailability of
promethazine is greater after intramuscular injection than after oral ingestion," it
is not possible to conclude that the better efficacy is due to a microgravity-induced
change in bioavailability.
Oral Route
Oral administration is best accepted by patients, and is therefore the most

common route of drug administration. However, it is also the most complicated
.anzymanc a a l u
Mlary excroth
LINTRAMUSCULAR AND SUBCUTANEOUS ROUT

1 I
RECTAL ROUTE
I O R A L ROUTE
J
Figure 2. Variability of drug absorption in function of routes of drug administration

and their site of loss.
route in terms of the physiological steps involved, each of which may be modified
in microgravity. The most important steps are dissolving of the drug in the
gastro-intestinal fluid, gastric emptying, intestinal motility, absorption through the
duodenal cell membranes, and passage through the liver. Gastric emptying is known
to be greatly influenced by the position of the body,'* the characteristics of the
pharmaceutical form of the drug, and the presence or absence of food in the
intestine. In microgravity, gastric emptying may be influenced by the weightless-
ness of the gastric contents.I2 The absence of gravity may have a further effect,
since gastric emptying can be seen as a probability occurrence with a random
chance that a particle in the stomach passes through the py10rus.'~Consequently,
some modification may occur in the rate of drug absorption. This may delay the
gastro-intestinal transit time and also the transit motility.
Intestinal absorption involves membrane crossing phenomena that may be dis-
turbed by modifications in local blood flow or transit time. The intestine is the main
site of absorption due to its large surface and the extended residence time. On Earth,
after gastric emptying, changes in intestinal absorption are mainly due to differ-
ences in the intestinal blood flow. If this flow is reduced by the fluid shift in
weightlessness, then intestinal drug absorption may be decreased or slowed down.
Such a mechanism has been described for digoxin in patients with cardiac decom-
pen~ation.~
However, some drugs are never fully absorbed, either because of a primary
decrease in absorption through the intestinal membrane, or as the result of local
metabolism by bacteria or enzymes, or by physicochemical interactions in the
intestinal lumen. Table 1 lists the main drugs for which variations of intestinal
absorption are classically observed on Earth. The most common example of drug
interaction before absorption is the complexation of the first generation of tetracy-
clines with divalent cations such as calcium or magne~ium.'~ The bioavailability
of this antibiotic may thus be significantlyreduced by concomitant administration
Table 1. Drugs Known to be Variously Absorbed from

the Gastro-Intestinal Tract
Aspirin Pentazocine
Aldosterone Pethidine
Chlorpromazine Phenacetin
Dexarnethasone Propoxyphene
L-Dopa Progesterone
Flurazeparn Rirniterol
Hydrocortisone Salicylarnide
lsoprenaline Sulfamides
Methadone Terbutaline
Metoclopramide Testosterone
Morphine
with milk or milk products. Tetracycline are present in the space pharmaceutical
kits. Since the astronaut diet may contain extra calcium to compensate the bone
loss with negative calcium balance occurring in space, it will be necessary to
dissociate the administration of the drug and the meal. Other examples are the
interactions between digoxin or warfarin with cholestyramine, of penicillamine
with aluminum or magnesium ions, of digoxin with metoclopramide and propan-
theline, and of penicillin with n e ~ m y c i n . ' ~
There is an important phenomenon which explains why the entire dose of an
orally administered drug does not reach the general circulation: the hepatic 'first
pass effect'. After being fblly absorbed from the gastro-intestinal tract, the drug
passes through the liver before reaching the general circulation. In the liver a
substantial fraction of the drug may be metabolized to an inactive product. This is
probably the most important phenomenon, both in terms of absolute amount and
of variability. It can be quantified by the extraction ratio (E,) which corresponds
to the fraction of the drug reaching the liver that is metabolized. The amount
escaping the liver, i.e., the maximum amount reaching the circulation after oral
administration, is given by:
where F represents the bioavailability.

The higher the extraction ratio, the lower will be the availability and the more
variable will be the absorbed amount of the drug. If enzymatic activities of the liver
vary due to diurnal changes or inductiodinhibition phenomena, even a large change
in E, (up to 100%) will have only little effect on the availability of drugs with a
low extraction ratio. On the other hand, for drugs with a large extraction ratio, a
small change in E, will lead to a large change in the amount of drug escaping the
liver. Table 2 shows the drugs most sensitive to this phenomenon. For example,
Table 2. Drugs Most Sensitive to Hepatic First Pass Effect

Alprenolol Nifedipine
Aspirin Nortryptiline
Cortisone Oxprenolol
Desimipramine Oxyphenbutazone
Dopamine Pentazocine
FIuorouraciI Pethidine
Hexobarbital Phenacetin
lmipramine Pindolol
lsoprenaline Propoxyphene
Lidocaine Propranolol
Metoprolol Salicylamide
Morphine Serotonin
propranolol not only requires an oral dose 8 times higher than that necessary by
intravenous route, but it also shows a higher variability after oral than after
intravenous administration. Among these drugs, some as aspirin, lidocaine, mor-
phine and nifedipine are usually included in the space medical kit. The upward fluid
shifts and hemodynamic changes observed in space may increase the blood perfi-
sion of the liver.599For drugs with a low extraction coefficient, being blood flow
independent, it is unlikely that microgravity will have a significant effect on their
hepatic first pass metabolism. However, flow-dependent drugs may be metabolized
more efficiently in space than on Earth, due to the higher hepatic blood flow in
space. Consequently, the bioavailability of these drugs and their circulating con-
centrations will be lower in space, which might necessitate an increase in dosage.
Sublingual and Buccal Routes
Sublingual administration is commonly used to prevent the hepatic first pass

effect. Factors that may influence absorption by this route in microgravity are
possible modifications of local conditions, e.g., dryness of the mouth and cephalic
fluid shift. The latter may be responsible for an increased local blood flow, which
may cause a rise in the rate or the amount of drug absorbed. Absorption from these
two sites depends on the blood flow, and thus the previously made remarks apply.
Rectal Route
On Earth, absorption after rectal administration is variable, particularly because

the hepatic first pass effect is different in terms of the veins involved in this mode
of drug absorption. In microgravity, changes in the hepatic first pass effect may
influence drug bioavailability, as previously described for the oral route.
Percutaneous Route
Percutaneous administration does not involve the hepatic first pass effect, but the
fluid shift may modify the local blood flow. Drug absorption may then depend on
the site of administration. At this time the importance of such changes is difficult
to predict. The local environment, such as dryness of the skin or cutaneous diseases,
may also affect the absorption.
Pulmonary and Nasal Route
Absorption through lungs and nose depends on the local blood flow, which will
probably be influenced by the occurrence of a fluid shift. The resulting effects are
again difficult to predict. After pulmonary administration, the drug must reach the
capillary membrane, which is achieved by microdispersion of the drug vehicle.
Microgravity may modify the characteristics of the dispersion, and thus also the
amount of drug reaching the pulmonary membrane.
111. DlSTRl BUTlON
Distribution is the process by which a drug is transferred from the blood to the
interstitial fluids and the various tissues of the organism. Many factors may
influence the distribution of a drug, and these could potentially be influenced by
microgravity. On Earth, the main factors are the physicochemical properties of the
drug, the membrane composition, the binding to tissue and plasma proteins, and
the blood flow in the tissues. Protein binding and blood flow, as well as the effect
of exercise, will be discussed in some detail.
A. Protein Binding
Protein binding is an important phenomenon, occurring with endogenous as well

as exogenous compounds. Protein molecules are always large compared to the drug
molecule, 100- to 1000-fold larger. Therefore, the drug may bind to different
specific sites on a given protein molecule. This binding may be saturable or
non-saturable and have a high or low affinity and specificity, depending on the
nature of the chemical link and the fit between drug and protein molecule. Binding
is generally reversible, so that the free and bound fractions are in equilibrium with
each other. The main proteins in blood, which bind circulatingdrugs or endogenous
compounds, are albumin, a 1-glycoprotein and lipoproteins (Table 3). There exist
a few proteins in blood plasma, which specifically bind a particular substance, like
the steroid transcortin.
Protein binding is an important phenomenon in pharmacokinetics, since only the
free fraction of the drug is able to diffise and is therefore likely to be active or to
be metabolized. The distribution of drugs is greatly influenced by binding to plasma
and tissue proteins. Some changes in protein concentrations during spaceflight have
been reported; these may influencedrug binding. If protein binding is non-saturable
in the usual ranges of plasma protein and drug concentrations,a decrease in protein
concentration will lead to little change in free and bound drug concentrations. If
the protein binding capacity is near saturation, a reduction of the concentration of
this protein may lead to saturation and consequently to an increase in the free active
drug fraction. A similar effect will be observed when the drug dosage is increased.
In microgravity, a decrease in total body water is observed, which is responsible
for an increase in the concentration of hemoglobin and other blood proteins through
hemoconcentration. Theoretically, the increase in protein concentrations will re-
duce the free fraction of drugs, but the hemoconcentration will in turn probably
increase the total drug concentrations. As a consequence, the free drug concentra-
tion may be virtually unchanged. However, for drugs that are highly bound in
tissues, the overall equilibrium can be modified and the bound drug in the tissues
could be larger than on Earth.
Table 3. Protein Binding of Drugs

NON-SATURABLE SATURABLE
WEAK ACIDS
to ALBUMIN: to ALBUMIN:
Warfarin Valproic acid
Acenocoumarol Salicylic acid
Furosemide Phenylbutazone
Diazepam Clofibric acid
WEAK BASES
to ALBUMIN: to a1 -ACID
G LYCOPROTEIN:
Quinidine Quinidine
Rifarnpicin platelet antiagregants
platelet antiagregants Lidocaine
Disopyramide
to LIPOPROTEINS: lmipramine
Erythromycin
Quinidine beta blockers
platelet antiagregants
beta blockers
Rifampicin
6. Blood Flow
Blood flow regulates the rate of entry in and the output of drugs from tissues. It
is more involved in the rate of distribution than in its intensity. As shown in Table
4, the tissue perfusion rates vary widely in the organism, from 0.025 ml.min-'.g-'
for peripheral fat to 10 ml.min-l.g-l for lung. Obviously, when comparing individ-
ual organ flows, the organ size must be taken into account. For example, the muscle
perfusion rate is low, 0.025 ml.min-'.g-l, but its total blood flow is as large as 750
ml.min-l. On the other hand, the cardiac perfusion rate of 0.6 ml.min-l.g-l is 24 x
that of muscle, but the total cardiac blood flow of 4 ml.min-' is 190 x lower than
in muscle.'6
The higher the perhsion rate of a tissue, the faster the equilibrium between drug
inflow and outflow will be reached. During spaceflight, the fluid volume may be
decreased by dehydration after vomiting induced by space motion sickness. The
fluid shift is estimated to be 1 liter from each leg." This is probably the most
important factor leading to changes in distribution,blood flow and tissue or protein
binding. A quantitative prediction of an eventual perturbation of the blood flow in
different regions of the body in space is not possible, because there is also a small
increase in heart rate and a slight decrease in stroke volume and blood pressure."
Table 4. Local Blood Flow and Perfusion Rates of Various Tissues

Body Volume Blood Flow Perfusion Rate
Organflissue % mI.min-’ rn1.rnin-l .g-’ of Tissue
Adrenal glands 0.03 25 1.2
Bone 16 250 0.02
Brain 2 700 0.5
Fat tissues 10 200 0.03
Heart 0.5 200 0.6
Kidneys 0.4 1100 4
Liver 2.3 1350 0.8
Lungs 0.7 5000 10
Inactive Muscle 42 750 0.025
Skin ia 300 0.024
Thyroid gland 0.03 50 2.4
Total body 100 5000 0.071
If there are changes in blood flow, an increase will probably induce a faster
distribution of drugs, while in areas with a decreased blood flow the distribution
will be slowed. Changes in tissue volumes will influence tissue distribution.
Therefore, in space the ratio of adipose to muscle tissue may increase due to muscle
atrophy. On Earth cardiac decompensation is known to reduce the volume of
distribution of several drugs, such as dihydroquinidine, disopyramide, lidocaine,
procainamide, and q~inidine.~
C. Physical Exercise
During spaceflight, crewmembers carry out physical exercise as a countermea-

sure to cardiovascular and musculoskeletal deconditioningduring the flight and to
orthostatic intolerance upon landing. Few studies have been performed on the
influence of exercise upon the pharmacokinetic disposition of drugs. The intensity
of the exercise increases the blood flow of the active muscles with simultaneous
reduction in the blood flow in inactive regions.” Therefore,the distribution of drugs
may be modified by exercise. Furthermore, physical exercise is known to mobilize
free fatty acids from adipose tissue, and thus to increase their concentration in the
blood plasma. As these compounds are known to displace drugs from their binding
sites on albumin, this process is likely to affect drug distribution.*’
IV. EllMlNATlON OF DRUGS

Drug elimination may occur by two mechanisms: metabolism and excretion. The
liver is the organ most frequently involved in drug metabolism. Excretion of drugs
and their metabolites is generallyperformed by the kidney. In both mechanisms the
unbound fraction of the drug is generally cleared. Therefore, protein binding of the
drug may be an important parameter. For drugs with a high extraction ratio, blood
flow will largely determine their elimination.
A. Hepatic Elimination
The liver acts on drug disposition through the hepatic first pass effect, metabolic
transformation,and biliary excretion.Metabolic transformation of a drug generally
leads to a more hydrophilic compound, which will be more easily cleared by the
kidney. Drug metabolites may be equally, less or more effective and toxic than the
parent compound. Drug transformations are enzymatic reactions, which are subject
to intra- and inter-individual variations. The intra-individual variations observed in
drug metabolism are induction or inhibition of the responsible enzymes by drugs
or environmental factors. Autophenomena have been described. Inter-individual
variations are due to genetic polymorphism involved in many enzymatic reactions.
As biotransformations are enzymatic processes, they follow the Michaelis-Menten
equation. When the plasma concentration is low, the reaction is roughly linear.
When it is high, saturation may occur, as has been observed for alcohol and
phenytoin kinetics.2' In that case, a small increase in dose will induce a large
increase in plasma concentration, and thereby in the drug effect.
Biliary excretion is generally a passive phenomenon, which corresponds to a
negligibleroute of excretion. An active secretion has been described for some drugs
such as tetracyclines and veralipride, for which very high concentrations were
observed in the bile. In that case biliary excretion becomes a significant route of
elimination. After gallbladder contraction, the excreted drug reaches the intestinal
tract and may there be re-absorbed. This is the entero-hepatic cycle which may
occur several times during the day.
Hepatic clearance represents the overall capacity of the liver to metabolize a drug.
It is a function of the intrinsic ability of the liver to metabolize the drug (intrinsic
Clearance),of the unbound fraction of the drug, and of the hepatic blood flow. When
drugs exhibit a high extraction ratio, their hepatic clearance depends only on the
hepatic blood In microgravity, this may be modified by the fluid shift.
Hepatic clearanceof drugs with low hepatic extraction ratio and low protein binding
are only influenced by induction or inhibition mechanisms,which are generally due
to the drug itself or to other co-administrated drugs. This problem is not specific to
the microgravity environment. Drugs with low extraction ratio and high protein
binding are influenced by the intrinsic metabolic capacities of the liver and the free
fraction of the drug. These two parameters may be modified by microgravity.
If the perfusion rate of the liver is modified, drugs with high extractionratio show
a variable hepatic clearance. During bedrest studies, no change was observed in the
hepatic blood f10w.23,24Inflight experiments are needed before definitive conclu-
sions about the situation in space can be drawn.
B. Renal Excretion
Renal excretion of drugs is always executed by glomerular filtration. modified
by tubular secretion and reabsorption. Glomerular filtration is a passive phenome-
non for small molecules, while proteins are not filtered. Therefore, only the free
fraction of a drug can pass through the glomerular membrane, which means that
changes in protein binding may affect glomerular filtration. Tubular secretion is an
active and saturable process, which may be subject to competitive interaction with
other compounds, including endogenous substances. Tubular reabsorption is essen-
tially a passive mechanism following the concentration gradient. The pH of the
urine is an important factor, since the only the unionized form is able to diffise.
During spaceflight the urine pH may be changed by the different way of eating and
drinking.
One of the consequences of weightlessness is a decrease in renal plasma
which may lower the glomerular filtration rate. The renal vascular resistance in the
kidney is decreased in the head-down tilt test without c~untermeasure.~’ These
changes have been shown to decrease the renal clearance. For a drug with low renal
extraction ratio these changes may be important, since they may lead to a higher
plasma concentration for the drug in space than observed on Earth at the same
dosage. Changes in the diuresis and the renal blood flow may also decrease the
excretion of some drugs.
The bone demineralization process increases calcium excretion with a conse-
quently raised risk of kidney stones. This can cause local injuries and infections.
The presence ofkidney stones in the urinary tract will decrease glomerular filtration
and may increase the ability of drugs to permeate through the glomerular mem-
brane. With the occurrence of infection, the renal pH may increase, which could
change drug reabsorption. In the case of a weakly acidic drug its reabsorption and
thus its plasma concentration will decrease. The opposite changes will occur with
weakly basic drugs.

The possible pharmacokinetic mechanisms affected by microgravity are listed in
Table 5. In studies of pharmacokinetics in humans, one has generally only access
to drug concentrations in plasma and urine which are the results of several
concurrent mechanisms. During weightlessness, different changes may occur in
each step of the drug disposition process. The most important changes need to be
identified and then predicted for the main drugs used in space.
The use of a drug as a probe (Table 6 ) will permit to estimate the changes in
specific pharmacokinetic parameters during spaceflight. However, this type of
studies is technically difficult to carry out in space, but simulation studies on the
ground are easier to perform. Two studies of hepatic blood flow showed no changes
in this parameter during b e d r e ~ t ? but
~ . ~a~more recent study showed changes in
Table 5. Pharrnacokinetic Mechanisms Possibly Affected by

Microgravit y
ABSORPTION
Absorption rate Gastric emptying (oral route)
Blood flow (all routes)
Amount Vomiting (space motion sickness)
Blood flow (First pass effect)
PROTEIN BINDING Fluid loss
DISTRIBUTION Modifications in muscular/adipose ratio
Modifications in blood flow
Modifications in protein binding
METABOLISM Blood flow
First pass effect
Enzymatic induction or inhibition
EXCRETION Modifications in blood flow
Urine pH
Table 6. Probes in Pharrnacokinetics

Mechanism Drugs
GASTRIC EMPTYING Acetaminophen, metoclopramide, labeled solid meals
HEPATIC FIRST PASS EFFECT Morphine, propranolol, trinitrate molecules, Nifedipin,
Nortryptiline
PROTEIN BINDING Valproic acid, clofibrate, warfarin, propranolol,
carbamazepine, disopyramide, phenytoin,
phenylbutazone, salicylic acid, lidocaine, beta
blockers, erythromycin
DISTRIBUTION Non-steroidal anti-inflammatory drugs, erythromycin,
propranolol
METABOLISM
High extraction ratio drugs Lidocaine, indocyanine green, propranolol, morphine,
nitroglycerine, pentazocine, pethidine, propoxyphene,
salicylamide
Low extraction ratio drugs
low protein binding Antipyrine, acetaminophen (glucuronidation),
theophyl Iine
high protein binding Warfarin, phenytoin, diazepam
RENAL EXCRETION
Glomerular filtration Creatinine, inulin
Tubular secretion
acidic drugs Para-aminohippuric acid, furosemide, penicillin,
salicylates
basic drugs Morphine, neostigmine, quinine
Tubular reabsorption Hydrochlorothiazide, salicylates, methylamphetamine
120 S . SAlVlN et al.
lidocaine disposition during a four-day head-down-tilt.26 Due to the large differ-

ences between individuals, pharmacokinetic changes must be fairly large (> 10-
20%) to be observed in studies with probes. To detect a small change in
weightlessness will require a number of subjects far higher than can be achieved in
spaceflight.
In summary, spaceflight is known to change many physiological parameters. The
pharmacokinetics of drug disposition is determined by the combination of several
complex phenomena. Each step of this process may be influenced by
physiopathological changes occurring in spaceflight. This review shows how from
a theoretical point of view absorption, distribution and elimination of drugs may
be affected by weightlessness. The physiological changes most frequently involved
in these modifications are the changes in blood flow due to the fluid shift.
ACKNOWLEDGMENTS
This work was supported by the PHARMEMSI Study and contract NO961 1/91/FL from the
European Space Agency.
REFERENCES
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Medicine. 2nd ed., pp. 13!&153. Lea & Febiger, Philadelphia, 1989.
2. Pavy-Le Traon, A,, Giiell, A., Saivin. S., Houin, G., Soulez-LaRiviere, C., Pujos, M. The use of
medicaments in space-Therapeutic measures and potential impact of pharmacokinetics due to
weightlessness. ESA Journal, 1833-50, 1994.
3. Lesne, M. Influence de la decompensation cardiaque sur les parametres pharmacocinetiques des
medicaments. SEMPER, 11:26-29, 1988.
4. Labaune. J.P. Pharmacocinbique. Principes Fondamentau. Masson, Pans. 1984.
5. Charles, J.B., Lathers, C.M. Cardiovascular adaptation to spaceflight. Journal of Clinical Phar-
macology, 31:101&I 023, 1991.
6. Lathers, C.M.,Charles, J.B., Elton, K.F., Holt,T.A., Mukai,C.. Bennett, B.S..Bungo, M.W. Acute
haemodynamic responses to weightlessness in humans. Journal of Clinical Pharmacology,
31:615-627, 1991.
7. Lathers, C.M., Charles, J.B., Bungo, M.W. Pharmacology in space. Part 1 Influence of adaptative
changes on pharmacokinetics. Trends in Pharmacological Science, 10: 193-200, 1989.
8. Leach, C.S., Cintron, N.M., Krauhs, J.M. Metabolic changes observed in astronauts. Journal of
Clinical Pharmacology, 31:921-927, 1991.
9. Leach, C.S., Inners, D.L., Charles, J.B. Changes in total body water during space flight. Journal
ofCIinical Pharmacology, 31:lOOl-1006, 1991.
10. Bagian, J.P. First intramuscular administration in the U.S. space program. Journal of Clinical
Pharmacology, 31:920, 1991.
11. Schwinghammer, T.L., Juhl, R.P. Comparison of the bioavailability of oral, rectal and intramus-
cular promethazine. Biopharmaceuticsand Drug Disposition, 5:185-194, 1984.
12. Backon, J., Hoffman, A. The lateral decubitus position may affect gastric emptying through an
autonomic mechanism: the skin pressure vegetative reflex. British Journal of Clinical Pharma-
cology, 32: 138, 199 1.
13. Amidon, G.L., Debrincat, G.A., Najib, N. Effects ofgravity on gastric emptying, intestinal transit,
and drug absorption. Journal of Clinical Pharmacology, 31:96W73, 1991.
14. Neuvonen, P.J. Interactions with absorption of tetracyclines. Drugs, 11:45, 1976.
15. Stockley, I.H. Drug Interactions.A Source BookofDrug Interactions, Their Mechanisms. Clinical
Importance and Management. Blackwell, London, 2nd ed., 199 1.
16. Rowland, M., Tozer, T.N. ClinicalPharmacokinetics. ConceplsandApplications. Lea and Febiger,
Philadelphia, 2nd ed., 1989.
17. Moore, T.P., Thornton, W.E. Inflight and postflight fluid shifts measured by legs volume changes.
In: Results of the lije sciences DSOS conducted about the Space Shuttle 1981-1986 (NASA
Editorial Review Board), pp. 59-65. NASA TM-58-280, NASA, Washington, D.C., 1987.
18. Mulvagh, S.L., Charles, J.B., Riddle, J.M., Rehbein, T.L., Bungo, M.W. Echocardiographic
evaluation of the cardiovascular effects of short duration spaceflight. Journal of Clinical Phar-
macology, 31:102&1026, 1991.
19. Iversen, P.O., Standa, M.. Nicolaysen. G. Marked regional heterogeneity in blood flow within a
single skeletal muscle at rest and during exercise hyperaemia in the rabbit. Acta Physiologia-
Scandinavica. 136:17-28, 1989.
20. Ylitalo, P. Effect of exercise on pharmacokinetics. Annals of Medicine, 23:28%294, 1991.
21. Winter, M.E., Tozer, T., Phenytoin. In: AppliedPharmacokinetics (W.G. Evans, J.J. Schentag, W.J.
Jusko, Eds.), pp. 493-539.2nd ed., Applied Therapeutics, Spokane, WA, 1986.
22. Wilkinson, G.R.. Shand, D.G. A physiological approach to hepatic drug clearance. Clinical
Pharmacology and Therapeutics, 18:377-390, 1975.
23. Putcha, L. Cintron, N.M., Vanderploeg, J.M.. Chen, Y., Habis, J., Adler, J. Effect ofantiorthostatic
bed rest on hepatic blood flow in man. Aviation Space EnvironmentalMedicine.59:306-308,1980.
24. Kates, R.E., Harapat, S.R., Keefe. D.L., Goldwater, D., Harrison, D.C. Influence of prolonged
recumbency on drug disposition. Clinical Pharmacology and Therapeutics,28:624428, 1980.
25. Arbeille, P., Gauquelin, G., Pottier, J.M., Pourcelot, L., Giiell. A,, Gharib, C. Results of a 4-week
head-down tilt with and without LBNP countermeasure: I1 Cardiac and peripheral hemodynamics-
comparison with a 25 day spaceflight. Aviation Space Environmental Medicine, 63:%13, 1992.
26. Saivin, S., Pay-Le Traon, A,, Cornac, A,, Guell, A,, Houin, G. Impact of a four-day-head-down
tilt (-6")on LidocaYne pharmacokinetics used as probe to evaluate hepatic blood flow. Journal of
Clinical Pharmacology, 35697-704, 1995; erratum 351059, 1995.
Chapter 6
REGULATION OF BODY FLUID

VOLUME AND ELECTROLYTE
CONCENTRATIONS IN SPACEFLIGHT
Scott M. Smith. Jane M. Krauhs. and Carolyn S. Leach
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
I1. Body Fluid Volume and Distribution . . . . . . . . . . . . . . . . . . . . . . . 125
A . Body Mass and Total Body Water . . . . . . . . . . . . . . . . . . . . . 125
B . PlasmaVolume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
C . Extracellular Fluid Volume . . . . . . . . . . . . . . . . . . . . . . . . . 127
D. Body Fluid Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . 127
111. Electrolyte Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
A . Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
B. Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
IV. Capillary Pressure and Osmotic Pressure . . . . . . . . . . . . . . . . . . . . 133
V. Fluid and Electrolyte Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
VI . Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
A . UrineVolume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

ISBN: 0-7623-0147-3
123
124 SCOTT M. SMITH, JANE M. KRAUHS, and CAROLYN S. LEACH
B. Electrolyte Concentrations in Urine . . . . . . . . . . . . . . . . . . . . . 137

C. Other Indices of Renal Function . . . . . . . . . . . . . . . . . . . . . . 139
VII. Endocrine Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
A. Antidiuretic Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
B. Renin- Angiotensin-Aldosterone System . . . . . . . . . . . . . . . . . .147
C. Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
D. Natriuretic Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
E. Catecholamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
F. Prostaglandins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
VIII. Mechanism of Spaceflight Effects . . . . . . . . . . . . . . . . . . . . . . . . 158
IX. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160
1. INTRODUCTION
Before the first human spaceflights, biomedical scientists predicted that space
travelers would experience alterations in body fluid regulation, including dehydra-
tion, reduced blood and plasma volume, diuresis, and urinary retention.' Some of
these predictions have proved to be true, some have not, and some appear to depend
on time of sampling (time of day as well as time in relation to the beginning of
weightlessness), individual differences, and mission-specific factors. Many of the
variables involved in fluid regulation have circadian rhythms, and adaptation takes
place at different rates for different physiologic systems. Individual crewmembers
may differ in metabolic variables, physiologic response to stress, ingestion of food
and pharmacologic agents, flight experience, and aspects of their behavior that
influence intake, distribution, and excretion of fluid. Mission-specific or flight
program-specific factors include the amount of time between donning the space
suit and launch, position of the body while awaiting launch, mission duration,
spacecraft temperature and humidity, availability of exercise equipment, and
method of landing.
The effects of returning to Earth's gravity must also be distinguished from the
effects of weightlessness. Although body fluid samples from astronauts have
usually been obtained as soon as possible after landing, the lack of control of the
time of day of landing and the ingestion of food and fluid near the time of landing
have introduced confounding factors. The number of variables involved; the
difficulty of performing metabolic, fluid volume, and renal function studies during
flight; and the small number of subjects on each mission complicate the interpre-
tation of the findings.
From the more than 300 different individuals who have now experienced space-
flight, we have learned much about regulation of body fluid volume and blood
electrolyteconcentrationsduring spaceflight. The contribution of decreased plasma
volume to orthostatic hypotension at landing is the most important clinical conse-
quence of alterations in body fluid and electrolytes during weightlessness, and some
Fluid and Electrolyte Regulation in Spaceflight 125
progress has been made in reducing orthostatic intolerance. These findings are
discussed in this chapter.
II. BODY FLUID VOLUME AND DISTRIBUTION

A. Body Mass and Total Body Water
Mild dehydration of astronauts on some ofthe earliest orbital flights (the Mercury
program) was deduced from high space suit inlet temperatures, weight loss,
reduction in urine volume, and hemoc~ncentration.~.~~~
After the earliest U.S. flights, body weight could not be determined until the
astronauts returned to dry land after splashdown in the ocean and retrieval by an
aircraft ~ a r r i e rDevices
.~ for measuring body mass during flight were developed in
the U.S. and Russian space programs. In the U.S. program, a body mass measuring
device was first used during the Skylab mission^.^
On short-term flights, body mass loss is thought to consist mainly of fluid loss.6
A loss of up to 5 kilograms of weight has been recorded for individual crew
members on flights of up to 2 weeks duration, but the amount of weight loss was
not related to flight duration.'.'
Direct measurement of the body fluid volume was accomplishedbefore and after
several Gemini flights (plasma and blood volume only),' the Apollo missions,' and
the Skylab missions: and before, during and after several Space Shuttle
Total body water (TBW) was measured by dilution of tritiated water (3H,0)12
(Apollo and Skylab) or "0-labeled water" (Space Shuttle). Saliva instead ofblood
samples were collected during these Space Shuttle missions.
Total body water of 12 Apollo astronauts decreased on the basis of absolute
volume, but on the basis of body mass at the time of measurement it increased by
1.6% (flight of 6 to 13 days duration).' Similar results were obtained from the
Skylab astronauts.'
Postflight total body water of 5 astronauts on two Space Shuttle flights (duration
4 and 6 days) was not significantly different from preflight values, when all values
were adjusted to a body mass of 70 kg, but during these flights after 1 to 3 days of
weightlessness the adjusted total body water values were significantly lower (3.4%,
P = 0.019) than before and after flight." However, total body water of 6 astronaut
subjects on the Spacelab Life Sciences missions SLS-1 and SLS-2 after 2 1 hours
or 7 days of flight was not significantly different from preflight values."
B. Plasma Volume
Before direct measurements were available, it was thought that the plasma
volume of astronautsdecreases during flight, because postflight increases in plasma
proteins and electrolytes appeared to indicate hemoconcentration.I
Plasma Volume
i f
-25 I I I
0 10 20 30 40 50 60 70 80
Flihl day
Figure 1. Percent change in plasma volume duringand after spaceflight. The radioio-
dinated serum albumin method was used for all measurements. Each point represents
the mean of an inflight or postflight plasma volume measurement (ml/kg) compared
with that subject's preflight measurementb),for 2 to 8 astronauts on one or more flights
from the Gemini, Apollo, Skylab, and Spacelab programs. Error bars represent
standard error of the mean.
The plasma volume has been determined by a single method, dilution of radioio-
dinated serum albumin (RISA), throughout the U.S. space program.13At first the
13'1 isotope was used, but during the Apollo program 1251was employed. The blood
volume was calculated by adding the red cell mass, determined by dilution of
"Cr-labeled red blood cells, to the plasma volume.'3
Plasma and blood volumes were first determined before and after spaceflight
during Gemini, the second U.S. flight program. Four Gemini astronauts lost plasma
volume between pre- and postflight measurements; only the two astronauts on the
1C&y Gemini-7 mission showed a gain in plasma vofume (Fig. l).'
Plasma volume of six Apollo astronautswas reduced after 9- to 13-daymissions,
while that of six other Apollo astronauts on missions of the same duration was
elevated, expressed as mlkg body weight.14 The change in plasma volume at
landing was not related to whether or not the astronauts had landed on the Moon
during their mission. Plasma volume of two astronauts on the shortest (28-day)
Skylab mission was also elevated at landing, compared to preflight measure-
ments." For all other astronaut subjects, plasma volume was found to be reduced
after landing. 1~15-17
On the SLS-I mission in 1991, inflight measurementsof the plasma volume were
made for the first time. The mean change in plasma volume on the two SLS missions
Fluid and Electrolfle Regulation in Spaceflight 127
(6 subjects) was a 17% decrease after 21 hours of flight, a much greater change
than the 10% reduction at landing.l 1 These calculationswere based on data scaled
to a body surface area of 1.73 m2.Plasma volume remained below preflight levels
throughout the mission.
Plasma volume is known to increase with temperatureand decrease with exercise.
In a recent study,” the change of the plasma volume with exercise was found to
depend on the hydration status of the subjects, with the largest decrease occurring
during euhydration. Apollo crew members were exposed to elevated ambient
temperatures on the spacecraft and during recovery;’ this may have caused them to
have a smaller decrease or even an increase in plasma volume compared to other
astronauts on missions of similar duration (Fig. 1). On the Gemini-7 mission,
astronauts had three 10-minute exercise periods per day, one before each meal.’
C. Extracellular Fluid Volume
The extracellular fluid volume, which comprises about 38% of total body water,
has been determined by dilution of 35S0,in Apollo, Skylab, and Space Shuttle
astronauts. Results for extracellular, intracellular, and interstitial fluid volumes
immediately after the Apollo flights resembled the total body water results: a
decrease in the volume, but a slight increase in the volume per kg body weight
(extracellular, 1.1%; intracellular, 1.9%, interstitial, 1.7%). It was suggested that
the increased extracellular fluid volume was compensating for tissue losses.’ In
nine Skylab astronauts an average decrease of 1.9% in extracellular fluid volume
was measured, but this was eliminated when expressed per kg body mass.’
“Bromide space,” approximating extracellular fluid volume, was variable in
cosmonauts after 18- or 49-day flights, sometimes increasing and sometimes
’’
decreasing. Extracellular volume of six cosmonauts after 96- to 175-day missions
on the Salyut-6 space station was reduced by 1.2 to 14.6%, without correlation to
the flight duration2’ The report did not mention whether these changes were based
on volume alone or on volume per kg body weight.
Inflight measurements have shown that the extracellular fluid volume decreases
considerably more during flight than can be determined by measurements after
landing. Extracellular fluid volume was reduced by 10% after 2 1 hours of flight on
SLS-1 or SLS-2 (6 subjects, data scaled to 1.73 m2 body surface area); it was still
significantlyreduced after 8 days (6 subjects) and 12 days of flight (3 subjects).
D. Body Fluid Distribution
Comparison of inflight measurements of total body water, extracellular fluid

volume, and plasma volume with preflight and postflight measurements indicates
that the volumes of these body fluid compartments undergo rapid changes during
spaceflight and re-adaptation to Earth’s gravity. However, the restoration of fluid
volume at landing apparently is not rapid enough to prevent orthostatic intolerance
and dehydration. The relatively large inflight decrease in plasma volume and
extracellular fluid volume, compared to the relative constancy of total body water,
suggests that in microgravity the intracellular fluid volume may increase at the
expense of the extracellular fluid.”
111. ELECTROLYTE CONCENTRATIONS

A. Sodium
Blood samples for conventional clinical laboratory determinationshave usually

been obtained from astronautson more than one preflight day, and a preflight mean
has been determined for comparison with inflight and postflight values. Pre-
flight samples were obtained at least three times for Apollo and Skylab missions
and at least twice for Shuttle missions. Astronauts in all U.S. flight programs
have fasted overnight before preflight antecubital venous blood samples were
obtained.
During the early space missions no inflight blood samples were obtained. Serum
electrolyte changes were unremarkable for the Mercury and Gemini m i s ~ i o n s ? ~ ” ~ ’
Serum sodium and chloride of Apollo astronauts (n = 33) were not significantly
different from preflight values after missions of 6 to 13 days.8 Since the high
temperature during capsule recovery in a tropical area should have tended to
increase serum sodium, the values may have been low during flight. The normal
serum sodium values were also taken as evidence that the Apollo astronauts were
not dehydrated.
The first equipment for inflight blood collection was developed for Skylab.22The
system included a centrifuge, automatic sample processors with an evacuation
regulator, syringes (containing anticoagulant), needles, and sample vials. Blood
was drawn into a syringe and ejected into an evacuated sample processor, which
was then placed in the centrifuge. Plasma and blood cells were separated by
centrifugation. Plasma was automatically transferred to a sample vial, which was
sealed and stored frozen until return to the laboratory on Earth.
The system used on the Space Shuttle consists of several trays containing
blood-collection equipment and supplies, one tray for each day on which blood is
to be drawn. The use of evacuated tubes made the automatic sample processor
unnecessary. The tubes, treated to withstand liquid nitrogen temperatures, contain
an anticoagulant (heparin) or disodium ethylenediaminetetraacetate(EDTA), and
a gel that keeps cellular and liquid phases separate after centrifugation. Although
electrolytes are usually measured in serum, it has sometimes been necessary to
measure them in plasma. Tests have shown that this does not affect the results as
long as a correction is made for the presence of sodium in EDTA. The separated
samples are stored in a freezer at -20°C. When hematocrit values are to be
determined during flight, a microcapillary centrifuge is included.
Plasma sodium was reduced during the Skylab flights. Data are shown in Fig. 2
and Table 1. The reduction was significant(P < 0.05) at half of the inflight sampling
-10 !
51 t Skylab
-10 !
0 10 20 30 40 50 60 70 80 90 100
Flight day
Figurez. Percent change in serum or plasma sodium during spaceflight. Each symbol
represents the mean of an inflight electrolyte concentration (meq/l) compared with
that subject's preflight value(s), for 3 to 6 astronauts on Skylab or 2 to 9 astronauts on
Shuttle flights, or the percent change for 1 cosmonaut on the Mir Aragatz flight. Data
are shown in Table 1 .
times from 3 to 82 days.' After landing, plasma sodium remained below preflight
levels for at least 3 days, though the reduction was not significant. Serum sodium
was also below preflight levels at most samplingtimes from 5 hours to 8 days during
Spacelab m i s ~ i o n s . ' 'However,
~~~ inflight serum or plasma sodium of SLS subjects
was not significantly different from preflight concentrations." At landing, serum
Table 1. Electrolyte and Osmolality Results from In-Flight Blood Sampling on Three Flight Programs
Variable FDf FD2 FD3 FD4 FD5 FD6 FD7 FD8 FD9 FDll FDf2 FDf3 FDf4 FDZO FD21 FD27 FD30 FD38 FD45 FD48 FDS8 FD59 FD73 FD82
Serum or plasma sodium, percent change
Skylabg
n 6 3 3 5 3 3 3 3 3 3 6 3 3 3 3 3 3
Mean -2.0 0 -3.7 -1.7 0.5 -6.0 -1.8 -1.1 -1.9 -5.0 -4.2 -3.9 -2.7 -2.8 -4.6 -1.1 -2.4
SE 1.8 1.1 1.7 1.0 0.9 1.1 2.0 1.9 1.5 3.6 1.3 1.3 1.3 1.6 1.0 2.1 0.9
Shuttlell.l7.23.63
n 9 5 2 2 7 6 3
Mean -0.1 -1.0 -2.1 4.4 4.1 -1.3 0.4
SE 0.5 1.0 2.1 1.1 0.3 0.6 1 .o
MirZ6
n 1 1
%change 1.8 -0.2
Serum or plasma potassium, percent change
Skylabg
n 6 3 3 5 3 3 3 3 3 3 6 3 3 3 3 3 3
Mean 5.1 0.9 19.9 -3.2 -2.4 4.2 4.2 8.5 3.1 4.8 1.0 7.6 7.3 2.5 6.8 -3.4 7.7
-L
3.5 2.7 7.0 1.2 2.7 5.0 3.6 6.0 2.1 1.6 4.1 2.9 5.1 5.7 5.6 4.8 0.1
w
0 SE
5hunle11.17,23,63
n 9 5 2 2 7 6 3
Mean 9.9 -4.1 -10.8 2.6 19.8 5.2 -0.4
SE 4.7 6.1 8.4 6.9 6.0 2.9 4.5
Mri2('
n 1 1
76 change I .4 -4.8
Serum or plasma csmolality, percent change
Skylabg
n 6 3 3 5 3 3 1 3 3 3 6 3 3 3 3 3 3
Mean -0.2 -0.3 -2.4 4.7 4.6 --2.3 -1.3 4 4 -0.8 -0.9 -4.0 -3.4 0.4 -2.4 -1.9 -2 5 -1.9
SE 0.4 0.4 0.5 0.5 0.3 1.6 1.0 0.3 0.2 0.1 1.4 0.7 0.5 1.5 1.2 1.1 0.4
5hunle1 1.1 7.23.30.63
n 11 4 7 8 3
Mean -0.6 4.1 2.5 -2.3 4.6
SE 0.5 0.6 1.1 0.4 1.I
Mif6
n I 1
%change ______ - 2.0 ~ ~~
-1.7 ~ .
Nore: FD = flight day
sodium of Shuttle astronauts (n = 133, flight duration 2 to 11 days) was unchanged

from preflight levels.24
The Russian equipment for inflight blood collection, Plasma 0125or the later
Plasma 02,26 consists of a blood-collection kit, a centrifuge, a freezer, and a
container for transporting samples. Blood is drawn into a syringe, and a test tube
with a stop valve is mounted on the syringe. When this assembly is centrifuged,
plasma remains in the syringe, while the sediment enters the test tube. Samples are
stored frozen until they are brought to the laboratory. A Reflotron@blood analyzer
system has been used by at least one crew (the ‘Aragatz’ 25-day mission) on the
Russian space station Mir to measure 11 clinical chemistry variables in capillary
blood.27The Reflotron analyzer is an electrochemical reflectance photometer that
analyzes reagent pads containing blood samples.
After 9 days ofweightlessness on the 25-day Mir Aragatz mission, a cosmonaut’s
plasma sodium was 2% above its preflight value (141 mM), but after 20 days it was
141 mM again.26It was elevated to 150 mM immediately after landing, but was
back to 141 mM three days later. A summary of results from Russian spaceflights
shows that serum sodium was significantly elevated after flights of 2 to 3 days
(n = 8) and of 6 to 8 days (n = 16).28However, after longer flights the differences
between preflight and postflight values were not significant.
The results of most U.S. inflight measurements of serum or plasma sodium seem
to indicate that, as expected from the Apollo results, sodium is generally reduced
during spaceflight and astronauts are not dehydrated during flight. The finding that
blood sodium increases during landing suggests that fluid leaves the bloodstream
or sodium enters it. The latter occurs when astronauts or cosmonauts use the saline
loading countermeasure. This countermeasure was not used in the U.S. program
before the onset of the Space Shuttle flights; the low plasma sodium values of
Skylab astronauts immediately after landing suggests that ingestion of salt tablets
before landing does raise this variable.
B. Potassium
Potassium is thought to be lost from tissues such as muscle during long-term

spaceflight? which might lead to increased serum potassium levels. The blood
potassium concentration is regulated by the hormone aldosterone, which reduces
the potassium level. If the aldosterone secretion were elevated, this could occasion-
ally produce an over-reduction of serum potassium.
Serum potassium of the Apollo crew members was 7.3% lower after landing than
before flight, a significant difference (P< 0.05, n = 33). It was also significantly
lower in cosmonauts after flights of most durations, except after short flights of 2
to 3 days.28
Plasma potassium of the cosmonaut on a 25-day Mir flight was slightly elevated
after 9 days, and reduced after 20 days (Fig. 3, Table l), returning to the preflight
value immediately after landing.26On the Skylab missions it was usually not
132 SCOTT M. SMITH, JANEM. KRAUHS, and CAROLYN S. LEACH
\ o l -
-15
g 0 - -
-5
-10
-15 !
-m- Skvlab
- o l -15
- 0 10 20 30 40 50 60 70 80 90 100
Flight day
Figure 3. Percent change in serum or plasma potassium during spaceflight. Each

symbol represents the mean of an inflight electrolyte concentration (meq/l) compared
with that subject’s preflight value(s), for 3 to 6 astronauts on Skylab or 2 to 9 astronauts
on Shuttle flights, or the percent change for 1 cosmonaut on the Mir Aragatz flight.
Data are shown in Table 1.
significantly changed, but it was significantly reduced after 38 days of flight.’

Serum potassium of Spacelab astronauts increased as much as 20% and decreased
as much as 11% at various times.1123
Total body potassium of astronauts was measured for the first time by gamma
spectrometricmeasurement of total body 4k before and after Apollo missions 12,
13, and 14. Decreases of 3 to 10% were observed in 7 out of the 9 crewmembers.8
Total body exchangeable potassium was first determined by using 42K in the
astronauts of the Apollo 15 mission. For the 9 astronauts on Apollo 15, 16, and 17
(flight duration 11 to 13 days) postflight total body exchangeable potassium,
expressed in milliequivalents potassium per kg body weight, was reduced by 6.2%
compared to preflight levels at a dilution time of 24 hours.8 Two days after return,
the reduction was 2.0%. For the 9 Skylab astronauts (flight duration 28 to 84 days),
postflight total body exchangeable potassium was reduced by 6.4% compared to
preflight levels.’
IV. CAPILLARY PRESSURE A N D OSMOTIC PRESSURE

The major processes through which fluid volume and blood electrolyte concentra-
tions are controlled are capillary bed filtration pressure, intake of fluid and electro-
lytes, and reabsorption of fluid and electrolytes by kidney tubules2’ These topics
will be discussed in the next three sections.
Capillary pressure is one of the four “Starling forces,” which tend to move fluid
across the membrane of blood capillaries. Capillary pressure moves fluid out of the
capillary into the interstitium. Plasma colloid osmotic pressure is the only one of
the four forces that tends to move fluid in the other direction. Indications of its
magnitude can be obtained by measuring blood osmolality and protein, especially
albumin. The latter variables have been measured during and after some space-
flights. Capillary pressure has not been measured during spaceflight.
During the Skylab missions, plasma osmolality was lower than preflight values
at almost all sampling times (Fig. 4, Table l), significantly at several times.’ After
landing it was at preflight levels again. Plasma osmolality was also unchanged at
landing for Shuttle astronauts (n = 133, flight duration 2 to 11 days), most of whom
used the saline countermeasures before landir1g.2~On an 8-day Spacelab flight
plasma osmolality of 4 astronauts was below preflight levels on days 2 and 7.30
Serum osmolality of 4 astronauts on SLS-1 and 3 astronauts on SLS-2 was above
preflight values on flight days 1, 8, and 12, but not significantly.” Plasma osmo-
lality of 3 cosmonauts was below preflight levels on days 2 17-2 19 of a 237-day
mission.28
Total blood protein of Gemini,’ Ap0ll0,~’Skylab,’ and Shuttle24astronauts was
elevated at landing. During the SLS missions serum protein was significantly
elevatedon flight day 1,but not on days 2,8, or 12.” During the Spacelab-1mission,
serum protein of 4 astronauts decreased slightly but not significantly.” Serum
albumin, which is responsible for 75% of the plasma colloid osmotic pressure, was
slightly increased (but not significantly) on day 1,but was at preflight levels on day
7.
These findings suggest that osmolality receptors in the cardiovascular system are
probably not stimulated more in microgravity than at 1G. The osmotic pressure,
provided by protein and sodium in serum and by other sources, probably yields
about the same q q n t of opposition to capillary pressure and the other Starling
t Skylab
10 20 30 40 50 60 70 80 90 100
Flight day
Figure 4. Percent change in serum osmolality during spaceflight. Each subject’s

inflight osmolality (mOsm/kg)was compared with that subject’s preflight value(s).Each
symbol represents the mean for 3 to 6 astronauts on Skylab or 3 to 11 astronauts on
Shuttle flights, or the percent change for 1 cosmonaut on the Mir Aragatz flight. Data
are shown in Table 1.
forces during spaceflight as on Earth. Osmotic pressure change does, therefore, not
seem to account for the large decreases in plasma volume during weightlessness.
Osmotic pressure probably depends more on other factors than it does on weight-
lessness per se. It is possible that on flights such as the Skylab missions, during
which plasma osmolality was sometimes significantly diminished, reduced colloid
osmotic pressure could account for some reduction in plasma volume.
V. FLUID AND ELECTROLYTE INTAKE

Fluid and electrolyte balance in astronauts has been studied since the earliest
spaceflights.’ After the first U.S. orbital flight an attempt was made to combat
dehydration by an increased fluid intake. Maintenance of body weight has been an
important goal of spaceflight meal planning in every U.S. flight program. However,
fluid and food intake are undoubtedly adversely affected by the lack of leisure time,
ready availabilityof food and fluid, and convenient facilities for elimination during
flight. The necessity of spending hours aboard recovery ships was an additional
complicating factor for U.S. flights before the advent of the Space Shuttle. Motion
sickness during recovery may have contributed to postflight intake reductions for
some astronauts. In addition to these factors, weightlessness by itself is believed to
decrease thirst. Crew members on Gemini missions 5 (8 days) and 7 (14 days), but
not those on mission 4 (4 days), were said to be adequately hydrated at the end of
the mission, but a report7 stated that “the crewmen must be reminded of their water
intake.”
Fluid intake of the 9 Skylab astronauts decreased by 700 ml/day during the first
6 flight days compared to preflight intake.’ Since urine volume decreased by only
400 ml/day during this time, there was a net fluid loss (even without including
insensible losses and sweat). Sweat loss was estimated to decrease 11% during
weightlessness, compared to preflight 10~s.~’
Daily fluid intake of seven SLS astronauts during the first 9 days of flight was
about 1 liter less than preflight, a statistically significant difference on several
days.” Fluid intake of three SLS-2 subjects toward the end of their 14-day flight
was not significantly different from preflight intake.”
The food intake of Apollo astronauts was described as variable and generally
below basal requirementsduring the first mission day.8This has probably been true
for many Shuttle astronauts, especially for those who experience space motion
sickness. Energy intake of 7 SLS subjects was somewhat below preflight levels on
the first flight day only.” Anecdotal evidenceindicates that spaceflightaffects crew
members’taste responses, and some astronauts like foods to be more salty and spicy
than they normally do.
VI. RENAL FUNCTION

A. Urine Volume
Interpretationof urinary excretion data obtained before, during, and after space-
flight is complicated by the lack of data regarding fluid intake on some missions
and extra-renal fluid loss. Operational constraintshave hindered collection of such
data, which are needed for accurate determination of fluid balance.
Diuresis was first reported after the second U.S. human orbital mi~sion.~ Al-
though fluid intake exceeded urinary output, the astronaut lost about 6 pounds in
less than 24 hours between preflight and postflight examinations, a period that
included 4.5 hours of weightless flight and 3 hours in a life raft. The conclusion
that diuresis had occurred was based on weight loss, hemoconcentration, and the
low specific gravity and electrolyte concentrations of an inflight urine specimen.
An effort had been made to ensure adequate hydration, but a high suit inlet
temperature had caused excessive sweating.
On the Mercury flights astronauts used urine collection devices that did not
permit separation of urine voided during flight from that voided on Earth.’ For the
Gemini program procedures were improved, and urine samples were collected
during flight for the first time on the 14-day Gemini-7 mission.’ The urine volume
was measured with a flowmeter and addition of a fixed amount of ’H,O to each
void, from which then a sample was taken and preserved with benzoic acid. The
device malfunctioned on a subsequent flight, hence, the urine volumes obtained on
Gemini flights were not considered reliable. Inflight urinary excretion was calcu-
lated using the assumptions that renal clearance was not significantly altered by
spaceflight and that creatinine excretion remained unchanged.
During the Apollo-17 mission, urine samples were collected in sampling bags,
containing boric acid as a preservative and 30 mg of lithium chloride to provide a
means of estimating the total volume of urine by measuring the final lithium
concentration. During that flight, urine volume of all three crew members was
elevated compared to preflight values, but for two of them, postflight urine volume
was reduced compared to preflight values.8The inflight results provided evidence
of the diuresis that was expected during flight.
During the Skylab missions urine was collected throughout with an automatic
device that used a positive airflow to convey urine to the collection apparatus.’ The
urine of each crew member was collected for each 24-hour period in a “pooling
bag,” which contained a known amount of lithium chloride for volume determina-
tion. An aliquot from each 24-hour pool was stored frozen until analyzed on Earth.
Urine volume was essentially unchanged during flight, except during the first 6
days when the volume for all 9 crew members was on the average 400 ml less than
the preflight volume.
Further urine collection experiments have been carried out in the Space Shuttle.
In a single experiment, in which the urine of one crew member was collected
throughout the flight, an apparent diuresis beginning on flight day 2 and lasting for
several days was observed.”
On the SLS missions the ‘Space Laboratory Urine Monitoring System’was used
for urine collection and volume mea~urement.’~ This device records the mass of
each void and retains an aliquot. For the seven astronauts the urine volume was
slightly reduced on flight day 1 and significantly reduced on flight days 2 and 3. l 1
On Russian missions inflight urine samples from cosmonauts are collected with
the ‘Diurese’ kit; the samples are stored frozen until a n a l y s i ~ ? The
~ , ~urine
~~~~
volume of two cosmonauts on an 18-day Soyuz mission was decreased during.the
first 2 days of flight, but not on the 18th day.36On a 7-day Mir flight the urine
volume of one cosmonaut was followed; it was reduced on days 1,5, and 6.37The
urine volume of one cosmonaut on a 25-day Mir flight was reduced from the
preflight value of 940 ml to 700 ml on day 5 and 600 ml on day 19 of the mission.26
On the other hand, the urine output of a French cosmonaut on the Mir Antares
mission increased from 1000 ml preflight to 1330 ml on day 8 and 1270 ml on day
11.35 The urine volume of a Russian cosmonaut on this mission did not change
during flight. The changes in urine volume and sodium for the French cosmonaut
were attributed to his use of the 'bracelets' countermeasure (thigh cuffs), which are
thought to have delayed adaptation to weightlessness. During a 237-day Soyuz
flight the urine volume of one cosmonaut was 20 to 40% of fluid intake on days
2 16-2 18, that of the other 60 to 70% of fluid intake on days 2 17-2 19, while the
preflight urine volume was 50 to 60% of fluid intake for both c o s m o n a ~ t s . ~ ~
Postflight water retention was reported for the first time for the Gemini-7 and
Gemini-9 a~tronauts.~'*~~ This has been a consistent finding for astronauts and
cosmonauts subsequently, even when they have used fluid-loading countermea-
sures before landing.937,4'The postflight urine volume of 30 Apollo astronautswas
significantlyreduced by 32% from preflight values.873'The 338-m1 average reduc-
tion in urine volume of 7 SLS subjects on the day of landing was not statistically
significant.'' There may be an effect of flight duration: reduction of urine volume
was less for cosmonauts on missions of 63 to 175 days than for those on missions
of 30 days or less?' Urine volume of cosmonauts on longer flights, up to 366 days,
was also reduced at landing!3
B. Electrolyte Concentrations in Urine

Urine is probably the main vehicle ofelectrolyte loss during spaceflight,although
a metabolic balance study performed during the Gemini-7 mission revealed that
sweat was a significantroute of sodium and potassium loss.44In general, conditions
that promote sweating are avoided during spaceflight, except that exercise is
encouraged on all flights and required on long-term flights.
A slight reduction in urinary sodium was noted during the Gemini-7 mission and
a marked retention was noted after landing.' This finding was one of the first
indications that some fluid and electrolyte variables begin to change as soon as
weightlessness ends. Pre- and postflight analysis of urine from Gemini-9 crew
members indicated that sodium, potassium, and chloride as well as water were
retained at landing.40Urinary sodium has been reduced at landing for all flight
durations from 2 to 366 days.28Statistical analysis of data from Apollo,8 Skylab,'
and Shuttle41crew members shows that postflight urinary electrolyteswere signifi-
cantly lower than preflight levels. After Apollo flights, for example, sodium in 30
astronauts had decreased by 49% and potassium by 47%.8
During the Skylab missions urinary excretion of sodium was significantly
elevated inflight and after the first 6 days postflightover preflight values.' However,
during and after the SLS missions urinary sodium was not significantly different
fiom preflight values."
Urinary electrolyte excretion by cosmonauts has generally been reduced during
weightlessness. However, urinary sodium of two cosmonauts on the Mir Antares
mission increased by 23% on flight day 9 and by 39% on flight day 11, but not on
days 4,5, or 8.35For two cosmonauts on the 18-day Soyuz-9 mission, the amount
of sodium excreted was greater on flight day 1 than on day 2 or 18?6 On days 1,5,
and 6 of a 7-day Mir flight, urinary sodium of one cosmonaut was significantly
reduced from its preflight On days 43-45 and 86-88 of a 150-day flight of
the Salyut-7-Soyuz-Torbital complex, urinary excretion of sodium and potassium
by one cosmonaut was red~ced.~' On days 2 16-2 19 of a 237-day flight on Salyut-7,
urinary sodium of two cosmonauts was only 39 to 59% and 49 to 76% of sodium
intake, while the preflight values for both cosmonauts were 80 to 90% of sodium
intake.38
Alterations in urinary chloride nearly always follow the same pattern as those in
urinary
Urinary excretion of potassium was found to decrease during many missions:
Gemini-7,' SLS," 7-day Mir?7 and S0yuz-9.~~ However, unlike sodium excretion,
it began to increase immediately after landing of Gemi11i-7~and the Mir flight.37
At landing, urinary potassium was significantly different from preflight values only
for the shortest Russian flights, 2 to 3 and 6 to 8 days.28Gemini-7 crew members
had a negative potassium balance during spaceflight.* On the 237-day Soyuz flight
urinary potassium as a percentage of potassium intake decreased slightly on days
2 16-2 19 from 8040% of intake to 55-76% for one cosmonaut and to 70-96% of
intake for the other?8
Urine osmolality was in many cases raised during flight. For the Skylab astro-
nauts it was significantly elevated during flight by more than 100 mOsd24 h.9 On
a ?-day Mir flight it also increased significantly for a cosmonaut on days 1.5, and
6?7 The mean urine osmolality of 6 Space Shuttle astronauts at landing was 80
mosd24 h higher than the preflight value of 643 mOsd24 h, but this change was
not statistically significant!6 The 30 Apollo astronauts showed a significantly
elevated osmolality of 833 mOsd24 h at landing compared to a preflight mean of
696 mOsd24 h.8 However, the opposite effect has also been found. The urine
osmolality of the French cosmonaut on the Mir Antares mission decreased by 19%
on flight day 8, while his urine volume increased by 33%.35At landing, the urine
osmolality of the Skylab astronauts was reduced by at least 50 mOsd24 h below
the mean preflight osmolality (650 mOsd24 h). Urine osmolality of cosmonauts
on flights of all durations from 2 to 366 days was reduced after landing, usually
significantly?*
Osmolal clearance (Cosm)depends on urine osmolality (Uosm), on urine flow rate
(Uflo,,,), and on plasma osmolality (Posm),according to the equation:
Free water clearance is the difference between urine flow rate (Uno,) and osmolal
clearance (Cosm),or the volume of plasma from which excess water is eliminated
by filtration per minute.
Free water clearanceincreased in cosmonauts after long-term flights (longer than
1 month), but not after short-term flights (7 or 8 days).*' On the other hand, during
the 28- to 84-day Skylab missions this parameter decreased slightly from preflight
levels, and increased somewhat at suggesting that in this case free water
was reabsorbed and diuresis did not occur during flight, while the opposite took
place after flight.
Use of countermeasures such as saline and calcium supplements would be
expected to elevate urine osmolality, but this cannot explain all differences in
osmolality at landing. There is no clear relation with flight duration. Elevated urine
osmolality, while urine volume and electrolyteswere reduced, indicates that other
osmotically active substances were elevated, but none of the other osmotically
significant substances measured in the urine of the Space Shuttle astronauts were
elevated!6
C. Other Indices of Renal Function
Blood Urea Nitrogen and Uric Acid
Blood urea nitrogen of three SLS astronaut subjects was reduced slightly on flight
day 1 but not on subsequent flight days and landing day." At landing, blood urea
nitrogen of 33 Apollo astronauts was significantly elevated by 12%.8,31This
finding, along with decreased serum sodium and increased aldosterone excretion,
suggests that renal blood flow decreases during weightlessness.8 Blood urea
nitrogen was elevated by 6.6% (not significantly) after Space Shuttle flights of 2
to 11 days48and after Russian flights of 18 to 24 days.36In the latter case this may
have been caused by occasional use of anabolic steroids by the cosmonauts,
accordingto the investigators. Blood urea nitrogen of 9 Skylabastronautsat landing
(no inflight measurements) was unchanged from preflight values.
As mentioned in the previous section,a significantincrease in urinary osmolality,
while urine volume and electrolytes are reduced, suggests that excretion of other
osmotic substances increases. This may have been the case for the Apollo astro-
nauts, where blood urea nitrogen and urinary uric acid were in~reased',~' However,
after the Skylab missions' and the first four Shuttlemissions46urinary uric acid was
significantly reduced in 9 and 6 astronauts, respectively. Uric acid in plasma or
serum was usually significantly reduced after Purine content of
the diet can affect levels of uric acid; e.g., if the inflight diet contained less meat
than the preflight diet, urinary and plasma uric acid levels would be reduced.
140 SCOlT M. SMITH, JANEM. KRAUHS, and CAROLYN S . LEACH
Creatinine Clearance and Glomerular Filtration Rate
The creatinine clearance of 29 Apollo astronauts was reduced by 12% immedi-

ately after flight. The inflight samples collected on Apollo 17 indicated that urinary
creatinine decreased on the first day of flight and then returned to preflight values.8
However, during and after the Skylab missions the creatinine clearance was
generally elevated, with some exceptions!’
The glomerular filtration rate of cosmonauts, as inferred from their urinary
creatinine excretion rate, was unchanged after short-term49as well as long-term5’
missions.
Direct measurements of the glomerular filtration rate and the effective renal
plasma flow were performed for the first time during the SLS Space Shuttle
missions. As measured by clearanceof a single injection of Inutest? the glomerular
filtration rate of six astronauts was significantly elevated by 16% over preflight
values on days 1 and 2, and by 18%on day 8. At landing, the glomerular filtration
rate was close to preflight values. Effective renal plasma flow was measured by
clearance of a single injection of para-aminohippurate. This parameter was gener-
ally higher, but not significantly,than the preflight value. Creatinine clearance was
elevated, but not significantly, on flight day 1 only. These results indicate that these
two parameters of renal finction are not impaired by spaceflight. However, reab-
sorption of certain electrolytes and other substances may be altered.
Clearance of Electrolytes
In Russian missions sodium clearanceis reported to have decreased significantly,

from 0.68 ml/min preflight to 0.24 ml/min after short-term flights (< 30 days) and
from 0.53 ml/min preflight to 0.30 ml/min after long-term flights (> 30 days).28
Since serum sodium was unchanged and creatinine clearance only slightly changed
after flight, these findings suggest that spaceflight may alter tubular reabsorption
of sodium.
Excretion of Water or Electrolyte Load
Russian investigators have used water or electrolyte loading tests as an index of

renal function before flight and after landing, in addition to clearance measure-
ments. In general, loading tests have indicated that renal finction is altered during
readaptation to Earth’s gravity, presumably because this function was changed
during spaceflight.
Water loading tests were performed 36 to 40 hours after landing, when body
weight has returned to preflight levels. A reduction of urinary excretion of water
was found after flights of 1 to 5 days5*and 30 days,52but it was unchanged after
an 18-day flight.5’ If the glomerular filtration rate is unaltered at this time, as
indicated by creatinine clearance, then a decrease in water excretion is likely to
result from increased reabsorption of water by the kidney tubules.
Grigoryev et al.” concluded from water loading tests, performed after a 96-day
flight on Salyut-6, that countermeasures used by the cosmonauts during flight
prevented some of the impairmentofrenal function that usually occurs immediately
after flight.
Urinary sodium excretion increased during water loading tests after flights of 1
to 63 but decreased during calcium lactate loading tests after long-term
mission^.'^ Russian investigatorshave proposed that the renal systemsthat reabsorb
calcium and sodium are uncoupled after long-term spaceflight,possibly because of
a change in calcium metaboli~rn.~~
Results of potassium loading tests and potassium balance studies suggest that
cells retain potassium less well after spaceflightthan they do b e f ~ r e . After
’ ~ flights
of less than 13 days, the potassium excretion rate during potassium loading tests
decreased, but after a 13-day flight this parameter increased.49The decrease in
potassium excretion after short flights was thought to provide compensation for
low blood potassium concentrations, but the increased excretion after 13 days was
considered evidence that extended weightlessness impairs the ability of tissues to
retain potassium. Urinary excretion of potassium also increased during calcium
lactate and potassium chloride loading tests after the Salyut-6 long-term mis-
sion~.’~*’~
VII. ENDOCRINE REGULATION

Reabsorption of water and electrolytes by the kidney, one of the most important
processes by which fluid volume and blood electrolyteconcentrations are regulated,
is controlled by a complex network of hormone Many of these
hormones have been measured before, during, and after spaceflight.
A. Antidiuretic Hormone
The amount of circulating antidiuretic hormone (ADH) increases when the

emetic reflex is ~timulated.’~ This could happen when astronauts experience space
motion sickness in the first few days of weightlessness, when they await recovery
after landing in the ocean, or when they remain aboard a recovery ship.
Reduction in plasma volume and an increase in blood osmolality are the main
physiologic stimulators of ADH secretion.Osmolality increases during spaceflight
do not seem to be large enough to stimulate ADH secretion (see Section IV),but
plasma volume decreases early in flight. Secretion of ADH probably helps to
prevent dehydration during flight, even though the plasma volume does not
immediately after landing return to preflight values. The lack of effectiveness of
ADH in elevating plasma volume may be due to changes in renal function during
weightlessness, especially after long-term missions.28
142 SCOTT M . SMITH, JANEM . KRAUHS, and CAROLYN S. LEACH
During Flight
Plasma ADH levels are sometimes elevated during flight (Fig. 5, Table 2). During
Spacelab missions of up to 14 days plasma ADH was highly variable after 5 h of
flight, but had increased by more than 100%after 24 h and remained elevated until
at least 5.4 days after l a ~ n c h . "However,
~ ~ ~ on the SLS missions plasma ADH was
not significantly elevated." On a Russian 25-day flight plasma ADH in one
cosmonaut had increased from a preflight value of 1.4 pg/ml to 7.8 pg/ml on day 9
and to 11.2pg/ml on day 20.26High ambient temperature and carbon dioxide levels,
as well as the fact that by day 20 the cosmonaut had been exercising for 75
minuteslday for a week, may have contributed to the increased ADH secretion in
this case.
+ Shuffle
4 Mir
-A- soyuz
E
0
n
_-
A
-1oo-l 7
0 5 10 15 20 25 30 217-219
Flight day
Figure 5. Percent change in plasma antidiuretic hormone during spaceflight. Each

symbol represents the mean of an inflight antidiuretic hormone concentration (pg/ml)
compared with that subject's preflight value($, for 1 to 8 astronauts on Shuttle flights
or 3 cosmonauts on the 237-day Salyut-7-Soyuz-T mission, or the percent change
for 1 cosmonaut on the Mir Aragatz flight. Data are shown in Table 2.
Table 2. Endocrine Results from In-Flight Blood Sampling on Four Flight Programs
Variable FDI FD2 FD3 FD4 FD5 FD6 FD7 FD8 FD9 FDll FDl2 FD13 FD74 FDZO FD27 FD27 FD30 FD38 FD45 FD48 FD58 FD59 FD73 FD82 217-219
Plasma antidiuretic hormne, percent change
Shualell 23.63
n 8 1 2 2 2 7 2 3
Mean 167 719 104 158 172 -14.0 92.0 145
SE 108 52 77 70 20.8 62.0 85
MirZ6
n 1 1
% change 45.7 700
Soyuz5’ 3
n -24.0
Mean 8.6
SE
Plasma renin activity, percent change
Skylab’
n 6 3 3 5 3 3 3 3 3 3 6 3 3 3 3 3 3
Mean 199 45.7 293 243 -16.8 -34.6 -7.4 318 -32.8 -68.6 79.2 169 31.9 118 106 56.4 120
A
SE 94.2 14.0 69 128 23.0 57.0 39.1 160 15.3 13.5 54.5 179 66.1 54.7 24.8 47.3 83.1
8 ~h~~l~11.23.63
n 7 5 2 2 7 4 3
Mean -38.8 -50.0 70.1 131 120 43.5 108
23.3 12.5 18.1 18.7 28 9.3 44
Zir26
n 1 1
% change -35.6 -51.0
Plasma or salivaly aldostemne, percent change
Skylab’
n 6 3 3 5 3 3 3 3 3 3 6 3 3 3 3 3 3
Mean -37.7 58.8 -69.3 9.8 64.1 42.8 115 -41.2 59.8 -57.3 -28.4 -20.5 -3.8 5.7 24.5 -17.6 -12.7
SE 23.1 29.9 12.0 37.7 24.7 94.1 168 25.4 47.2 29.2 18.5 27.3 16.7 59.7 54.8 45.0 21.22
Shunlell 23.63
n 9 5 2 2 2 7 6 3
Mean 4 8 . 2 -15.0 8.9 -34.4 -23.9 -22.5 3.8 9.2
SE 10.9 8.5 43.4 9.8 15.9 9.7 11.5 17.7
Mir26.35
n 1 2 1 1
Mean 39.5 -2.8 56.3 11.9
SE 82.6 (continued)
99 19
C IP
A N n o. D
r .N
9'4
0 rN
. W
ln
'4". 19
n r
n a
N N mI -P
'41
n m z
9 9?
-a n r l.n
w
-00
nr.m
P
1?
N lOn
* N
n
2
144
Plasma ADH was also raised in a cosmonaut near the end of a 241-day mission
on the Mir tati ion,^' but during flight days 216219 of the 237-day Salyut-7-
Soyuz-T flight plasma ADH tended to decline in two cosmonaut^.^'
Urinary ADH excretion often decreases during spaceflight. It was almost always
below preflight levels on the two longest Skylab flights (59 and 84 d), but on the
28-d flight it was sometimes raised, probably because of the elevated temperature
in the spacecraft.’ Urinary ADH excretion was also decreased in one cosmonaut on
days 43-45 and 86-88 of a 150-day flight on Salyut-7-Soy~z-T.~~
However, increased urinary ADH excretion has been observed in some cases: in
one cosmonaut on days 9 and 20 of the 25-day flight,26in two cosmonauts on days
216-219 of the 237-day Salyut-7-Soyuz-T mi~sion,~’ and on the first flight day
of the SLS missions.’’ This may be partly explained by a decreased renal respon-
siveness to the hormone, caused by increased serum calcium and decreased serum
potassium levels during flight.2835o
After Landing
The diuresis noted in the astronaut after the second U.S. human orbital flight has
been ascribed to suppression of ADH by fluid loading and supine posture, although
no measurements of ADH were made.3
Plasma and urinary ADH were first measured in the Gemini program. Both
parameters were elevated in the first postflight sample from one a~tronaut.”~’ After
the Apollo flights urinary ADH had increased in 26 astronauts by an average of
152%.8After the Skylab’ and Space Shuttle46missions it was not changed. After a
week of recovery from the Skylab missions urinary ADH decreased significantly.’
Immediately after Spacelab flights of up to 10days duration, plasma ADH was 49%
abovepreflight levels,6Obut after the SLS flights it was not significantlyincreased.”
Three cosmonauts on the 237-day Soyuz mission had higher plasma ADH at
landing than before flight, and for two of them it was still higher 8 d after landing.
Plasma ADH was elevated 2- to 3-fold in 16 cosmonauts after missions of at least
several months on Mir!3 Increased circulating ADH was thought to affect the
results of water loading tests; the water load apparently did not suppress ADH
sufficiently to permit an increased excretion of water.52
Urinary ADH was elevated after Russian spaceflights of 4 to 14 days,61 150
and 237 days.59
The evidence does not support the early idea that ADH secretion was suppressed,
either during weightlessness or after landing. Rather, its secretion usually seems to
be increased at landing. This is consistent with the findings of fluid retention at
landing and with the need to increase the plasma volume. Differences in endocrine
results from different flight programs may be attributed at least in part to environ-
mental conditions, use of countermeasures during some flights, and differences in
assay methods and sample handling during almost 30 years of research in space
medicine.
146 S C O T M. SMITH, JANE M. KRAUHS, and CAROLYN S. LEACH
-80 ' -
+ Skylab
7
0 10 20 30 40 50 60 70 80 90 217
Flight day
Figure 6. Percent change in plasma or salivary aldosterone during spaceflight. Each

symbol represents the mean of inflight data (pglml) compared with that subject's
preflight value(s), for 3 to 6 astronauts on Skylab, 2 to 9 astronauts on Shuttle flights,
or 3 cosmonauts on the 237-day Salyut-7-Soyuz-T mission. The Mir data represent
percent change in plasma aldosterone on days 9 and 20 for 1 cosmonaut on the Mir
Aragatz flight and percent change in salivary aldosterone on days 5, 9, and 12 for 1
cosmonaut on the Mir Antares flight (the correlation coefficient for plasma and salivary
aldosterone before and after flight was 0.83). Data are shown in Table 2.
Fluid and Electrolfle Regulation in Spaceflight 147
+ Mir
0
Flight day
Figure 7. Percent change in plasma renin activity during spaceflight. Each symbol
represents the mean of inflight data (ng/ml/h) compared with that subject’s preflight
value(s), for 3 to 6 astronauts on Skylab or 2 to 7 astronauts on Shuttle flights, or the
percent change for 1 cosmonaut on the Mir Aragatz flight. Data are shown in Table
2.
B. Renin-Angiotensin-AldosteroneSystem
The renin-angiotensin-aldosteronesystem is another important regulator of body
fluid and electrolytes. Its activity during and after spaceflight has been monitored
by measuring plasma renin activity and plasma and urinary aldosterone. The
enzyme renin catalyzesthe conversion of angiotensinogento angiotensinI, the most
abundant component of the renin-angiotensin-aldosteronesystem in the blood.
Angiotensin I is the precursor of angiotensin 11, the highest concentrationsof which
are found in the adrenal gland. Although there may be other active components of
the system,62angiotensin I1 is thought to be the main physiologically active
hormone of this cascade. It stimulates the secretion of aldosterone, but also acts
independently as a vasoconstrictor in the kidney.
During Flight
Aldosteronewas slightly elevated in two astronauts during and immediately after

the 14-day Gemini-7 mission, while urinary sodium was decreased.' These results
are consistent with the fimction of aldosterone as a sodium-retaining hormone.
During the Skylab flights urinary aldosterone of the astronauts was significantly
elevated 2- to 3-fold; however, in contrast to the Gemini results urinary sodium of
these astronauts was also significantly elevated.' Plasma aldosterone during the
Skylab missions varied from above to below preflight levels (Fig. 6, Table 2).9
During the Spacelab missions plasma aldosterone was at or below the preflight
levels for about 8 days, then it began to increase."'23 Plasma aldosterone was
significantly reduced in 7 astronauts on the first flight day of the SLS missions,''
and urinary aldosterone on the second and third flight days.
On the Mir Aragatz mission plasma aldosterone was elevated about 80%on day
9 in one cosmonaut, but only 12% on day 20.26On the Mir Antares mission salivary
aldosterone in one cosmonaut was elevated on days 5 (40%) and 12 (56%), but it
was reduced by 85% on day 9; urinary aldosterone remained ~nchanged.'~ On the
Mir Aragatz mission urinary aldosteroneexcretion was raised on days 5 and 19. On
the 150-day Salyut-7-Soyuz-T mission it was below preflight levels on days
43-45, but it was elevated on day 88.45 On the 237-day flight it was elevated on
days 2 16-2 19, while the urinary excretion of the aldosterone precursor 11-deoxy-
corticosterone was d e ~ r e a s e dThe
. ~ ~ investigators proposed that the synthesis of
aldosterone was increased. Salt consumption was reported to have been elevated at
the time of the increased aldosterone excretion.
Plasma renin activity has been variable during flight (Fig. 7, Table 2). During
Spacelab missions it was at or below preflight values for the first 2 days, but from
3 to 12 days it was elevated by as much as 140%."'23 During the SLS missions
plasma renin activity in seven astronauts was somewhat reduced on flight day 1,
and significantlyelevated on flight day 8. During the Skylab missions plasma renin
activity was above preflight levels for the first 14 days and below the preflight mean
from day 20 to day 48; on days 58 and 59 it was significantly elevated,but it returned
to low levels on days 73 and 82.9 On the 25-day Mir flight plasma renin activity
was below its preflight value (20.2 ng/ml/h) on days 9 and 20?6 On the 237-day
Salyut-7 mission it was elevated on days 2 1 6 219.59
The renin-angiotensin-aldosteronesystem seems to remain in a dynamic state,
even during long-term spaceflights. Aldosterone and plasma renin activity do not
seem as closely associated during weightlessness as they are on Earth. During a
Spacelab mission no significant correlation existed between plasma renin activity
and plasma aldosterone. However, plasma aldosterone and serum potassium levels
were significantly positively correlated (r = 0.78, P < 0.05), and plasma renin
activity and plasma atrial natriuretic peptide were significantly negatively corre-
lated.63Secretion of aldosterone, an important regulator of serum potassium, may
be activated by the release of potassium due to tissue breakdown during long-term
spaceflights.
The reduction in serum potassium and the lack of change in serum sodium
observed in postflight samples from the Apollo astronauts were attributed in part
to the increased urinary aldosterone ~ecretion.~' On the first day after recovery,
urinary aldosterone of 28 Apollo astronauts was significantly increased by 57%
over preflight values, while urinary sodium was decreased by 49%.*
Urinary aldosterone of Skylab' and Shuttle46astronauts was also significantly
elevated on the day of landing. Plasma aldosterone of Shuttle astronauts was
significantly elevated by 24% at but in the Skylab astronauts it was not
significantly elevated until the next day.9
Plasma aldosterone was elevated after Russian flights of 4 to 14 days, but it was
reduced after a series of Salyut flights of 1 to 8 months6' The day after landing
from 151 or 24 1 days aboard the Mir space station,plasma aldosteronewas elevated
in one cosmonaut in each case.27However, in two cosmonauts, who stayed on Mir
for 366 days, aldosteronewas increased only a week postflight.w After return from
73- to 185-day Salyut missions urinary aldosterone was significantlyelevated one
day p ~ s t f l i g h t . ~ ~
Plasma renin activity was measured in blood samples from 21 astronauts before
and after the Apollo missions. It was significantly elevated by 488% at landing.'
After the Skylab missions, plasma renin activity was at preflight levels on the day
of landing, but like plasma aldosterone it was significantly elevated (threefold
increase) on the next day.'
After short-term(4 to 14 days) Russian flights plasma renin activity was elevated,
but after Salyut missions of 1 to 8 months it was reduced at landing.61It was elevated
after the 151- and 241-day Mir flights.27After the 366-day Mir mission, one of the
two cosmonauts had an elevated plasma renin activity at landing, while in the other
cosmonaut it was elevated a week later.64
After a 13-day Salyut flight reduced excretion rates for potassium and sodium
and increased aldosterone excretion were found during a potassium chloride
loading test. This was interpreted to suggest that other factors besides aldosterone
may be important in the regulation of potassium.49At least some of the inconsis-
tencies in the results from long-term missions can probably be attributed to
differences in countermeasure use and dietary intake.
C. Corticosteroids
Corticosteroids are known to show a circadian rhythm of secretion. This may
complicate the interpretation of changes in these hormones during and after
spaceflight, unless hormone levels are determined in 24-hour urine pools.
At first, corticosteroids and catecholamines were measured in body fluids of
astronauts in order to obtain an indication of stress. Since corticosteroidspromote
retention of sodium and excretion of water, these hormones play a role in fluid and
electrolyte regulation.
Plasma 17-hydroxycorticosteroids of the Gemini-7 astronauts were elevated
immediately after landing.39After the Apollo missions plasma cortisol was reduced
by 27% in 30 astronauts, and adrenocorticotropic hormone (ACTH) by 24% in 12
astronauts, but urinary cortisol was elevated by 24% in 27 astronauts. The plasma
results would suggest that the reentry stress was insufficient to cause increased
secretion of these hormones. However, the difference in time of day for preflight
and postflight blood sampling may have confounded the interpretation of the blood
values: the preflight sampling time of 8:OO a.m. is closer to the daily peak ofcortisol
secretionthan the later postflight samplingtimes.8The postflight increase in urinary
cortisol may provide a better indication of the effect of reentry stress.
After Shuttle flights of 2 to 11 days plasma cortisol in 133 astronauts was on
average 3% (significant) below preflight levels?4 The landing-day samples were
obtained at various times of the day. However, in an earlier study, when 29
astronauts who had used the saline-loadingcountermeasure were excluded from a
group of 37 subjects, plasma cortisol was 7 1% above preflight levels at landing!'
Urinary cortisol in Shuttle astronauts was elevated at landing, by 52% in 4 subjects
who did not use the saline-loadingcountermeasure, and by 37% in 2.5 subjects who
used !ti' Plasma ACTH was raised by 98% in the former group and decreased by
5.8% in the latter group.
For the 9 Skylab astronauts (no countermeasure) plasma cortisol was increased
at landing, but not significantly.' Urinary cortisol was significantly elevated for
more than 2 weeks after landing. Plasma adrenocorticotropic hormone at landing
was below preflight levels, significantlyat some sampling times.
After 151 or 241 days on Mir, plasma cortisol was elevated 19 to 37% in 3
cosmonauts and adrenocorticotropic hormone was reduced.*' After a 150-day
Salyut mission urinary cortisol was elevated in one cosmonaut.45After a 366-day
Mir flight urinary cortisol in two cosmonauts was unchanged," but plasma adreno-
corticotropic hormone was 10-fold greater than its preflight c~ncentration.~~
During the 28- to 84-day Skylab flights, plasma cortisol was increased, signifi-
cantly at some sampling times (Fig. 8, Table 2).' Each month, urinary cortisol was
significantly increased. Plasma adrenocorticotropic hormone was reduced at all
sampling times (Fig. 9, Table 2), sometimes significantly.' Results from Spacelab
flights showed more variability: plasma cortisol first increased, then decreased;
plasma adrenocorticotropic hormone was elevated at most sampling times.'''23
Plasma Cortisol
lE01
150
t soyuz
t Mir
-60 ! , -
-60 ! 1 -
0 10 20 30 40 50 60 70 80 90 217-219
Flight day
Figure 8. Percent change in plasma or salivary cortisol during spaceflight. Each

symbol represents the mean of inflight data (pg/dl) compared with that subject's
preflight value(s), for 3 to 6 astronauts on Skylab, 2 to 9 astronauts on Shuttle flights,
or 3 cosmonauts on the 237-day Salyut-7-Soyuz-T mission. The Mir data represent
percent change in plasma cortisol on days 9 and 20 for 1 cosmonauton the Mir Aragatz
flight and percent change in salivary cortisol on days 5,9,and 12 for 1 cosmonaut on
the Mir Antar& flight (the correlation coefficient for plasma and salivary cortisol before
and after flight was 0.89). Data are shown in Table 2.
'"1
110-
55-
-A- Mir
0-. -- ------_---
55-I
c Skylab
0 10 20 $I 40 50 60 70 80 90
Flight day
Figure 9. Percent change in plasma adrenocorticotropic hormone during spaceflight.
Each symbol represents the mean of an inflight hormone value (pg/ml) compared with
that subject's preflight value(s), for 1 to 6 astronauts on Skylab or 2 to 7 astronauts on
Shuttle flights, or percent change for 1 cosmonaut on the Mir Aragatz flight. Data are
shown in Table 2.
On the Mir Antares flight salivary cortisol increased in one subject from 4 nmol/l
preflight to 7.3 nmol/l on flight day 12, but on flight days 5 and 9 it was not changed
(adrenocorticotropichormone and urinary cortisol were not measured).35During
the 25-day Mir flight plasma cortisol was decreased and adrenocorticotropic
hormone increased in one subject on days 9 and 20.26During the 7-day Mir flight
salivary and urinary cortisol were unchanged.37During the 150-day Salyut mission
urinary cortisol was reduced on days 43-45, while on day 88 both urinary cortisol
and aldosterone were elevated; an increase in the proportion of bound cortisol
indicates that the steroidogenesis pathway had been altered.45On the 237-day
Salyut flight plasma cortisol in two cosmonauts was increased on days 2 16-2 19,38
but urinary cortisol was unchanged. On the 241 -day Mir flight both plasma cortisol
and adrenocorticotropichormone were unchanged before landing day.27
Anegative feedback effectcauses adrenocorticotropic hormone to decrease when
the plasma cortisol level increases. This effect has been noted on the 25-day Mir
and the S p a ~ e l a and
b ~ ~Skylab' flights.
Corticosteroidmetabolites were measured in plasma or urine of astronauts in the
Mercury, Gemini, Apollo, and Skylab programs. On Apollo 17 urinary hydrocor-
tisone decreased between the last day of flight and the first day after landing for all
three crew members, but for only one of them did most inflight values differ from
preflight values (higher).8 After the Mercury flights plasma 17-hydroxycortico-
steroid levels were similar to those measured during preflight astronaut training,
when the highest concentration was noted after a 5-mile run.66During the Gemini-7
mission urinary excretion of 17-hydroxycorticosteroidwas reduced, but immedi-
ately after landing it was increased, probably due to reentry stress.44The latter
occurred also after the Gemini-9 mission!'
After the Apollo missions total urinary 17-hydroxycorticosteroidsin 6 astronauts
were reduced by 30% immediately after landing.8 The two crew members of
Apollo-17 who landed on the Moon, had very high urinary 17-hydroxycortico-
steroid levels on the last flight day and reduced levels after return, but the crew
member who remained in the command module had lowered 17-hydroxycortico-
steroid levels during flight and slightly elevated values postflight. Total 17-hy-
droxycorticosteroids and total 17-ketosteroids were generally somewhat low
during flight.' On the basis of these results, urinary cortisol excretion would have
been expected to decrease during flight, but it increased. This divergence was
attributed to possible sensitivityof 17-hydroxycorticosteroidsto storage conditions
or liver blood flow changes that altered the rate of conjugation of cortisol to its
metabolite.'
After the Skylab missions total 17-hydroxycorticosteroids were significantly
reduced, but during the flight they were unchanged.' Total 17-ketosteroids were
significantly elevated during flight, but returned to preflight levels at landing. As
with the Apollo missions, urinary cortisol was elevated at landing. As mentioned
earlier in this section, urinary cortisol was also elevated during the Skylab missions.
Urinary sodium excretion of the two Gemini-7 astronauts was significantly
correlated with urinary excretion of 17-hydroxycorticosteroids,but not with urinary
aldosterone excretion (all flight phases included).44However, during flight urinary
sodium increased moderately, while urinary 17-hydroxycorticosteroidsdecreased.
In Skylab astronauts total 17-hydroxycorticosteroid excretion was not correlated
with urinary sodium.' On S o y - 9 urinary 17-hydroxycorticosteroids of two
cosmonauts also decreased during flight, but on the last day these metaboliteswere
close to their preflight levels.36
The consistence of the effects of spaceflight on 17-hydroxycorticosteroidssug-
gests that early findings of low values were not the result of storage conditions.
These compounds do not seem to increase in response to stresses like acceleration
and disorientation. A further discussion of stress and spaceflight is provided in
section VII E on catecholamines.
D. Natriuretic Hormones
A reduced plasma volume and an increased urinary sodium excretion have been
observed during several spaceflights. Natriuresis would normally follow a reduc-
tion in plasma volume. These observations have led to the search for a natriuretic
hormone that might regulate fluid volume and serum electrolyte concentrations
during weightlessness. A possible candidate is atrial natriuretic peptide, which has
now been measured in blood samples from a small number of astronauts and
cosmonauts.
Atrial natriuretic peptide was first measured in blood samples from 4 astronauts
on an 8-day Space Shuttle mission.30p63 About 30 hours after launch, its level was
82% above the preflight level, but by day 7 it had decreased to about 50% below
preflight level. On landing day, it was still reduced, but 3 days after landing it had
returned to the preflight level. During the 9- and 14-day SLS missions, blood
samples were obtained several times during flight from 4 and 3 astronauts, respec-
tively. Atrial natriuretic peptide was significantly lowered 3 to 5 h after launch and
on flight days 8 and 12."
During the 25-day Mir Aragatz mission, atrial natriuretic peptide in one cosmo-
naut increased from 4 1.5 pg/ml preflight to 47.5 pg/ml on day 9 and on day 20 it
had returned to the preflight level, while after landing it increased to 56.0 pg/m1.26
During the Mir Antares flight, the peptide level was determined in saliva, since
preflight measurements showed a strong correlation between salivary and plasma
levels. No changes were observed on flight days 5,9,and 12.35
Urodilatin is another natriuretic hormone that has so far been detected only in
urine.67 During a 7-day Mir flight it was determined in one cosmonaut. No
significant changes were found, but the investigators reported that the correlation
between urinary sodium and urodilatin was considerably altered during weightless-
ne~s.~'
Cyclic AMP (adenosine 3',5'-monophosphate) and cyclic GMP (guanosine 3 ' 3 -
monophosphate) are the 'second messengers' for hormones that cannot cross a cell
membrane. After interaction of the hormone with its receptor in the cell membrane,
cyclic AMP or cyclic GMP is released in the cell and there effects the hormone
action. Cyclic GMP is the second messenger for atrial natriuretic peptide and
urodilatin. During the Mir-Antares mission cyclic GMP was measured in urine and
saliva of the French cosmonaut. The only change noted was a several fold increase
in salivary cyclic GMP on the 1st and 3rd day after landing.3' In the urine of SLS
astronauts cyclic GMP did not change significantly with time in weightlessness,
but the results for the two SLS missions differed in SLS-1 cyclic GMP was below
preflight values, and in SLS-2 it was usually above preflight values."
Insufficient data are available to provide a clear picture of the effects of space-
flight on atrial natriuretic peptide and other natriuretic hormones. However, since
central venous pressure appears to decrease in weightlessne~s,6~~~ stimulation of
atrial secretion of atrial natriuretic peptide probably does not usually occur.
E. Catecholamines
Catecholamines were the first hormones measured in biological samples from
astronauts; they were considered to provide a measure of short-term response to
stress. Measurements of the catecholamines epinephrine and norepinephrine and
their metabolites in the urine of the Mercury astronauts indicated that epinephrine
was elevated most often after spaceflight, although some increases were observed
for other catecholaminesand their metabolite^.^' Intersubject variability was high,
and some of the samples may have been compromised because of delays in
preserving them. The catecholamine responses of these individuals to spaceflight
did not exceed their responses to stressful training procedures. The investigators
concludedthat these astronauts may have had a high resistance to stress that usually
activates the sympathoadrenal system.
Since the catecholaminesshow circadian rhythms of excretion,'* it is desirable
to consider results from 24-h urine pools and the plasma levels of these hormones
and neurotransmitters.
During Flight
During the Skylab missions neither epinephrine nor norepinephrine in urine in

the nine astronauts changed from preflight values.' During the SLS flights plasma
epinephrine and norepinephrine, as well as urinary epinephrine, did not change
significantly in 7 astronauts from preflight concentrations. Urinary norepinephrine
of 4 subjects on SLS-1 was insignificantly reduced on flight days 1 and 4/5."
During a 6-day Space Shuttle mission urinary epinephrine in one subject did not
change from the highly variable preflight baseline, but urinary norepinephrine
decreased on day 1.33 There is some evidence from Space Shuttle flights that
adrenergic receptor activity is decreased in weightlessness: although plasma con-
centrations of epinephrineand norepinephrine measured in the supine and standing
positions were greater at landing than before flight, and levels of both catecholami-
nes increased in response to the stand test, total peripheral vascular resistance did
not change in response to the test.73A diminution of the vasoconstrictor response
to sympathetic stimulation, which involves beta adrenergic receptors, may have
contributed to this result.
156 S C O l l M. SMITH, JANEM. KRAUHS, and CAROLYN S. LEACH
Catecholamines and their metabolites have been measured in blood and urine
samples obtained from cosmonauts during several flights. It was concluded that
flight duration may be an important factor in determining the levels of these
hormones. On the 25-day Mir Aragatz flight norepinephrine in plasma from the
cosmonaut was elevated from 250 pg/ml before flight to 46 1 pg/ml on day 9, and
epinephrine from 16 pg/ml before flight to 66 pg/ml on day 20.26Dopamine was
reduced from 46 to 30 pg/ml on day 9. There was no change in the plasma levels
of catecholamine sulfates, which are influencedby stress.74Urinary catecholamines
and their major metabolites (vanilmandelic acid and homovanillic acid) did not
change during this flight. The authors proposed that the slight activation of the
sympathoadrenal system, indicated during this short-term flight, was caused by
anxiety over blood withdrawal, physical exercise, or some other weak stimulus of
the system rather than by weightlessness. During weightlessness simulationby bed
rest or water immersion, epinephrine and norepinephrine are usually significantly
reduced.
During the Mir Antares flight all catecholamine variables measured in the urine
were elevated during and after flight, compared to their concentrations 30 days
before flight and compared to the same variables during and after the Aragatz
mission. No measurements of catecholamines were made in the inflight plasma
samples.
On the 237-day Salyut-7 mission samples obtained from 3 cosmonauts on days
2 17-2 19 showed slightly increased plasma epinephrine and norepinephrine levels.
Urinary excretion of these catecholamines did not change, while excretion of their
metabolites (normetanephrine, metanephrine, and vanilmandelic acid) decreased
from preflight values.25
After Landing
Although the sympathoadrenal system does not seem to be activated during

spaceflight, the catecholamines have usually been elevated in plasma and urine
sometime after landing in other flight programs since Mercury. A short-lived
granulocytosisobserved after the Gemini missions was considered to indicate that
epinephrine was released during reentry.' The 24 Apollo' and 3 Apollo-Soyuz7'
astronauts showed little change in urinary epinephrine and norepinephrine imme-
diately after landing.
After 4- to 10-day Space Shuttle flights plasma epinephrine and norepinephrine
were significantly elevated in 24 astronauts, and plasma norepinephrine was still
significantly elevated 3 days after landing.76Urinary norepinephrine in 4 subjects
on SLS-1 and 3 subjects on SLS-2 was elevated after landing, as was urinary
epinephrine of subjects on SLS-2. However, their plasma catecholamine levels
were not changed significantly after landing."
After Russian flights of 4 to 14 days urinary excretion of epinephrine, nor-
epinephrine, dopamine, and catecholamine metabolites in 20 cosmonauts was
elevated.77Norepinephrine levels were highest immediately after flight, but epi-

nephrine excretion was slightly higher two days later. The ratios between these
compounds indicate that synthesis and secretion of catecholamines, especially
epinephrine, were stimulated, and that epinephrine was being metabolized more
slowly than norepinephrine.
After the Skylab flights urinary epinephrine was not significantlydifferent from
preflight values, but urinary norepinephrine was significantly elevated for at least
14 days after landing.'
Plasma and urinary catecholamineswere elevated after Russian flights of 25 days
to 12 months d u r a t i ~ n . ~ 'In~ one
~ , ~cosmonaut
~ , ~ ~ on the 25-day flight urinary
norepinephrine, dopamine, and their metabolites vanilmandelic acid and ho-
movanillic acid, were highest on the first and second days after landing, while
plasma levels of sulfates of norepinephrine and dopamine were highest on the first
and third days after landing.74These results indicate that adrenergic, noradrenergic,
and dopaminergic neurons in the sympathoadrenal system of this individual were
activated. After the 2 11-day Salyut-7 mission urinary excretion of catecholamines
and their metabolites by 2 cosmonauts was reported to be highly elevated for at
least 8 days after landing, and not to have returned to preflight levels until 45 days
after landing.79
After the 237-day Salyut-7 flight an apparent delay in activation ofthe sympatho-
adrenal system occurred in 3 cosmonaut^.^^ One day after landing the plasma
catecholamineswere unchanged from preflight levels, but on day 8 after landing
plasma epinephrine and norepinephrine were above preflight levels.
The second messengers cyclic AMP and cyclic GMP (see section VII D) were
measured before and after short-term and long-term Russian space missions.61380
After short-term flights,the cyclic AMP level and the cyclic AMP/cyclic GMP ratio
increased in cosmonaut blood samples?' After one series of long-term flights,
CAMPdid not change from its preflight level and the ratio decreased. This was
considered to indicate that a fimctional blockade of adrenoception occurs during
long spaceflighk6' However, after another series of long-term flights cyclic AMP
was increased and cyclic GMP decreased, so that their ratio increased and activation
of adrenergic mechanisms was indicated.80
The launch and landing phases of spaceflight are physically stressful, and the
novelty of the weightless condition might be expected to cause some psychologic
stress. Currently available information includes measurements of cortisol and
corticosteroid metabolites, as well as measurements of catecholamines and their
precursors and metabolites. Although plasma and urinary cortisol are elevated
during weightlessness most of the time, other endocrine evidence indicates that
weightlessness is not physically stre~s-inducing.~~ Landing, on the other hand,
stimulates the sympathoadrenal system, though after long-term spaceflights the
response may be delayed.
F. Prostaglandins
Prostaglandins are a group of hormones, some of which have pressor effects

(prostaglandin F23 or depressor effects (prostaglandins A and E) on blood vessels.
Prostaglandins have been measured in blood samples from cosmonauts on short-
and long-term flights. The concentration of depressor prostaglandins A and E in
blood samples was significantlydecreased after flights of4 to 14 days, but not after
longer flights.6’ After the 366-day Mir flight prostaglandin E, in blood plasma of
the two cosmonauts was below preflight concentrations, but the pressor pro-
staglandin F,, was elevated 7 days after landing. This was considered a response
of cardiovascular regulatory mechanisms to pressor effects of catecholamines,
which were increased after this mission.81
VIII. MECHANISM O F SPACEFLIGHT EFFECTS

The importance of distinguishingbetween physiologic responses to weightlessness
and responses to the return to Earth has been recognized since the beginning of
human spaceflight. However, measuring these responses during and immediately
after flight is often difficult or even impossible with current technology. Attempts
have been made to explain the processes by which alterations in body fluid
compartments and blood electrolyte concentrations occur during both phases of
spaceflight.
Gemini7andApol10~~ crew members reported a sensation of ‘fullnessin the head’
during the first day of flight that has been attributed to the headward shift of blood
in weightlessness. After measurement of body fluid volumes before and after the
Apollo missions, the hypothesis was proposed that circulating blood and other
extracellular fluids become distributed equally throughout the vascular space and
this change is detected by receptors as a volume expansion.8 This would cause
inhibition of the secretion of antidiuretic hormone and aldosterone, causing fluid
and weight
Although there is little question that the legs lose volume, while the volume of
some fluid compartments in the head and neck the connections
between these changes, weightlessness, fluid volume status, electrolyte concentra-
tions, and endocrine regulators of fluid and electrolytes are still in the process of
being elucidated. Space travelers lose weight, much of which is fluid: but whether
diuresis occurs is still debatable and secretion of antidiuretic hormone is not
inhibited. An increase in central venous pressure was a key part of the hypothesized
me~hanism,~’ but recent direct measurements of central venous pressure during
spaceflight6%” have not confirmed such an increase. Echocardiographic measure-
ments of the cardiac volume86suggest that the central blood volume is increased
on the first day of weightlessness, and then it decreases to a level below that of
supine subjects on the ground.87 Until these contradictory findings have been
resolved, the proposed chain of events cannot be determined.
The rapidity of the plasma volume reduction in the first 21 h of weightlessness"

suggests that the observed hormone changes, such as increases in antidiuretic
hormone, plasma renin activity, and cortisol, are not causing the reduction of the
plasma volume but maintaining it, or preventing it from decreasing further. Atrial
natriuretic peptide seems to play little or no role in causing the loss of electrolytes,
though more data are needed, especially for the second day of flight.
At least two of the three major factors controlling fluid and electrolytes may be
involved in reducing plasma volume soon after the onset of weightlessness: reduced
colloid osmotic pressure, and a decreased fluid and electrolyte intake. Inhibition of
hormones that increase reabsorption of water and sodium might be expected to
occur, and this seems to be true for plasma renin activity and aldosterone on the
first day of weightlessness. Antidiuretic hormone and cortisol, however, do not
seem to be inhibited at this time. Atrial natriuretic peptide, whichmight be expected
to increase, may do so for a short time after the first day.
After the initial reduction of plasma volume, colloid osmotic pressure may still
be reduced, but fluid and electrolyte intake are not so sharply reduced. Endocrine
factors (elevated antidiuretic hormone, plasma renin activity,and aldosterone)seem
to favor increased retention of fluid and electrolytes at least part of the time; urine
volume and sodium data from different flights are not consistent.
The increased antidiuretic hormone level and the water retention at landing, as
observed during the Gemini program, were considered to indicate that cardiovas-
cular stretch receptors are important for adaptation to weightlessness and re-adap-
tation to Earth's gravity.39Pooling of blood in the lower extremities after landing
would make the volume receptors in atria and thorax sense an apparent decrease in
blood volume. Secretion of antidiuretic hormone and aldosterone would then be
stimulated, leading to retention of water and electrolytes. Current evidence does
not contradict this early explanation, although more hormones are probably in-
volved and flight duration plays a role in determining the order ofendocrine events.
IX. CONCLUSIONS AND SUMMARY

Despite a number of difficulties in performing experiments during weightlessness,
a great deal of information has been obtained concerning the effects of spaceflight
on the regulation of body fluid and electrolytes. Many paradoxes and questions
remain, however. Although body mass, extracellular fluid volume, and plasma
volume are reduced during spaceflight and remain so at landing, the changes in
total body water are comparatively small. Serum or plasma sodium and osmolality
have generally been unchanged or reduced during spaceflight, and fluid intake is
substantially reduced, especially during the first week of flight. The diuresis that
was predicted to be caused by weightlessness, has only rarely been observed as an
increased urine volume. What has been well established by now, is the occurrence
of a relative diuresis, where fluid intake decreases more than urine volume does.
Urinary excretion of electrolytes has been variable during spaceflight, but

retention of fluid and electrolytes at landing has been consistently observed. The
glomerular filtration rate was significantly elevated during the SLS missions, and
water and electrolyte loading tests have indicated that renal function is altered
during readaptation to Earth’s gravity. Endocrine control of fluid volumes and
electrolyte concentrations may be altered during weightlessness, but levels of
hormones in body fluids do not conform to predictions based on early hypotheses.
Antidiuretic hormone is not suppressed, though its level is highly variable and its
secretion may be affected by space motion sickness and environmental factors.
Plasma renin activity and aldosterone are generally elevated at landing, consistent
with sodium retention, but inflight levels have been variable. Salt intake may be an
important factor influencing the levels of these hormones. The circadian rhythm of
cortisol has undoubtedly contributed to its variability, and little is known yet about
the influence of spaceflight on circadian rhythms. Atrial natriuretic peptide does
not seem to play an important role in the control of natriuresis during spaceflight.
Inflight activity of the sympathetic nervous system, assessed by measuring catecho-
lamines and their metabolites and precursors in body fluids, generally seems to be
no greater than on Earth, but this system is usually activated at landing.
Collaborativeexperiments on the Mir and the International Space Station should
provide more of the data needed from long-term flights, and perhaps help to resolve
some of the discrepancies between U.S. and Russian data. The use of alternative
methods that are easier to execute during spaceflight, such as collection of saliva
instead of blood and urine, should permit more thorough study of circadian rhythms
and rapid hormone changes in weightlessness. More investigationsof dietary intake
of fluid and electrolytes must be performed to understand regulatory processes.
Additional hormones that may participate in these processes, such as other natri-
uretic hormones, should be determined during and after spaceflight.
Alterations in body fluid volume and blood electrolyte concentrations during
spaceflight have important consequences for readaptation to the 1-G environment.
The current assessment of fluid and electrolyte status during weightlessness and at
landing and our still incomplete understanding of the processes of adaptation to
weightlessnessand readaptation to Earth’s gravity have resulted in the development
of countermeasures that are only partly successful in reducing the postflight
orthostatic intolerance experienced by astronauts and cosmonauts. More complete
knowledge of these processes can be expected to produce countermeasures that are
even more successful, as well as expand our comprehension of the range of
adaptability of human physiologic processes.
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Chapter 7
MEDICAL MONITORING IN
LONG-TERM SPACE MISSIONS
A.I. Grigoriev and A.D. Egorov
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
I1. Principles of Medical Monitoring during Spaceflight . . . . . . . . . . . . . . 168
I11. Health Changes to be Diagnosed . . . . . . . . . . . . . . . . . . . . . . . . 169
A. Normal State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
B. Pathology and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
C. Unfavorable Conditions in Spaceflight . . . . . . . . . . . . . . . . . . . 172
D. Contingency-Related States . . . . . . . . . . . . . . . . . . . . . . . . 175
IV. Current Practice in Prolonged Missions . . . . . . . . . . . . . . . . . . . . . 176
A . Criteria for Selection of Physiological Parameters . . . . . . . . . . . . . 176
B. Medical Monitoring and Extensive Examination . . . . . . . . . . . . . 177
C. Statistical Methods in Diagnostic Data Processing . . . . . . . . . . . . 183
V. Medical Monitoring in Interplanetary Flights . . . . . . . . . . . . . . . . . . 186
A . Interplanetaryvs. Orbital Flight . . . . . . . . . . . . . . . . . . . . . . 186
B . Mission to Mars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
VI. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

.
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ISBN:0-7623-0147-3
167
168 A.I. GRlGORlEV and A.D. EGOROV
1. INTRODUCTION
Among the measures providing for the safety of the crew onboard a spacecraft an
important role is assigned to the system of medical monitoring and diagnosis. In
addition to the familiar changes occurring normally in the human body during
spaceflight, there are other medical hazards: possible occurrence of occupational
injuries, malhctioning of life support systems, physical illness, and other unpre-
dictable emergency situations. The experience from both short-termand long-term
manned missions has provided sufficient evidence indicatingthat there is always a
potential risk of medical problems in crew members.
The on-going manned space programs comprise short-term missions aboard
multiple-use space vehicles and long-term missions on a space station. Moreover,
studies of prospective interplanetary missions, such as a journey to Mars, are in
progress. This chapter addresses a system of medical monitoring used for orbital
space station missions, which could be adapted for use during future interplanetary
missions. The discussion is focussed on the problems of medical diagnosis and
observation, while countermeasures and medical care subsystems are not consid-
ered.
II. PRINCIPLES OF MEDICAL MONITORING DURING

SPACEFLI C HT
Medical monitoring should be based on a set of principles formulated from an
analysis of the types of medical support needed during past extended spaceflight^.^
These principles are discussed here.
Planned screening investigations. The investigations are aimed at: 1. pre-
nosologic diagnosis; 2. detection of deviations from the norm; 3. detection of
diseases; 4. detection of risk factors which promote or increase the probability of
unfavorable conditions or diseases; 5. identification of crew members who require
additional medical examinations and therapeutic or prophylactic treatment.
Screening investigations within the medical monitoring program employ stand-
ard approachesand common methods of thorough medical investigation. They must
be carried out periodically, even if there are no complaints.
Screening of body systems and subsystems. If integral characteristics in one of
the body systemsexhibit signs of impairment or deviation from the norm, a directed
diagnostic study of the system’s hierarchy is to be conducted. Such study is carried
out in several stages, and is aimed at defining changes in the subsystem(s) respon-
sible for the observed disturbances. Special attention must be given to alterations
that may have unfavorable effects on controlling mechanisms in other body
systems.
Individualization of investigation. An individualized approach to the medical
examinationsis needed, which takes into account the data obtained during the crew
selection phase and during training and preparation for the flight. The data contain
Medical Monitoring in Space Missions 169
information about specific physical properties, reactivity to any type of environ-

mental stress, and the existence of potentially vulnerable systems or organs (loci
minoris resistentiae), noticed before or during flight.
Adaptation of the investigation program. Methods of medical examinations are
adapted on the basis of inflight responses of body systems and deviations observed.
Evaluation and interrelationsof parameters. This principle envisages a wide use
of complex indices obtained from the baseline data by the application of statistical
methods, such as multi-dimensional statistics, principal components method, dis-
criminant analysis, and method of canonical correlations. This approach permits to
decrease the number of analyzed parameters and to study changes in various
parameters in their totality expressed by one or several linear combinationsand the
canonic correlation factor.
Evaluation in terms of environmental adaptation. This principle includes inter-
pretation of data from the standpoint of adaptation to the given environment,when
inflight changes in various body systems (skeletal demineralization, physical and
orthostatic deconditioning, functional erythrocytopenia, etc.) may be viewed as
typical for spaceflight and for countermeasures used.
Continuity of investigations. The principle of continuity of medical investiga-
tions during flight and clinical-physiological studies in the pre- and postflight
periods is ensured by the use of identical methods.
Medical history. It is important to carry out information analysis which will
include medical records in the database from medical examinations and observa-
tions during cosmonaut selection and training for the mission.
Confidentiality. This principle requires strict observance of medical ethics and
confidentialityto secure close medical contact with the space crew during and after
flight.
111. HEALTH CHANGES TO BE DIAGNOSED

A. Normal State
Medical monitoring during long-term space missions should detect and diagnose
the diseases, unfavorable states, and psychological disorders that are listed in Table
1. The psychological disorders become apparent as borderline psychoneurological
disorders, such as neurotic reactions and n e ~ r o s e s . ~
In the process of diagnosis, including automated diagnosis, medical data are
compared with limits for each index. In broad terms the norm is an averaged
statistical standard with deviation limits, characterizing healthy human subjects.
Essentially, it is a sum of limits established by way of systematic comparisons and
various measurements which allow to elicit more or less stable indices for longitu-
dinal observations. In the general physiological sense the norm is defined as the
state of the body in dynamic equilibrium with ambient conditions achieved by
compensatoryadaptiveresponses, which acquired their functional and morphologi-
170 A.I. GRIGORIEV and A.D. EGOROV
Table 1. Health States to be Diagnosed in Long-Term Spaceflights

Unfavorable conditions inherent in spaceflight
Psychological disorders due to:
psychological stress,
psychotraumaticor extreme factors,
somatic disease
Occupational injuries (e.g., traumata, burns, electric shock)
Health disorders during contingency situations, e.g.,
malfunction of life support systems
partial depressurization of living modules
fire
lnflight diseases with or without symptoms
cal properties in the process of phytogenesis and ontogene~is.~ Norm is the interval
within which oscillations of psycho-physiological processes are able to maintain
the living system at a functional optimum, i.e., the optimal zone wherein the
organism does not overstep the pathological level of self-control.6In relation to
medical monitoring special significance is attributed to the data about the norm for
each crew member and its specific features. These data can be obtained by statistical
single-dimensional or multidimensional analysis of results of medical examina-
tions and observations during selection, training and other tests.
B. Pathology and Disease

In the light of current medical knowledge pathology can be defined as any
deviation from the norm, including structural and functional symptoms of disease.
Apathological process, which is a more general category than disease, is a regularly
emerging succession of body responses to the damaging effects of a pathogenic
factor.'* Or, according to the definition by Ado and Ishimova,13it is a combination
of physiological and protective adjusting reactions in injured cells, tissues or
systems which may not yet be clinically observable. The simplest form of a
pathological process is a reaction of the body to a pathological stimulus, which
leads to impairment of homeostasis. A pathological state is a relatively stable
deviation from the norm, which is biologically negative for the body and develops
more slowly than the pathological process. Existence of a pathological state or
process does not necessarily imply a disease of the whole body, though this may
occur.as a result of other i m p a ~ t s .The
' ~ evolution of a pathological process into a
disease of the whole body is a quantitatively and qualitatively new form of
disturbance in the body.
In general, a disease is a deviation from or an impairment of the normal structure
or function of any part of an organ or system (or their combination) that is
Table 2. General Signs of Disease

Impaired efficiency and/or deteriorated socially productive activities
Changes in body functions, e.g.,
reactivity,
metabolism,
energy exchange,
adaptation
Changes in cellular structures in specific organs and systems
Typical set of symptoms
Decreased adaptability to permanently changing ambient conditions
Instability of autonomous functions
Changed forms of activation of compensatory mechanisms
Limitation of subject’s actions
manifested by a characteristic set of symptoms and signs and whose etiology,

pathology and prognosis may be known or unkr~own.’~ This definition covers the
whole spectrum of diseases, including all sorts of injury. However, the definition
does not reflect common signs of any type of disease. According to Veselkin,”
disease is the life whose course has been disturbed by structural and functional
damages of the body inflicted by external and internal factors with qualitatively
peculiar reactive mobilization of its compensating-adjusting mechanisms. Disease
is characterizedby general and partial losses of adaptability and restricted freedom
of the patient’s life. Amosov defines disease from the standpoint of cybernetics as
‘the state of instability of a self-controlling system resulting from excessive or
unusual external impacts or from defects in its own programs’.16
Viewing disease as a disturbance of the normal life of the human body, which
has been damaged by various harmfbl factors, and employing definitions proposed
by Ado and I~himova,’~ Alpern,”Amo~ov,’~ Petrov and Lemus,18 the main general
signs of disease are listed in Table 2.
It should be emphasized that these signs of disease are not absolute. With regard
to impaired efficiency, it can be said that in early phases professional efficiency
may be preserved although there are signs of work decrements in other areas, but
it will be affected as disease progresses. The decreased adaptabilityto permanently
changing ambient conditions and the impairment of controlling functions will lead
to a disrupted interactionbetween the body and the environment. On the other hand,
the reduced performance of healthy subjects, due to fatigue or to the temporal
indisposition and work deterioration following a transcontinental airtrip, are by no
means signs of disease. The same is true with respect to mountaineers at moderate
altitudes. The symptoms observed are rather a pathological reaction (according to
the above definition) or, if their manifestation is severe, a pathological process,
whkh gradually subsides as adaptation to a normal environment progresses.
C. Unfavorable Conditions in Spaceflight
Effects of Spaceflight Factors and Protective Body Reactions
Currently, it is assumed that microgravity is the principal etiological factor

responsible for inflight shifts in the human body, which can be described by definite
syndromes in various body systems. The effects of microgravity are the result of
the interaction between this factor and the physiological systems, which have the
purpose of maintaining homeostasis. The key link in the mechanism of micro-
gravity effects is elimination of gravity-dependent deformations and mechanical
strain of the body structures. This underlies changes in afferent input, removal of
hydrostatic blood pressure and other body fluid pressures, and unloading of the
musculo-skeletal system. Each of the inflight physiological states has its particular
signs or symptoms associated with a definite etiological factor. There is also a
possibility of simultaneous advent of non-specific reactions typical for many states
of health or disease.
Nowadays it is understood that the sympatho-adrenal system is the first to react
to an environmental impact, followed by the pituitary-hypophyseal-adrenalsystem.
These systems are capable of sustaining the non-specific protective stress-reaction
for an extended period.' Long-term manned flights and experiments with animals
on the Bion Program showed the absence of typical signs of chronic stress in
conditions of weightlessness."" However, during the most important flight stages
(insertion into orbit, extravehicular activity, descent to Earth) and in contingency
situations there still remains the possibility of stress. The severity of such stress
depends on emotional factors, individual reactions and intellectual (cognitive)
judgments of risk and coping strategies. It should be emphasized that, even if there
is no occurrence of disease or unfavorable states induced by flight factors, micro-
gravity will in the first place inevitably and specifically modify the general
character of non-specific reactions. Therefore, special consideration should be
given to diagnostic methods that will allow differentiation between specific and
nonspecific reactions to long-term spaceflight.
hterrelation Between Manifestations of hjury and Defense and Latent

Pathology
As is known, diseases caused by some etiological (pathogenic, damaging) factor

involve the development of morphological changes and protective reactions to
stabilize or compensate altered functions. Compensatory adaptive reactions in
response to injury from a pathological factor are manifested on any level (cellular,
tissue or organ) by primarily the same structural compensation, namely, new
formation of organelles and increased number of cells so that these cells can
implement additional functions to replace damaged or dead cells.' 'Gradual'
increase of structural changes in a "sick" organ, although its function is essentially
preserved, may continue for a long time and a patient is thought to be in good
health’.’’ In other words, actual protective reactions of the body provide counter-
action to pathogenic factors long before the patient voices complaints or other
clinical manifestations appear. Structural changes may at all times precede hnc-
tional shifts, or arise simultaneously. The pre-morbid period is the result of
’’
subclinical morphological changes. For the task of inflight medical care this
means that it is important not to overlook at the stage of cosmonaut selection the
initial (asymptomatic)phases of morphological changes in the body, and to assess
their significance,the level of compensation and the expected flight effects for the
possible development of decompensation and clinical signs during flight. On the
other hand, it is also of primary concern to detect timely any tendencies towards
decompensation of asymptomatic diseases, which have been known before flight,
and to diagnose early any asymptomatic diseases developing during flight.
Main Syndromes Associated with Spaceflight Effects
The medical experience of manned missions shows rather good adjustment and
performance in microgravity for 12 months, although regular syndromes of shifts
in various body systems are exhibited.%” These syndromes are listed in Table 3.
Qualification of Space-Induced Syndromes
It is important to qualify changes in the human body developing during extended

spaceflight in an adequate way, using current physiological and pathophysiological
criteria. Appropriate qualification will facilitate the selection of rational strategy
and tactics of diagnosisand of appropriateprophylactic treatment of these change^.^
According to current knowledge, functional shifts in the human body in micro-
gravity represent a combination of events. These events result from direct effects
of this factor and from adaptive reactions that follow general biological rules.’ The
most marked evidence of health and performance deterioration and a variety of
specific symptoms is observed during the initial stage of spaceflight. Crew mem-
bers then develop space motion sickness(SMS)against the background of the acute
cephalic shift of body fluids, accompanied by subjective sensations of blood rush
to the head, bunged-up nose, facial edema, etc. In this case there is no decreased
adaptability, since humans enter microgravity in a healthy state. In this situation
cosmonauts are able to cope with the SMS symptoms within a few days of flight
through mobilization of the early protective reaction of the body with simultaneous
development of long-term adaptive reactions. We suggest that the marked and
subjectively distressing SMS syndrome may be qualified as a brief pathological
process. However, we have documented a case of SMS lasting 2 weeks with highly
expressed symptoms. Such a case of SMS can be qualified as a disease.
Other shifts in the human body in weightlessness after the first phase of adapta-
tion have no subjectivemanifestations, and the crew members feel well. This means
that physiological mechanisms of protection against adverse spaceflight effects,
Table 3. Syndromes Occurring in Spaceflight

First week of flight
Subjective symptoms related to shift in body fluid
Space motion sickness
Changes in motor activity pattern and coordination of motor acts
Prolonged exposure to spaceflight conditions

Changes in the motor and muscular systems:
decreased forcdvelocity characteristics
development of subatrophy or atrophy of antigravity muscles
changes of proprioceptive sensory inputs and spinal automatisms
decreased effectiveness of motor control
Negative calcium balance and reduction of bone density.
Changes in metabolism and its controls:
prevalence of catabolic processes,
negative balance of some ions,
changes in hormone secretion,
changes in blood enzyme levels
Changes in the digestive tract status
Cardiovascular deconditioning:
decreased tolerance to orthostatic and physical loading
Functional erythrocytoenia
Decreased immunological responsiveness
Other conditions
Neuro-emotional stress may grow with increasing mission length,
particularly during interplanetaryflights
Fatigue, asthenization and sleep disturbances
primarily microgravity, appear to be sufficient to provide equilibrium in the

body-environment system and to ensure adequate efficiency of crew member^.^.'^
It is clear that long-term spaceflight changes the functioning of the main physi-
ological systems, and introduces some modifications in the structure of various
organs. Examples are: decreased volume of the lower extremities, atrophy or
subatrophy of antigravitational muscles, bone demineralization, and expansion of
some viscera. Also affected are: body energy exchange, homeostatic parameters of
water-salt balance, cardiovascular and other systems, and neurohumoral controls,
including those related to the hormonal status of the body. All these changes appear
to indicate the establishment of a new level of functioning that is optimal in the
spaceflight situation.
It should be emphasized that these microgravity effects can be compensated to a
considerable extent by means of suitable countermeasures. Factual shifts in the key
body systems are just residual deviations that cannot be eliminated so far by
available prophylactic means.’ In studying the reactions of these systems to
long-term flight, we essentially investigate the concurrent actions of microgravity

and opposing countermeasures. In addition, we must allow for a lack of under-
standing about some intimate controlling mechanisms and histo-morphological
alterations in the body systems. All this means that we do not yet know all clinical
implications of the shifts occurring in the human body during spaceflight.
Qualification of Readaptation Syndromes
Immediately after return to Earth from a long-term space flight the human body
responds acutely to the reimposed gravity. Distinct symptoms are observed, includ-
ing a decreased capacity for work during the early period of readaptation. Imme-
diately after touch-down some cosmonauts demonstrate subjective and objective
signs of vestibular dysfunction, various degrees of disruption in the motor system
and its controls, and noticeable reduction of physical and orthostatic tolerance.
These effects are the result of deadaptation processes due to decreased fhctional
loading of some body systems during a prolonged stay in weightlessness. They
depend more on amount, type and intensity of physical exercise and other counter-
measures employed during flight and on individual characteristics of crew members
than on the duration of the flight.
The reduced hctional loads on the body systems in microgravity thus appear
to give rise to the establishment of a new level of functioning in a relative
equilibrium of the body-environment system. This level is characterizedby stable
deviations from preflight levels in some systems. deconditioning and deadaptation
of underloaded systems, decreased functional reserves of the body, impairment of
tolerance for different loads and unfavorable external effects. All this has a negative
biological implication for the human body during readaptation to Earth gravity after
long-term microgravity. The combination of these changes in different body
systems, resulting from re-exposure to gravity after prolonged spaceff ight, may be
qualified as a post-weightlessness gravity-induced syndrome, which is sometimes
clinically n~ticeable.~.'~ The syndrome can probably be qualified as a transient,
progressively decreasing process that starts in the first hours after return to Earth.
Risk Factors
It is also important to recognize and assess risk factors associated with space-
flight, which may not actually cause disease or an unfavorable state but may
increase its probability. There are many risk factors in long-term flight, but it is not
always easy to identi@them. Some that are known are listed in Table 4.
D. Contingency-RelatedStates
The most dangerous states, which require urgent diagnosis and medical aid, may
arise as a result of the following emergency situations:20
Table 4. Health Risk Factors in Long-Term Spaceflights

Continuous exposure to weightlessness
Inadequate use of countermeasures
Abnormal resvwork cycle
Insufficient or defective sleep
Non-physiological deviations in gas and microclimate parameters
Increased microbial levels in body tegrnental tissues and station interior
Inadmissible contaminant and particle levels in station atmosphere
Abnormal nutrition regime and diet with development of intestinal
dysbacteriosis
Prolonged stressful situations
partial depressurization of a spacecraft or one of its modules, leading to: acute

hypoxia, boiling syndrome, decompression sickness, aero-otitis and aero-
sinusitis;
0 failure of life support systems, leading to hypoxia, hyper- or hypothermia,or
carbon dioxide poisoning;
0 micrometeorite bombardment, leading to bums, mechanical injuries,traumas;
0 spacecraft contamination by solid or liquid particles, leading to mechanical
or chemical effects;
0 acute radiation damage.
IV. CURRENT PRACTICE IN PROLONGED MISSIONS

A. Criteria for Selection of Physiological Parameters
A review of the medical information accumulated from past manned space

missions made it possible to enunciatethe key principles for selectingphysiological
parameters for inflight medical monitoring. In addition, it was necessary to select
suitable methods for non-invasive fbnction testing and for data processing, without
neglecting other sources of useful information. All this is needed to provide an
adequate system for the diagnosis of deviations in the health of crew members
during flight.'.22' The selected parameters and tests must be experimentally and
clinically proven, whether existing or specially designed.
The functional tests must allow to:
0 diagnose the main body conditions associated with standard or contingency
flight situations and with the diseases that can be expected to occur;
0 evaluatethe dynamics of physiological parameters and of controllingsystems;
0 predict potential developments in the body status as a fbnction of flight
duration, and detect asymptomatic diseases
0 determine the effectiveness of inflight countermeasures;
0 assess data of medical examinations on board the spacecraft on-line on Earth

by means of telemetry;
0 diagnose and predict changes in the main physiological functions and per-
formance of crew members during flight;
0 differentiatebetween physiological states corresponding to the current envi-
ronmental situation and pathological states resulting from the inability of
defensive and adaptive mechanisms to maintain dynamic balance between
body and environment;
0 differentiatebetween specific and non-specific manifestations of responses;
0 differentiatebetween defensive, adaptive or compensatory events and patho-
logical manifestations (‘physiological measure against diseases’ in Pavlov’s
terminology).
It must be admitted that at present it is not possible to select and utilize methods
which are sufficient to diagnose all conditionsor diseases in spaceflight.Therefore,
it is important to ensure diagnosis of the most likely inflight conditions (syn-
dromes), and prediction of the probable diseases. Yet, there remains the possibility
of unpredictable conditions or diseases.
The medical monitoring system should be designed detect expected conditions
and diseases. The system should permit periodical medical examinations of the vital
body systems, specifically cardiorespiratory system, visceral organs (including
biochemical investigations), thermoregulation, neurophysiological status, and
work efficiency. The examination should include evaluation of the key regulatory
processes by means of functional tests and assay of the main endocrine indices.
B. Medical Monitoringand Extensive Examination

The main components of the health care system used for long-term Russian
spaceflights are listed in Table 5 . Medical monitoring in long-term flight includes:
a. on-line medical monitoring during active flight phases and extravehicular activity
(EVA); b. on-line medical monitoring and periodical profound medical examina-
tion as scheduled or when necessary (Figure 1).
On-line Medical Monitoring
During insertion into orbit and return to Earth as well as during docking,
re-docking and EVA the on-line medical monitoring on the station is conducted
through registration of heart rate and electrocardiograms in bipolar chest lead DS
(ECGDS) with the use of other sources of information shown in Figure 2.
Additionally, during EVA spacesuit parameters are measured for subsequent
calculation of the body thermal status. Medical examinations are carried out in the
period prior to an extravehicular activity and immediately before egress to deter-
mine cosmonaut physical fitness, as shown in Table 6.3”6
Table 5. Components of Health Care System for Long-term Spaceflights

Somatic state
medical diagnostic subsystem
subsystem for medical monitoring of life/work conditions
countermeasure subsystem
medical service subsystem
Psychological state
psychological diagnostic subsystem
subsystem of the group activity control
psychological support subsystem
medicinal aid subsvstem
Source: Reference 3
Analysis of cosmonauts' health reports and subjective sensations, video obser-

vations, and telemetric data on heart rate, ECG, respiratory rate and temperature
are simultaneouslydisplayed on the station and in the space control center (medical
control panel). This permits on-line assessment of the crew members' health status.
Permissible limits are set for each of the tested parameters, e.g., body temperature
is termed normal in a range of 34,5-37,5OC with an optimal value of 36 f 0,5"C.
Any deviation from these limits may lead to temporal termination or reduction of
work load, changes in thermal management, or other actions.
Thermal balance is identified on the basis of data about metabolic rate and heat
removed by the liquid cooling garment (LCG).34335 The latter value is calculated
from water flow per time unit and temperature difference between inlet and outlet
of LCG. The amount of water required in the thermal control system is estimated
INCIDENTAL
MEDICAL
MONITORING
EXTRAVEHICULAR WHEN IT IS
ACTIVITY NECESSARY
Figure 1. Medical monitoring in space flight

OPERATIONAL MEDICAL MONITORING
ACTIVE FLIGHT EXTRAVEHICULAR
REGISTERED REGISTERED
PARAMETERS PARAMETERS
Figure 2. Parameters of the medical monitoring program
from preflight tests of the spacesuit, and water flow can be controlled by the
cosmonaut by means of a compact tester. The temperature gradient is registered via
telemetry. Heat removed by ventilating air is quantified in terms of ventilation
expenditure, temperature difference and gas humidity at the inlet and outlet of the
heat exchanger.Along with subjectivesensationsofthe space crew, these data allow
a quantitative assessment of the performance of the thermal control system.
On-line medical monitoring includes permanent subjective assessment of health
by crew members and environmental control and supervision by controllers on
Table 6. Medical Examinations Before and During Extravehicular Activity (EVA)

Before EVA
Evaluation of physical fitness:
Manual veloergometer test, 8-1 2 days before event
Functional loading on leg veloergometer, 6-8 days before event
ECGDS at rest, arterial pressure, body temperature on day of EVA
Health reports (talk with physician) on day of EVA
During EVA
Physiological monitoring:
Telemetric recording of ECGDS, pneumogram and temperature
Analysis of health reports
Spacesuit monitoring:
Telemetric recording of pressure and temperature in spacesuit
Pressure changes in 0 2 tank
C 0 2 concentrations
Inlet and outlet water temperatures in liquid cooling garment
Metabolic rates calculated from 0 2 uptake and C 0 2 release
b DAILY MEDICAL EXAMINATION
IEVALUATION OF
SUBJECTIVE
IAND OBJECTIVE
I MONITORING OF
ENVIRONMENTAL
PARAMETERS AND
STATUS OF OBSERVATIONS BY
THE CREWMEMBERS EXPERTS
MORNING HEALTH CONTROL OF THE MAIN

REPORT LIFE SUPPORT
PARAMETERS
STATUS REPORT RADIATION MONITORING

ACCORDING CONTROL OF THE
TO THE MEDICAL WORWREST CYCLE
QUESTIONNAIRE
EVALUATION OF
RADIOCOMMUNICATI PSYCHONEUROGICAL
WITH THE FLIGHT STATUS BASED
SURGEON ON THE ANALYSIS OF
RADlOCOMMUNlCATlONS
WITH THE SPACE CENTRE
CONTROLLERS
Figure 3. Daily medical examination
Earth. Evaluation of subjective and objective status of crew members includes

morning health reports, status reports through the medical questionnaire, and
confidential radiocommunications with the flight surgeon. The main life support
parameters (gas and microclimate parameters in the spacecraft modules) are con-
trolled by experts on the basis of monitoring of environmental parameters and
observations. Additionally, there is radiation monitoring, control of the workhest
cycle, and evaluation of neuropsychological status based on the analysis of radio-
communicationswith the space control center (Figure 3).
The program of medical monitoring in long-term flights on board the ‘Mir’
station comprises the following investigations:
0 anthropometric measurements;
0 cardiovascular investigations at rest and during functional tests;
0 clinical and biochemical analyses of blood and urine by means of the Reflo-
tron device and reagent strips (some flights);
0 evaluation of microbial contamination of the internal surfaces of the station
(2 days before the end of flight).
These medical diagnostic examinationsare also a source of scientific information
and an integral part of the Medical Research Program. Conversely, results of
medical examinations, which are not included in the regular medical monitoring
program, are used for diagnostic purposes.28
Cardiovascular System
Cardiovascular monitoring is carried out with the onboard stationary medical

equipment. Data records are transmitted to Earth in the analog form via radiotele-
metry. Examinations are performed which include simultaneous recording of six
parameters in various combinationsat rest and during functional tests with graded
physical load (veloergometry) and lower body negative pressure (Table 7).
Extensive cardiovascular examinations by Holter-monitoring and echocardio-
graphic investigations (Table 8) have been carried out with autonomous equip-
ment.30Measurements were transmitted during video contacts or, if a video contact
was too short, registered by the video tape-recorder and then transmitted to Earth
at a later occasion.
Table 7. Periodic Extensive Medical Examination with Onboard Equipment

Myocardial bioelectric activity
Parameter. ECG (1 2 routine leads) Interval: 1.5-2 months
Cardiac cycle and peripheral circulation at rest
Parameters: thigh sphygmogram (SPG)
radial artery SPG; kinetocardiogram (KCG) of right and left cardiac cavities; leg SPG; ECGl
Circulation at rest
Parameters: venous-arterial pulsogram; ECCDS; rheoencephalogram (REG) of displacement
(WRo) and velocity (Wdt) in the bimastoidal lead; rheogram (R/Ro) in the arm-arm lead;
tachyoscillogram; pressure in the cuff
hterval: 1.5-2 months
Circulation during lower body negative pressure (LBNP)
Procedure: -25 mmHg for 1 min; -35 mmHg for 3 min; -45 mmHg for 3 rnin in pneu-
movacuum suit
Parameters: venous-arterial pulsogram; ECGDS; rheoencephalogram (REG) of displacement
(WRo) and velocity (R/dt) in bimastoidal leads; tachyoscillogram; pressure in the cuff; pres-
sure in the pneumovacuum suit
Interval: 2-3 months and before LBNP training
Circulation during functional test with leg ergometer
Procedure: 125 W for 5 min, 1 min rest; 175 W for 3 min or 125, 150 and 175 W for 3 min
each without rest
Parameters: venous-arterial pulsogram; ECGDS; temporal pulsogram; rheogram of displace-
ment in the arm-arm lead tachyoscillogram; pressure in the cuff; physical loading
Interval: 1.5-2 months and a few days before EVA
Circulation during functional test with manual ergometer (acc. to Kozlovskaya)
Procedure: manual pedalling at 150 W as long as possible
Parameters: ECGDS; blood pressure; loading
Interval: 8-1 2 days before EVA
Table 8. Periodic Extensive Cardiovascular Examination

Monitoring of bioelectrical cardiac activity (Hotter-monitoring)
Test equipment cardiorecorder
Parameter. 24-hour ECG recording from two leads
Echocardiographic examination (onedimensional heart echography)
Test equipment echocardiograph ‘Argument’; video tape-recorder
Parameters: left ventricle dimensions; myocardiac contractile function
Muscular System
Periodic examinations of the state of the muscular system are made, which
include measurements of the body mass and the leg volume, as detailed in Table
9.31
Biochemical Parameters
Biochemical and immunological investigations of blood, supervised by I.V.

Konstantinova,B.V. Morukov and V.B. Noskov, have been implemented only in a
few Mir missions. The Reflotron system, based on the dry chemistry technique, and
reagent strips for semiquantitativeurine analysis were employed (Table 10).
The Microvzor system, which integrates a microscope with an onboard TV
transmitter;* was used by the flight-physician to conduct blood analyses of the
crew members of the 3rd and 4th resident expeditions (Table 11). The system
permitted to perform blood counts, and to investigate morphological properties of
erythrocytes both by the flight-physician and in ground-based facilities where
video-taped microscopicpictures of blood smears were analyzed. During long-term
Mir missions a complete blood count was performed 2-3 times per flight. The
results were recorded in the flight log, and transmitted to Earth during the next radio
contact.
Table 9. Periodic Examination of Muscles

Body mass measurements
Test equipment mass-meter
Parameters: inherent oscillation period of mobile part of device; total mass oscillation period
(cosmonaut plus mobile part)
Interval: 1 month
Leg volume measurements
Test equipment individual leg volumemeter
Parameters: leg parameters on specified levels
Interval: 1 month
Table 10. Biochemical Analysis of Blood and Urine

Blood analysis
Equipment: Reflotron system (dry chemistry technique)
Parameters:
protein metabolism: protein, creatinine, urea, uric acid
lipid metabolism: cholesterol, triglycerides
carbohydrate metabolism: glucose
liver function: total bilirubin, aspartate-aminotransferase, alanine aminotransferase
amylase, hemoglobin
Interval: 2 months .
immunoglobulins A, M, and C (in blood serum), from diameters of opacity rings
Interval: on indication
Urine analysis (semi-quantitative)
Equipment: reagent strips
Parameters: pH, leucocytes, protein, blood, nitrites, ketone bodies, glucose, bilirubin, uro-
bilinogen
Interval: 2 months
Environmental Parameters
In addition to the physiological and clinical examinations,sanitary hygienic tests

have been made to monitor environmentalparameters ofthe habitat modules. These
included evaluation of cosmonauts’auditory function, noise levels, automicroflora
compositions, microbial contaminationof the spacecraft interior, trace contaminant
gases in the habitat atmosphere, and assessment of processes related to the forma-
tion of these microadmixtures.
C. Statistical Methods in Diagnostic Data Processing
This section summarizes potential areas for the utilization of some multivariate
statistical methods to analyze data of medical diagnostic examinations during
long-term manned mission^.^'-^^ Some of the approaches discussed below have
been described by Egorov et al.29
Table 11. Hematological Investigations in Long-Term Mir Missions

Equipment: Microvzor system incl. microscope, video taperecorder, micropipets for blood
sampling, slides, reagents
Parameters: blood count, hemoglobin, erythrocyte morphology
Interval: 2-3 times per flight
Equipment: M-1100 unit, N-Hematocrit packing
Parameter: hematocrit
Intervakon day 1 of flight, monthly thereafter
184 A.I. GRlCORlEV and A.D. ECOROV
Individual and Group Physiological Standards
Concurrently measured physiological parameters can be referred to as the pa-

rameter vector that enables the application of multivariate statistical methods for
data processing. These methods permit obtaining integrated characteristics of
interconnected physiological parameters in the shape of multivariate confidence
limits (ellipsoids) to describe norms for individuals (for each crew member) or a
group (for a spacecrew), and to draw up integrated characteristicsas values of the
average vector and covariant matrix of the pre-, in- and post-flight physiological
parameters.
Multivariate Multifactorial Analysis of Variance
The methods allow to determine the effects of the qualitative factors on the
multitude of parameters studied (the parameter vector) and the magnitude of
differences among vectors at various flight stages.
Multi- or Monodimensional Analysis of Covariance
This method allows to delineate relationships between changes in a parameter

(parameter vector) and qualitative and quantitative factors (including environ-
mental parameters), and to express this relationship in an equation. Moreover, the
so-calledreduced mean values, which do not depend on the microclimate parameter
as this is excluded by covariance analysis, can be calculated and their dynamics
studied.
Reduction of the Vector Dimension
Factor analysis incorporates methods and models which permit the compact
display of empiric information and to express n measured parameters as linear
combinations of m general (unknown) factors, where m < n. These linear combi-
nations can be interpreted as new important characteristics of the conditions under
study. In this event the role of factors responsible for psychological disturbances
can also be determined. It should be born in mind that shifts in psychological
parameters during extended spaceflight cannot be measured. However, the method
of expert assessment allows to range them, to a certain degree of probability, in
numbers and thus include these parameters into the model of statistical analysis.
Rao’s criterion of additional information permits to determine significance of one
or several parameters in the multitude of physiological parameters and to decrease
the number of parameters analyzed (dimensionality of the vector) in case their
contributions in the multitude of parameters is in~ignificant.~’
The essence of the principal component method consists in obtaining linear
combinations of the baseline parameters with the largest dispersion (reduction of
the number of parameters) for integral evaluation of effects of flight factors or

provocative tests at various mission stages.23
Smoothing of the Mean Parameter Values
The mean parameter values can be smoothed for multidimensionality of the

principal components by using the smallest square method. In this way tendencies
of changes of the parameter or linear combinationswith the largest dispersio-he
principal components depending on flight duration are objectively studied.
Methods of Correlation
The method of paired and multiple correlations and regressions consists in the
establishment of a linear relationship between pairs of parameters and/or between
one parameter and a multitude of others. The correlation ratio and index allow to
’
establish a non-linear relationship between pairs of parameter^.^'.^ The method of
canonical correlation is intended to delineate relationships among multitudes
(vectors) of physiological parameters, e.g., multitudes of parameters of electrical
and physiological hnctions of the heart.
Cluster Analysis and Identification
The cluster analysis is used to identify groups or clusters of identical elements in

the multitude of elements to classify multitudes of physiological parameters (vec-
tors) that are specific for a body system or for the body as a whole. The method of
identification allows to refer, on the basis of measurements of parameters, charac-
terizing a specific system or a number of systems, a new individual (a candidate
cosmonaut) to one of the groups defined during an earlier classification.The areas
of application of these methods include:
0 assignment of cosmonauts to groups with similar individual reactivity, and

establishment of relationships between changes of responses during the flight
and the readaptation period and the individual pre-flight reactivity;
0 assignment of cosmonauts with flight experience to groups on the basis of the
‘image’(the parameter vector) of a body system or individual responsiveness
on the whole, and identification of crew members assigned to a flight with
one of the classification groups in order to give a preliminary prognosis for
their flight tolerance;
0 correction of the preflight prognosis according to the results of inflight
examination and of the group to which the subject belongs.
Proceedingfrom the above, a series of statisticaloperationsis carried out to analyze

the diagnostic data (Table 12).
186 A.I. GRlGORlEV and A.D. ECOROV
Table 12. Sequence of Statistical Treatment of Diagnostic Data

1. Calculation of confidence limits to compare mean vector values with confidence limits of
a group of healthy subjects or of an individual crew member.
2. If confidence limits from inflight and ground-based data coincide, analysis is finished.
3. Comparison of confidence limits for physiological parameters recorded at various flight
stages with individual and group norms, and with each other.
4. Identification of differences between stochastic pre- and inflight physiological parameters
by means of Hotelling’s T2-statistics or multidimensional analysis of variance.
5. Assessment of contributions of quantitative factors (on board atmospheric composition,
temperature, humidity, etc.) by multidimensional analysis of covariance. Calculation of
the vector of reduced average values, which exclude variability associated with the
factors studied.
6. Reduction of dimension of physiological parameter vector and calculation of main
components with largest dispersions.
7. Determination of trends of main component changes by smoothing through application
of least-square method.
8. On-line evaluation and prognosis of body system conditions by means of classification
and identification methods.
Results of the statistical analysis will afford grounds for a more thorough
assessment of the health status of the space crew and for the acquisition of more
detailed information on what causes the dynamics of physiological parameters and
how these parameters would tend to change with increasing length of flight.
V. MEDICAL MONITORING IN INTERPLANETARY FLIGHTS

A. lnterplanetary vs. Orbital Flight
The conditions of medical monitoring in orbital and interplanetary space flights

differ ~ubstantially.~
Orbital Flight
The main features of orbital flights are:

0 remote telemetric recording of medical information, radioexchange and re-
ception of required recommendations in real time,
0 the possibility, if necessary, to return the space crew rapidly to Earth or to
return an injured crew member for admission to a hospital.
lnterplanetary Flight
These missions are characterized by:

0 a unique flight length of at least 2 years;
0 effects of G-loads during a descent to and a launch from the Martian surface
and on return to Earth on the body unconditioned by an extremely long stay
in microgravity;
0 a potential risk of exposure to heavy ions and other components of the galactic
cosmic rays due to inadequate radiation protection or unscheduled situations;
0 long-term habitation in confinement and isolation with an artificial microcli-
mate, an unusual microbial flora and the possible accumulation of biological
and chemical trace contaminants in the air to which sensitivity may be
developed;
0 possible changes in gas composition and physical parameters of artificial
atmospheres of the spacecraft or spacesuit which may violate comfort and
safety limits through failure or malfunction of the life support systems, faulty
design or unjustified requirements to standards;
0 an increase of the space crew;
0 inclusion in the crew of middle-aged and elderly persons with possible health
problems and decreased adaptive potential, resulting from the necessity to
enlist experiencedpayload specialistsand spacecraft control experts who have
special experience in spaceflight or in some specific research discipline;
0 risks of alterations in the reactivity and neuropsychological status of the space
crew, and development of psychoemotional stress under conditions of an
interplanetary mission and life aboard the space vehicle.
B. Mission to Mars
The most important stress factors in an expedition to Mars are likely to be:
0 the prolonged isolation and confinement, and the absence of direct contacts
with Earth;
0 the communicationproblems due to the impossibility of real-time information
exchange between the spacecraft and Earth and bilateral direct-mode commu-
nications, as a signal may be delayed by more than 20 min;
the impossibility of speedy return of the space crew or an ill person to Earth
and of replacement of an evacuated crew member;
0 psychological incompatibility may develop due to the necessity for life,
intercourse and joint work of the crew members in isolation and confinement
for a very long period;
0 sleep disturbances, anxiety, agitation, depression, irritability, and impaired
spatial perception may develop.
During a stay on Mars humans are likely to be influenced by such factors as:
0 hypogravity of 0.38 G;
0 hypokinesia due to the small size of the lander;
0 extended extravehicular activities under 0.38 G, involving intensive physical

loads against inevitable deconditioning as a result of long-term microgravity,
reduced body adaptability and altered reactivity;
0 fairly long and repeated hyperoxic exposure during extravehicular activities;
0 possible unfavorable effects of Martian physical factors on the skin and the
mucosal endomicroflora;
0 the possible existence of Martian microorganisms that differ from the terres-
trial species and thus may impose undesirable effects upon exposure;
0 psycho-emotional stress due to the contact with an unknown world of another
planet.
The following principles, derived from the experience of medical support during
past spaceflights, can provide a basis for a system of health monitoring and
diagnosis during long-term and interplanetary missions:
0 a system of preflight medical screening;

0 medical screening on a systemic basis, which may include purposeful diag-
nosis in subsystems following the method of hierarchic structure;
0 use of an individual approach;
0 correction of the medical program with respect to the space crew status;
0 assessment of the interrelations of the entire complex of parameters;
0 utilization of the methods of correlation, classification and identification to
elicit interrelations between different functions;
0 evaluation of shifts in body functions and their adequacy to ambient condi-
tions
0 continuity of medical examinations during all pre-flight stages, during flight
and after completion of flight;
0 analysis of information and anamnestic data by means of data bases;
0 confidentiality of medical conclusions.
Discussed are a classification of unfavorable microgravity-related syndromes,

possible impairments due to abnormal situations, and some approaches to the
prediction of the risk of various diseases in relation to the construction of a
conceptual diagnostic model for interplanetary missions.
In the interest of medical monitoring special significance is attributed to the
knowledge of individual norms for each crew member and of his unique peculiari-
ties. Such data can be compiled by means of statistical analysis (single and
multidimensional analysis) of the results of medical examinations and of observa-
tions during selection, training and tests.
Selection of the necessary physiological parameters, functional loads and data

processing techniques which can be used in combination with other data sources
for inflight diagnosis should be based on the following principles:
the use of informative, non-invasively registered parameters and functional

tests to reveal adverse states or most probable diseases;
the possibility to assess the dynamics of physiological parameters and the
status of the regulatory systems, and to predict possible developments in the
body;
the possibility to check the efficiency of countermeasures
the possibility to differentiate a physiological state adapted to the current
environment from a pathological state;
the possibility to differentiate between specific and non-specific reactions;
the possibility to differentiate defensive, adaptive or compensatory phenom-
ena from pathological manifestations.
This paper describes the application of single- and multidimensional, statistical

methods to process diagnostic information, to reduce the vector dimension of the
chosen parameters, and to classify and identify individual and crew physiological
standards providing the ability to assign an individual to a suitable team. Thus it
will be possible to acquire comprehensive and statisticallyreliable information in
compact format, and thus to perform a more incisive analysis and diagnosis of
various states by comparingthem with the baseline preflight data. These theoretical
considerationsconstitute the basis for a conceptual model of medical diagnosis in
long-term orbital and interplanetary missions.
The paper contains a section dealing with the practical aspects of medical
monitoring and diagnostic examinations during Mir missions. The program of
medical monitoring for long-term Mir missions incorporates on-line monitoring
during intravehicular and extravehicular activities, routine daily examinations,
periodic extensive medical examinations accomplished on schedule or on indica-
tion. The Mir program includes cardiovascular investigations at rest and during
functional loading, testing of the cosmonaut muscular system, validation of the
standardtraining protocols, occasional blood and urine analysis, incl. serum immu-
noglobulins, and blood cell counts. In addition, environmental parameters of the
station modules are monitored, including assessment of the acoustic analyzer
function, noise levels, automicroflora composition,microbial contaminationof the
spacecraft interior, trace contaminants in the air, and assessment ofpocesses related
to formation of the microbial flora.
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2 1. Nefedov, Yu.G., Egorov, A.D., Kakurin, L.I. Theoretical approachesto selectionof physiological
parameters for medical monitoring in manned spaceflight. Kosmicheshya Biologia i Aviakos-
micheskaya Meditsina, 2(6):47-5, 1968. In Russian.
22. Aivazyan, S.A., Buchstaber, V.M., Yenyukov, I.S., Meshalkin, L.D. Classification and Reduction
of Dimensionality. AppliedStutistics.Reference Edition (S.A. Aivazyan, Ed.) Finansy i Statistika,
Moscow, 1989. In Russian.
23. Anderson, T. Introduction to Multivariant Statistical Analysis. Physmathgis, Moscow, 1963.
Russian translation.
24. Ahrens, H., Laurn, J. Mehrdimensionale Varianzanalyse.Akademie-Verlag. Berlin, 1981. Russian
translation.
25. Kendall, M.G., Stuart. A. Advanced Theory of Statistics. Volume 3. Design and Analysis, and
Time-Series. 2nd edition. Charles Griffin and Co. Ltd, London. Nauka, Moscow, 1976. Russian
translation.
26. Kullback, S. Information Theory and Statistics. Nauka, Moscow, 1967. Russian translation.
27. Rao, C.R. Linear Statistical Methods and Applications. Nauka, Moscow, 1968. Russian transla-
tion.
28. Scheffe. H. Analysis of Yariance. Nauka, Moscow, 1960. Russian translation.
29. Egorov, A.D., Egorov, B.B., Kiselyov, A.A., Shandrinsev, I.S. Problems ofautomation ofoperative
medical control in spaceflight. Kosmicheskaya Biologia i Aviakosmicheskaya Meditsina, 1(2):7-
14. 1967. In Russian.
30. Ezekiel, M., Fox, K.A. Methods qf Correlation and Regression. Mir, Moscow, 1966. Russian
translation.
3 1. Mils, F. Statistical Methods. Mir. Moscow. 1958. Russian translation.
32. Kalandarova, M.P., Polyakov, V.V., Goncharov, I.B. Hematological Parameters of Cosmonauts
during Spaceflights of the 3rd and 4th Main Expeditions Onboard Orbital Station Mir. In: IXth
All-Union Conference on Space Biology and Aerospace Medicine, Kaluga. 19-21 June, 1990.
Abstracts, Moscow-Kaluga. pp. 77-79, 1990. In Russian.
33. Abramov, I.P., Barer. A.S.. Vacar, M.I.. Golovkin, L.G.. Zinchenko, V.P., Fillipenkov, S.N.,
Sharipov, R., Schigolev, V.V. Physiologico-hygienic aspects of cosmonaut performance mainte-
nance in orbital flight. Kosmicheskaya Biologia i Aviakosmicheskaya Meditsina, 16(6):16-22,
1982. In Russian.
34. Barer, A.S., Fillipenkov, S.N. Cosmonaut work in spacesuit. In: Space Biology and Medicine (O.G.
Gazenko, Ed.), p. 146-176. Nauka, Moscow, 1987.
35. Barer, AS., Vacar, M.I., Fillipenkov, S.N.. Schigolev, V.V., Kovalenko, E.A., Kasyan, I.].,
Zinchenko, V.P., Golovkin, L.G., Osipov, Yu. Medical support of cosmonaut work in open space.
In: Physiological Problems of Weightlessness (O.G. Gazenko, Kasyan, L.L., Eds.), pp. 1 7 w 97.
Meditsina, Moscow, 1990.
36. Severin G.I., Abramov, I.P., Barer, AS., Sverschek, V.I. Space Suits. Ten Egresses from the Salyut
7 Station to Open Space. X x y v h IAF Congress. Lausanne, Switzerland, 7-13 October: 1984.
Preprint.
€hapter 8
FROG EXPERIMENT ONBOARD

SPACE STATION MIR
Akem i IzumLKurotani. Yosh ihiro Mogami.

Makoto Okuno. and Masamichi Yamashita
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
I1 . Experimental System and Operation . . . . . . . . . . . . . . . . . . . . . . 194
A . TreeFrog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
B . Transportation of Frogs to Mir . . . . . . . . . . . . . . . . . . . . . . . 195
C . Experiment in Orbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
D. Recovery of Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
E. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
F. Ground Control Experiment . . . . . . . . . . . . . . . . . . . . . . . . 201
111. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
A . Experimentinorbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
B. Control Experierntn on Ground . . . . . . . . . . . . . . . . . . . . . . 208
C. Postflight Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
IV. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

ISBN: 0-7623-0147-3
193
194 IZUMI-KUROTANI et al.
1. INTRODUCTION
Living creatures on earth have evolved in the earthly environment from birth.
Gravity is one of the important parameters in the environment. Behavior of various
animals on this planet is influenced by gravity. Some space experiments have been
aimed at the study of the role of gravity in animal behavior, for instance in the
construction of beeswax combs in a colony of honey bees,' in the mating behavior
of parasitic wasps,' in the web-building behavior of garden cross spiders? in the
swimmingbehavior ofjelly fish' and killifi~h,'.~ in the swimming behavior of larva
and adult of African clawed frog? and in the behavior of space-hatched Japanese
quaii.5
In December 1990, we sent six Japanese tree frogs to the Russian space station
Mir to investigate their posture and behavior in microgravity. Tree frogs present
several types ofbehavior, i.e., walking,climbing,swimmingandjumping. Principal
objective of this space experiment was to study how the frogs would respond to the
condition of weightlessness. When a frog is briefly exposed to microgravity in free
fall (1-2 seconds) or parabolic flight (about 20 seconds)? they arch their back and
stretch out their four limbs. Spaceflight can greatly extend the duration of exposure
to microgravity, in this mission to 8 days.
In this experiment we observed the posture and behavior of Japanese tree frogs
during a prolonged stay in the microgravity environment. Questions studied were,
for example: When frogs try to change their position, do they select any original
mode of behavior in microgravity?; Can they create a new way of locomotion?
When an external stimulus is presented to the animals in orbit, is the stimulus
strength changed from that observed on earth?
II. EXPERIMENTAL SYSTEM AND OPERATION

A. Tree Frog
The Japanese tree frog, Hylujuponicu,is an arboreal animal, i.e., it normally lives
on leaves and twigs of small grasses or shrubs. During the breeding season it appears
in or near the water, such as in a wet rice field. Its fingers have round adhesive discs
and poorly developed webs.'
From a group of about 400 tree frogs, caught in fields in the Kanto area of Japan,
100 healthy specimens were selected for the experiment. One of the criteria for
selection was the ability of the animals to change their body color relatively rapidly
(within 10 minutes; see section III).' Of these animals 18 were transported to the
training site Star City, Russia, in advance and kept there. The remaining 82 animals
were transported to the launch site, 10 days before launch, and were added to the
advance group. The ability to change body color rapidly was rechecked at the launch
site. The age of the animals ranged from 7-1 2 months, and their body weight from
3-1 u
Frog Experiment in Mir 195
Table 1. Selection Criteria for Animals

1 . Absence of visible injuries
2. Health condition expressed by posture
Frogs in poor health often crouch.
3. Normality of vestibular functions
Optokinetic nystagmus:
Head rotation was observed when the stage on which a frog sits turns on dorm-ventral
axis with angular velocity of 20-45 degreedsec.
Gravity response:
Reorientation of head was observed when stage on which frog sits was tilted to 3 0 4 5
degrees from horizontal. Two direction (right-left and anterior-posterior axis) were
tested.
Righting response:
Righting response was observed after a frog was made to lie on its back by an opera-
tor’s hand.
The day before launch 12 frogs (six males and six females) were selected from
the group of 100 animals by means of the criteria listed in Table 1. The 12 frogs
were divided into two groups, six flight animals and six ground control animals.
All animals were starved during the 10-day period preceding launch. This was done
to prevent clogging of the air vents of the Life Support Box with feces. In orbit the
frogs were not fed routinely, but they could eat meal worms, which were brought
to serve as food in the experiment on feeding behavior. The intestinal microflora
of the Japanese tree frog was analyzed in advance to ensure that the safety
requirements for the cosmonauts would be met.’
B. Transportation of Frogs to Mir

A Life Support Box, shown in Figure 1, was used for transportation of the living
specimens to Mir. It travelled on the Soyuz, which stayed in transfer orbit for two
days before docking to Mir. The Life Support Box has a three-layered structure.
The top layer has seven compartments: six for one flog each, and one for 30 meal
worms (living larvae of Tenebrio obscurus) serving as food inflight. The frog
compartment snugly fits a sitting frog and is lined with polyurethane foam, so as
to protect the animal against the vibrations and shocks during launch. The foam is
water-soaked to keep the animal from dehydrating. The compartment for meal
worms is not lined with polyurethane foam and is kept dry. During transportation
the frogs cannot approach the meal worms. Zeolite in the top layer absorbs odor
and urine from the living specimens. Each frog was installed in its compartment no
less than 7 hours before launch.
The middle and bottom layers of the Life Support Box house a small air pump
and air ducts. The small air pump is powered by a Ni-Cd battery. The air ducts are
made of soaked polyphenol foam to keep the air moist for frogs. There are two air
Figure 1. Life Support Box used for transportation of frogs on Soyuz, which stayed in
transfer orbit for two days before docking to Mir.
inlets in the bottom layer and two outlets in the top layer. Membrane filters with
sub-micron pores are present in the air inlets and outlets for the purpose of
bioisolation. Fresh cabin air was drawn into the Life Support Box by the air pump
for 2 days during transportation to Mir. The temperature in the box was passively
kept at the level of the crew cabin.
C. Experiment in Orbit
The Frog Observation System was used for the experiment in orbit in Mir. It is
composed of a glove bag for the animals (Figure 2), a tool bag with two sets of
experiment tools (Figure 3), a CCD camera and an 8 mm videorecorder. It was
shipped to Mir on the unmanned Progress, about 2 months prior to the anival of
the frogs.
Of the two sets of experiment tools, one set was for use inside the glove bag, the
other for use outside the glove bag. A complete list of the two sets of tools is
presented in Table 2. Tools were kept in pockets of the tool bag to prevent their
scattering in weightlessness.
On the day after rendezvous of S o p and Mir (three days after launch = day
L + 3) the frogs were transferred from the Life Support Box to the glove bag. First,
the Life Support Box with the living specimens, and the tool band with experiment
tools to be used inside the glove bag, were placed inside the glove bag. They were
Figure 2. Glove bag for observation and handling of frogs in bioisolation.
fixed on the inner face of the glove bag by Velcro tape. Second, the glove bag was
sealed by a seal clip bar, and inflated by air released from a small air cylinder. A
polyurethane foam on the tool band was soaked with water from a water reservoir
tube to keep the inside moist. Then the frogs were set free in the glove bag for
observation, and were kept in this bag during the entire mission. Frogs and
experiment tools in the glove bag were handled through gloves. The health
condition of the frogs (alive, weakened, or dead) was checked daily by visual
inspection for a few minutes.
From that point observation of the animals was started. The experiment with
observation and recording of the frogs was performed twice, on days L+3 and L+5,
in order to determine whether any form of adaptation occurred. The subjects of
observations are listed in Table 3. On the experiment days the behavior of the frogs
was recorded with a small CCD camera or 8-mm VTR camera by a Japanese
mission operator or a Russian cosmonaut.
D. Recovery of Frogs
AFrog Recovery Box (Figure 4) was installed in the tool band, which was placed
inside the glove bag at the beginning of the experiment. On the last day of the
mission (day L+8) an appropriate volume of water was poured on a calcium
peroxide tablet in the Frog Recovery Box to generate suficient oxygen gas for life
support during the recovery phase. The polyphenol foam covering the interior
198 IZUMI-KUROTANIet al.
Table 2. List of Tools

a. Tools used outside Glove bag
Name of Tool Quantity Use for
Seal clip bar 3 Sealing of glove bag
Hand-powered Air Pump 1 Air withdrawal from glove bag at end of expt.
Air cylinder 1 Inflating glove bag at start of expt.
Pressure regulator and valve 1 Regulation of releasing pressure of air from air
cylinder
Audio cassette tape 1 Voice stimuli (not used)
Supporting band 1 Fixing position of observation window to the
cosmonaut
Camera holder 1 Fixing CCD camera head on observation window
Scissors 1 Cutting off part of glove bag for frog recovery
Mending tape 1 Mending of damage of glove bag (not used)
Inner gloves 1 Absorption of sweat of hands in gloves of glove
bag (not used)
Gloves in exchange 1 Gloves of glove bag for back-up cosmonaut (not
used)
b. Tools used inside Glove bag
Name of Tool Quantity Use for

Water reservoir tube 4 Container of water
Air blower 2 Positioning of frog (not used)
Suction pipette 1 Positioning of frog (not used)
Tweezers 1 Handling of vinegar-immersed filter paper (not
used). Mechanical stimuli (not used) Picking up
meal worm
Scissors 1 General use
Wrench-driver 1 Screwing off of fuse of LSB
Mending tape 1 Mending of damage of gloves (not used)
Vinegar bottle 1 Experiment of chemical stimuli (not used)
Imitative snake 1 Experiment of visual stimuli (not used)
Imitative meal worm 1 Experiment of visual stimuli
Imitative willow sprig 1 Experiment of visual stimuli
Needle (in Needle holder) 1 Euthanasia (not used)
Paper for wipe 3 Wipe of observation window (not used)
Paper for white balance 1 Adjusting white balance (not used)
Burial bag 5 Keeping carcass
Burial box 1 Keeping burial bag with carcass
Polyurethane foam 1 Humidifier
Joint tube 1 Connecting between LSB and outlet of glove bag
to use Zeolite in LSB as deodorizer for gas from
glove bag
Frog recovery box 1 Recovery of frogs
Figure 3. Tool bags with tools used outside (a) and inside (b) glove bag.
surface of the frog compartment in the Frog Recovery Box was soaked with water
to provide moisture for the animal. The six frogs were placed in the individual frog
compartments.
The Frog Recovery Box was removed from the glove bag without breaking
bioisolation by the technique shown in Figure 5 . The box was placed in the entrance
port of the glove bag. The port was parted by two seal clip bars, which were placed
Table 3. Subjects of Observations of Frogs

1. Behavior and posture in microgravity
2. Response behavior to various external visual stimuli
real food (a living meal worm)
small moving object (imitation willow sprig with leaves, imitation worm or tweezers)
large moving object (hand of operator)
3. Orientation behavior on rotating object (water reservoir tube)
4. Ability to change body color
between the box and the body of glove bag. After cutting the space between the two
seal clip bars by a scissors, the Frog Recovery Box was isolated from the glove bag
and encapsulated. Each cut end was wiped with a sterilizing cloth. The encapsulated
Frog Recovery Box was returned to the ground in the Soyuz recovery capsule.
E. Postflight Experiments
Behavior and Vestibular Function
Observation and recording of behavior after recovery were performed in a hall

of the airport closest to the landing site, as early as two hours after landing. The
process of jumping, landing, walking, and climbing a wall by the frogs was
observed and recorded. The vestibular function was also tested. Test items and
procedures were the same as those at the launch site before launch (see Table 1).
These observations were repeated again at 12 hrs after landing.
Figure 4. Frog Recovery Box used for recovery of frogs upon return to Earth.
figure 5. Procedure for isolation of Frog Recovery Box from the glove bag.
Preparation of Organs and Tissues

From 2.5 hours after landing, two spaceflight frogs and two ground-control frogs
were dissected and their organs and tissues were fixed by a procedure suitable for
later analysis. The outline of the entire experimental procedure is diagrammatically
represented in Figure 6 .
F. Ground Control Experiment
Ground-control frogs were kept for about 2.5 days in an identical Life Support
Box as used for the transport of the flight animals. They were then released in an
air-opened box (200W x 125D x 140H mm) at the same time as the flight frogs
were released in the glove bag on the Mir station. Temperatureand humidity in the
LSB
LSB
e
N
SOYUZ
?3b
R LSB : Life Supporl Box 2.5 days -4- h g q-- FRB :Frog Recovery Box lday -1
Mays
e3 @ 1st EXD.L+3 davs

LSB 2nd Ex'p. L+5 da'ys !9 e!B
LSB
FRB FRB Observation Disection
B C CI l>l >
Lafe Access Recovery R+(-lh) R+(-2 hn) R+(-2.5 hrs)

Early Access
Figure 6. Outline of operation of the Frog Experiment.

two containers did not exactly simulate those in the flight Life Support Box and the
glove bag in the Mir station. The control frogs were then placed in a Frog Recovery
Box identical to that used inflight in reserve, and kept there for about 1 day.
Behavior and vestibular function were observed and recorded on days L+3 and
L+5, and after return of the flight animals.
111. RESULTS AND DISCUSSION

A. Experiment in Orbit
Recorded video tapes were handed to us ten days after return (day R+10). The
Japanese mission operator was interviewed about one month after his return. From
the tapes and the interview the following was learned.
Behavior of Floating Frogs
Frogs floating in the air inflight showed a posture similar to that during the few
seconds of microgravity in a parabolic flight or free They arched their back,
inflated their abdomen, and extended the four limbs with opened fingers and toes
(Figure 7). During floating, synchronous movements (like breaststroke swimming
or scissors-kick) and asynchronousmovements of both hind limbs were observed
sometimes. These movements lasted for such a short period that they could not
provide a system of coordinated motility for locomotion. Floating frogs tried to
catch whatever object one of their four limbs touched. The limbs pointed in a rather
Figure 7. Behavior of a frog floating in weightlessness. Arched back, inflated abdo-

men, extended limbs with fingers and toes opened and hind limbs extended in lateral
position.
lateral direction, although the direction was not stable. The typical posture during
floating in microgravity resembled, except for the position of the limbs, the posture
observed during jumping on the ground (Figure 8).
It has been reported that at dawn arboreal sub-tropical forest frogs parachute from
a canopy of rain forest to the ground.lo The parachuting frogs “move their front and
hind limbs lateral to their bodies and spread their fingers and toes during aerial
descent, adjusting limb position slightly during flight....... this lateral position of
the limbs is quite different than that observed during the trajectoryphase of a regular
jump where the forelimbs are anterior and lateral but the hind limbs remain
extended posterior to the body.”” According to this description, the parachuting
posture appears to bear a close resemblance to the floating posture in orbit.
However, it is not known whether the Japanese tree frog makes a retarded descent
like the subtropical forest frog. They may do it in a small way from shrubs or
grasses. Further observation and analysis are required to clarify this point.
This parachuting-like posture in orbit is not a common response to microgravity
among frogs and toads. Recently we have been investigating the behavioral
response of various frogs and toads to an abrupt decrease in gravity in free fall and
parabolic These experiments suggest that the response can be related to
Figure 8. Jumping posture of frog postflight. Frog was made to jump from a platform
(15 mm height) and land on polyurethane sheet. Recorded in darkness by 35-mm
camera with multiple exposures in strobe light (0.1 sec. intervals). Frog extended its
hind limbs backwards during the iump.
their way of life.14In microgravity these frogs and toads, who are non-arboreal and
move two-dimensionally on the ground surface (Rana rugosa, Rana nigromacu-
lata, Xenopus laevis, Lepidobatrachus budgetti and Ceratophryssp.),tend to rotate
around their rostral-caudal axis by means of scissor-like kicks. This long axis
rotation is similar to their righting reflex when the animal is inverted in normal
gravity, while arboreal or sub-arboreal frogs (Hyla japonica and Rachophorus
schlegefii) often show a similar posture during parachuting.
Behavior of Stationary Frogs
Though some frogsjumped off spontaneously,most of them did not move, stayed
on a surface, or hid themselves under some object during the mission. Frogs
frequently stayed on a moist polyurethane foam. Some frogs returned to a compart-
ment in the Life Support Box in the glove bag. When a frog was sitting on a surface,
like the glove bag, they frequently (four out of six frogs) assumed a particular
posture. The neck was sharply bent backwards (at nearly right angle), the back was
arched, the hind limbs were not folded completely, and the abdomen was pressed
against the substrate (Figure 9). In this posture they would walk backwards.
Posture in Microgravity
We have studied what this posture might express. The effects of selective
neurectomy in the labyrinth on motor function has been investigated by several
groups.15-'* This procedure might simulate the behavior experiment in micro-
gravity, since the input of the gravity signal can be prevented by this neurectomy.
Figure 9. Typical posture of a frog sitting on surface of glove bag. Arched back,
incompletely folded hind limbs, abdomen pressed against glove bag.
Figure 10. Posture during emesis induced by CuSO4 solution. 10 pI CuSO4 solution
(0.2 mg per g body weight) was administered by gastric cannula (photograph by Dr.
T. Naitoh).
The posture and behavior observed after neurectomy do not directly explain the
particular posture observed in Mir. Naitoh et aLt9 have reported that emetic
behavior (vomiting and retching: emetic behavior in the absence of any regurgita-
tion) can be induced in frogs by certain drugs. The particular posture in orbit
resembles the posture during retching behavior induced by emetic drugs (Figure
10). We have subjected several species of frogs to parabolic flights in order to test
whether frogs can suffer from motion sicknessby mechanical acceleration.12Some
frogs showed signs of emetic behavior, like cyclical mouth opening and closing,
blinking, and walking backwards during the flight, some frogs actually vomited
after the flight. Thus we have concluded that frogs, including Hyla japonica, can
suffer motion sickness from parabolic flight. Cyclical mouth opening and closing
of the frogs was also observed by the mission operator in Mir.20The frogs showing
the particular posture observed in Mir may thus have been in an emetic state,
possibly due to motion sickness.
Response Behavior
The body color of each frog was observed soon after their release from the Life
Support Box into the glove bag. The color of the polyurethane foam lining of three
compartments in the box was white, of the other three black. On the ground the
frogs respond to the white color by a change of their body color to yellow-green
within 10 min., to the black color by a change to green-black. According to the
report of the mission operator,21one of the frogs from a white compartment turned
dark soon after its release into the glove bag. This observation suggests that the
body color change response to the background color may be inhibited in space. It
was not clear whether the response was only delayed, since the mission operator
made no later report about the body color. Another point is that the body color
change depends on the light intensity, and it was not known whether the light
intensity at the site of the experiment in Mir was sufficient to induce the body color
change. The mission operator did not report about the colors of the frogs that had
been transported in black compartments. When the mission operator brought his
hand to the glove bag, he noticed an avoiding response as usually made from a large
object.
When the mission operator placed a meal worm in front of a frog sitting on the
surface of the glove bag, the frog tried to catch it by a forefoot, but it failed to catch
the worm. This failure seemed to be due to an instability of the footing when the
frog put out its forefoot. When a small (95 mm length) pair of tweezers floated
while rotating along its long axis, one frog looked toward it and oriented its body
to it, however it could not approach the tweezers after jumping off. It seems that
frogs in microgravity succeed in making a response behavior only when they keep
a stable contact with a surface by means of the round adhesive discs on their fingers.
Once they left the surface and floated, they could not control their movements
sufficiently to respond to a stimulus. They could only approach a food object and
eat it by keeping a steady contact with a surface. The hvo frogs, who were dissected
after recovery, were found to have some food (meal worms?) in their stomach,
although the mission operator could not observe eating behavior in orbit. Pre-
viously, it has been reported that space-hatchedJapanese quails could not approach
their food without the help of a c~smonaut.~ Quail chicks might not be able to hold
on to the substrate while moving toward their food.
When a frog rode on a rotating water reservoir tube (220 mm length, 32-50 mm
diameter), it walked along the circumference toward the opposite direction. The
orientation behavior observed on the ground was also shown in orbit, however it is
not clear whether the frogs oriented themselves by a visual standard, or by angular
acceleration sensing, or both.
Adaptation to Microgravity
After a frog jumped off in orbit, it would try to land or perch on some object, but
often failed to do so. The frequency of failure to land or perch after ajump decreased
with time: from an average of 2.7 random contacts with an object or surface per
jump before proper landing on day L+3 to an average of 1.4 contacts per jump
before landing on day L+5. This finding suggests that frogs can adapt to micro-
gravity in the matter of landing after a jump. However, the typical posture while
sitting on a surface and the parachuting-like posture during floating were main-
tained until the end of the experiment.
Adaptation to microgravity in the behavior of other animals has been reported.
A cross spider could build a better web after 3 weeks in microgravity than in the
208 IZUMI-KUROTANIet al.
beginning of its stay in orbit.2 The frequency of looping behavior of killifish in

microgravity gradually decreased after staying in orbit for several days.'33
B. Control Experiment on Ground
When the ground-control frogs were released from the Life Support Box or the
Frog Recovery Box, their walking, climbing, swimming and jumping behavior and
vestibular function (optokinetic nystagmus, gravity response and righting response)
were normal.
C. Postflight Experiments
Behavior of Recovered Frogs
All six frogs were recovered and returned alive after eight days of spaceflight.
Upon the first observation of the animals, two hours after return (R+2 hrs.), all six
frogs walked slowly and climbed a wall toitteringly. After landing from a jump, the
folding of the hind limbs was delayed. Vestibular function did not appear clearly.
Half an hour later (R+2.5 hrs.), their behavior and vestibular function began to
return to normal. At R+12 hrs. behavior and vestibular function were normal again.
Abnormal swimming behavior (looping) in Xenopw larvae has been reported to
continue for 1-2 days after their recovery from a 7-day pacef flight.^ We cannot
conclude that readaptation to 1-G condition in adult tree frogs was faster, since there
are few reports about the behavior of adult frogs and toads after recovery from
spaceflight.
Analysis of Organs and Tissues
The organs and tissues of recovered and ground-control specimens were distrib-
uted to several research groups for a histological and biochemical analysis of the
effect of an 8-day spaceflight. Their findings are summarized below.
Kashima et al. observed the weakening of a vertebral bone.22The spongy bone
in the caudal articulatio vertebra was less dense in the spaceflight animals than in
the ground-controls.In a quantitative analysis by means of a bio-imaging analyzer
they found 20% less calcium density in the posterior joint of the seventh vertebra
of a spaceflight frog.
Ohira and coworkers found a decrease in the P-adrenoreceptor activity, which is
thought to correspond to mitochondria1biogenesis, in the gastrocnemiusof space-
flight frogs.23
Yamazaki and coworkers found a decreased collagen content in the skin and a
lowered protein synthesis in the liver of spaceflight
Suzuki and coworkers studied the morphology of the vestibular sensory epithe-
lium by means of scanning electronmicroscopyand light micro~copy.~~ No changes
were found in the sensory cilia of the utricular macula and the semicircular canal
cristae in spaceflight frogs.
Feuilloley and coworkers examined in heart and brain of spaceflight frogs the
distribution of atrial natriuretic factor (ANF)-like peptides, which regulate water
and electrolyte balance and blood pressure.26327 The density and distribution of the
staining were identical in the hearts of control and spaceflight frogs. In the
ground-control frogs ANF-positive cell bodies were found in the parium and
striatum of the telencephalon, the lateral forebrain bundle of the diencephalon, and
the nucleus reticularis isthmi in the mesencephalon. These were absent in the
spaceflight frogs. Conversely, ANF-immunoreactivity was observed in the poste-
rior nuclei of the posterolateralis thalamus in spaceflight frogs, but in the ground-
control frogs these nuclei were scarcely stained. The authors suggested that
prolonged exposure to microgravity affects biosynthesis and/or release of ANF-
related peptides in discrete regions of the frog brain.
IV. CONCLUSIONS AND SUMMARY

Japanese tree frogs (Hylajaponica) showed unique postures and behavior during
an 8-day flight on the Russian space station Mir. When floating in the air, the
animals arched their back and extended their four limbs. This posture resembles
that observed during jumping or parachuting of the animals on the ground.
Frogs sitting on a surface bent their neck backward sharply, did not fold their
hind limbs completely, and pressed their abdomen against the substrate. They
walked backwards in this posture. This typical posture resembles that adopted
during the emetic behavior process on the ground, although the posture in space
lasts much longer. The possible mechanism of induction of this unique posture in
orbit is discussed. Frogs in this posture might be in an emetic state, possibly due to
motion sickness.
Response behavior to some external stimuli was observed in orbit. Body color
change in response to the background color appeared to be delayed or slowed down.
Response behavior to other stimuli showed little change as long as the animal
maintained contact with a substrate. Once it left the surface, the floating frog could
not control its movements so as to provide coordinated motility for locomotion and
orientation.
Adaptation to microgravity was observed in the landing behavior after jumping.
Readaptation of the frogs to the Earth environment took place within a few hours
after return. Postflight histological and biochemical analysis of organs and tissues
showed some changes after the 8-day spaceflight. Weakening and density loss in
vertebrae was noted. The P-adrenoreceptor activity of the gastrocnemius was
decreased. Skin collagen and liver protein synthesis were lowered. The distribution
of the atrial natriuretic factor-like peptides in the brain was changed.
ACKNOWLEDGMENTS
We thank the persons i n Japan and Russia, whose efforts made the “Frog I n Space” project
possible. A partial list of names is provided in reference 29. T h e extended studies on the
postflight behavior of frogs and toads would not have been possible without the cooperation
of Dr. R.J. Wassersug and Dr. T. Naitoh.
REFERENCES
1. Stark, R.E. Ethology in Space, a Unique Opporfunity for Behavioral Science. ESA STM-246,
ESA, Paris, 1993.
2. von Borstel, R.C., Smith, R.H., Whiting, A.R., Grosch, D.S. Mutational and physiologic responses
of Habrobracon in Biosatellite 11. In: The Experiments of Biosatellite 11 (J.F. Sanders, Ed.), pp.
17-39. NASA SP-204, NASA, Washington, D.C., 1971.
3. Scheld, H.W. Baky, A., Boyd,J.F.,Eichler,V.B.,Fuller,P.M.,Hofhan,R.B., Keef, J.R., Kuchnow,
K.P., Oppenheimer, G.M., Salinas, G.A., von Baumgarten, R.J. Killifish hatching and orientation.
In: Apollo-Soyw Test Project Surnmaiy Science Report (NASA Editorial Review Board), Vol. I ,
pp. 281-305. NASA SP-412, NASA, Washington, D.C., 1977.
4. Briegleb, W., Neubert, J., Schatz, A., Klein, T., Kruse, B. Survey of the vestibulium and behavior
of Xenopus laevis larvae developed during a 7-day spaceflight. Advances in Space Research,
6(12):151-156, 1986.
5. The quails that flew in space In: USSR Space Life Sciences Digest, issue 30 (L.R. Stone, R. Teeter,
J. Rowe, Eds.), pp. 37-40. NASA CR-3922(36) NASA, Washington, D.C., 1991.
6. Izumi-Kurotani, A., Yamashita, M., Kawasaki, Y., Mogami, Y., Okuno, M., Oketa, A. Behavior of
tree frogs under microgravify. Biological Sciences in Space, 5(3):185-1 89, 1990.
7. Maeda, N., Matsui, M. Frogs and Toads of Japan, Bun-Ichi Sogo Shuppan, Co., Tokyo, 1989
(Japanese).
8. Akimoto, M., Kawamura, K., Namiki, H., Kikuyama, S. Evaluation ofadaptability of“space frog”
candidates to background color. Biological Sciences in Space, 5(3):212-2 14, 1991.
9. Benno, Y., Izumi-Kurotani, A,, Yamashita, M. Intestinal microflora ofjapanese tree frog (Hyla
japonica). Biological Sciences in Space, S(3):182-1 84, 1991.
10. Stewart, M.M. Arboreal habitat use and parachuting by a subtropical forest frog. Journal of
Herpetology, 19(3):391-40 1, 1985.
11. Emerson, S.B., Koehl, M.A.R. The interaction of behavioral and morphological change in the
evolution of a novel locomotor type:“flying” frogs. Evolution, 44(8):193 1-1946. 1990.
12. Wassersug, R.J., Izumi-Kurotani, A., Yamashita, M., Naitoh, T.Motion sickness in amphibians.
Behavioral and Neural Biology, 60:42-5 1, 1993.
13. Izumi-Kurotani, A., Wassersug, R.J., Yamashita, M., Naitoh, T., Nagaoka, S. Frog Behavior under
Microgravit)cPossibility of Motion Sickness in Amphibia, Proceedings of rhe 9rh ISAS Space
UtilizationSymposium:1 12-1 14, 1992.
14. Wassersug, R.J., Pronych, S., Izumi-Kurotani, A., Fejtek, M. The Behavioral Responses of
Vertebrates to Microgravity: A Comparative Approach. Proceedings of Space Bound ’93:73-74,
1993.
15. McNally, W.J., Tait, J. Ablation experiments on the labyrinth of the frog. American Journal of
Physiology, 75:155179, 1925.
16. Gray, J., Lissrnan, H.W. The effect of labyrinthectomy on the coordination of limb movements in
the toad. Journal of Experimental Biology, 24:34-40, 1947.
17. Suzuki, M., Harada, Y., Omura, R., Fujii, M., Hirokawa, H., Mori, N. Effect ofselectivevestibular
neurectomy on frog motor function. Otologia Fukuoka, 36(2):25%263, 1990 (Japanese).
Frog Experiment in Mir 21 1
18. Ikeda, T., Sekitani,T., Kido,T., Kanaya, K., Tahara, T., Hara, H. Study on EquilibriumoftheSmall
Animal Using Drop Shafi-Usability of the Frog for the Vestibular Neurectomy and Experimental
maintenance-Proceedings of the Ninth ISAS Space Utilization Symposium: 9W02, 1993.
19. Naitoh, T., Wassersug, R.J., Leslie, R.A. The physiology, morphology, and ontogeny of emetic
behavior in anuran amphibians. Physiological Zoology, 62(3):81W 4 3 , 1989.
20. Akiyama, T. Interview one month after the recovery, 1991.
21. Akiyama, T. Report on the third day in space, 1990.
22. Kashima, I., Nishimura, K., Okamoto, Y., Kanno, M. Image analysis of bone changes in Hyla
japonica exposed to microgravity on the MIR orbital station. Biological Sciences in Space,
S(3):190-193, 1991.
23. Ohira, Y., Wakatsuki, T., Saito, K., Kuroda. A., Tanaka, H., Izumi-Kurotani, A,, Yamashita, M.
Responses of P-adrenoreceptors in frog and rat hind limb muscles to gravitational unloading and/or
creatine depletion. Biological Sciences in Space, 5(3):194-199, 1991.
24. Yamazaki, H., Kita, F., Ikuma, K., Ohnaka, H., Koike, K., Takahashi, S., Shiraishi, A., Ohashi. S.
Effects of gravity and oriental medicine, tochu (Eucommia ulmoides Oliver) leaves, on tree frog
Hylajaponica. .Biological Sciences in Space, 5(3):202-207, 1991.
25. Suzuki, M., Harada, Y., Takumida, M., Sekitani, T., Tahara, T., Kanaya, K. Vestibular sensory
Epithelia of the tree Frog returned from space. Biological Sciences in Space, 5(3):20%21 1, 199 I.
26. Feuilloley, M., Yon, L., Kikuyama, S., Okuno, M., Kawamura, K., Gutkowska, J., Vaudry, H.
Effect of space flight on the distribution of atrial natriuretic peptide (ANP)-like immunoreactivity
in the heart of the tree frog, Hyla japonica:-Preliminary Report-. Biological Sciences in Space,
5(3):215-2 17, 1991.
27. Feuilloley, M., Yon, L., Kawamura, K., Kikuyama, S., Gutkowska, J.. Vaudry, H. lmmunochemical
Localization of atrial natriuretic factor (ANF)-like peptides in the brain and heart of the tree frog,
Hyla japonica: -Effect of weightlessness on the distribution of immunoreactive neurons and
cardiocytes. Journal of Comparative Neurology, 330:32-47, 1993.
29. FRIS Experiment Group, Report ofFrog Experiment OnboardSpace Station MIR, Space Utiliza-
tion Research Center, Institute of Space and Astronautical Science. Kanagawa. 199 1 (Japanese).
Chapter 9
PLANT CRAWTROPIC RESPONSE
A. Merkys and J. Darginaviciene
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 13
11. Induction of the Gravitropic Response . . . . . . . . . . . . . . . . . . . , . 2 15
A. Stimulation and Perception of the Gravitropic Signal . . . . . . . . . . . 215
B. Transduction and Transmission of the Gravitropic Signal . . . . . . . . . 2 16
111. Expression of the Gravitropic Response . . . . . . . . . . . . . . . . . . . . 2 18
A. IAA Controls Transition from Induction to Gravitropic Curving . . . . . 2 18
B. Role of IAA Translocation in Growth Regulation . . . . . . . . . . . . . 222
C. Lateral Polarization of the IAA Receptor System . . . . . . . . . . . . . 224
IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
V. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
1. INTRODUCTION
The spatial orientation of plants is determined by their gravitropic response, i.e.,
by the functioning of systems which perceive gravitropic stimulation and respond

ISBN: 0-76234147-3
21 3
214 A. MERKYS and I. DARGlNAVlClENE
to this by growth movements. This was proved early last century by Knight when
on the basis of methodically valid experiments he showed that plant orientation is
determined by the gravity vector.' There are two theories for the mechanism of
gravitropic response. The first one, the Nemec-Haberlandt theory dating back to
1900, assigns a role in this process to the amyloplasts, mobile cell particles whose
specific weight exceeds that of the cytoplasm: when the direction of the axial organ
with respect to the gravity vector changes, the amyloplasts shift their position,
which causes gravitropic ~timulation.~-~
The second theory-the Cholodny-Went theory-considers the growth sub-
stance auxin to be of crucial i m p ~ r t a n c eAccording
.~~ to this theory, the anions of
auxin, currently thought to be p indoleacetic acid (IAA), accumulate through
cataphoresis in the lower part of a horizontally oriented axial organ (Fig. 1). Owing
to a different sensitivity to auxin of plant shoots and roots, the growth of the lower
part of the stem increases (negative gravitropic response), making it grow upwards,
while the growth of the lower part of the root is delayed (positive gravitropic
response), making it grow downwards. This theory is supported by Thimann's
finding that roots are more sensitiveto IAA than stems," and also by Dolk's studies
demonstrating that a greater amount of auxin always accumulates in the lower part
of horizontally oriented axial overground organs than in the upper part."
For many years the Nemec-Haberlandt and Cholodny-Wenttheories appeared to
describe the mechanism of the gravitropic response quite satisfactorily. Currently,
however, increasing evidence is accumulating concerning a role of calcium in the
early stages of the gravitropic response. The contribution of IAA in the regulation
of the growth process is also understood in a new way, since the main growth
regulating unit appears to be not the molecule of IAA itself, but rather its complex
with a receptor
Since the expression of the gravitropic response involves different growth rates
of the upper and lower parts of horizontally oriented segments, the question arises:
A B
figure 1. Scheme of Cholodny-Went.A stem; B root; + positive potential; - negative

potenti a I
Plant Gravitropic Reaction 215
how does the growth regulating function ofthe IAA-protein complex manifest itself
during the gravitropic response, and how can the receptor protein permit the
phytohormone to affect the growth of the cell? In this chapter we review the
evidenceavailablefrom experiments carried out in the microgravity of spaceflight14
and from the responses of plants to gravity vector changes on the ground in order
to arrive at a more detailed understanding of the sequence of processes operating
in the plant gravitropic response.
11. INDUCTION OF THE GRAVITROPIC RESPONSE

A. Stimulation and Perception of the Gravitropic Signal
The gravitropic response may be conditionally divided into an induction phase

and an expression phase, each consisting of a number of successive steps.ls17 The
sequence of processes during the gravitropic response is presented in Figure 2 for
the case of a horizontally placed stem and root. The induction of the gravitropic
response comprises the following steps. The gravitropic signal consisting of a
change in the direction of the gravity vector is perceived by the gravireceptorsystem
(perception). The physical signal caused by a shift in the statolith location is
transduced into a biochemical signal. The biochemical signal is then transmitted to
the system which determines the lateral polarity of the tissue.
The expression of the gravitropic response comprises a change in growth rate
with a laterally polarized growth of the axial organs. Nemec and Haberlandt
assumed that the gravitropic signal is perceived by the cells of root cap, coleoptile
tip and starchy leaf sheath, all of which contain statoliths or amyloplasts.When the
orientation of the axial organ is changed with respect to the gravity vector, there is
a displacement of the statoliths due to the fact that their density is higher than that
of the surrounding cytoplasm. The displacement of the statoliths would cause the
gravitropic stimulation.
The time required for perception of the gravitropic signal must be in the order of
seconds. This is concluded from calculations of the sedimentation rate v of particles
in the cytoplasm by means of Stoke's law:
v = 0.222 . G . ? (d, - df)/q C ~ / S
where G = 982 cm.sP2;r = particle radius in cm; d, = particle density (g . cmP3);d,
= medium density (g . ~ m - ~ q ) ; = cytoplasmic viscosity (g . cm-' . s-I). For
amyloplasts the deposition rate amounts to 5.5 . lo4 cm-s-l. This means that the
time for passage over a distance of 10 pm (approximatelythe distance across the
cell) is 3 minutes. The time required for a particle to shift over a distance equal to
its diameter is 36 s, which would then be the time minimally required for the
perception of the gravitropic signal.
On the basis of such calculations, Audus concluded that amyloplasts may act as
the operative agents in gravitropic stimulation.'8 Not only amyloplasts, but also
21 6 A. MERKYS and 1. DARClNAVlClENE
Gravitropic stimulation
4
Perception
J.
Transduction
JI
Transmission
JI
Lateral polarization of tissue
4
Expression: polarized growth
Figure 2. Sequence of gravitropic reaction processes.
inclusions like crystals of calcium oxalate or barium sulphate could act as particles
perceiving gravitropic stimulation. The threshold value for stimulation by gravity
was determined on board space station Salyut-7 for lettuce seedlings. The value
corresponds to 3.9 x 1OP3 G for hypocotyls, and 0.15 x 10-3 G for roots. l 9
Gravitropic induction is determined by the product of stimulation duration and
gravity force: A = t . G, where t is stimulation duration in seconds, and G is the
stimulation or gravity force. Its duration (t) in plants under Earth conditions ranges
from 0.>30 s.2G22This means that statolithswould cause stimulationwhile passing
a distance close to their diameter. On their way they would reach cisterns of the
endoplasmic reticulum, which are likely to play an important role in the gravitropic
stimulation process. Evidence for this assumption has been presented in several
papers dealing with this ~ u b j e c t . ” . How
~ ~ . ever,
~ ~ during sedimentation the statoliths
may also encounter structures other than the endoplasmic reticulum. Evidence has
accumulated for the existence in plants of actin-like proteins, which seem to play
a role in plant movement^?^^*^ Recent experiments have shown that during their
migration the amyloplasts or statoliths may interact with these actin-like proteins
of the cytoskeleton.22
B. Transduction and Transmission of the Gravitropic Signal
After the stimulation process, described in the previous section, the plant cell
passes into the excitation state. Here begins the transduction and transmission of
the gravitropic signal. It now appears that a release of free calcium ions to the
cytoplasm is an essential part of the transduction process. In the plant endoplasmic
reticulum calcium is sequestered. The increase in the ratio between the free calcium
Plant Cravitropic Reaction 21 7
-
A B
Stat01 i t b Stimulationand
excitation
R-Cd
colcoptilc
i
caz+
mot
Cistern of ER I
Figure 3. Mechanical stimulation by statoliths leading to calcium release. A -

stimulation and excitation; B - induction of gravitropic reaction in axial organs.
ion level in the cytoplasm and the sequestered calcium concentration in cisternae
of the endoplasmic reticulum alters the functional state of the cell.
In cells of plant axial organs, not affected by gravitropic stimulation, the cyto-
plasmic concentration of free Ca2+ions is low, in the order of 10-6 M.26 Upon
gravitropic stimulation this concentration is greatly increased due to the release of
calcium from a compartment in the endoplasmic reticulum. as schematically
represented in Figure 3A.
In the absence of gravitropic stimulation there will exist a balance between free
and sequestered calcium. This balance is governed by the Ca2’, Mg2+-ATPase
system present in the endoplasmic reticulum, which maintains an ATP-dependent
transport of ~alcium.~’*~* Similar systems of calcium transport are also known to
exist in other cell 0rganelles.2~This assumption is fixther supported by the obser-
vation that treatment of maize roots with a chelating agent like EDTA (ethylened-
initrilotetraacetate) makes them insensitive to gravitropic stimulation and the
reversal of this effect by subsequent treatment with CaC12.30
How can the released calcium ions transmit the information of the gravitropic
stimulation event to provide the further development of the plant response? The
increased free calcium concentration may modify cytosolic proteins, either en-
zymes whose activity changes, or receptor proteins whose binding characteristics
change. This may lead to the excitation of certain structures in the cell resulting in
gravitropic curvature realization. Although the exact nature of this excitation
process is not yet understood, there is experimental evidence for its occurrence.
For many years it has been known that decapitation of coleoptiles deprives them
of the capability to express phototropic and gravitropic curvature^.^^^^ This
phenomenon has been used by us for the study of gravitropic induction in the
following way. Wheat coleoptiles are decapitated and subjected to gravitropic
stimulation.Then agar blocks containing IAA are applied to the cut apical surfaces,
and the coleoptiles are placed on the rotating clinostat in a condition of simulated
weightles~ness.~~ Although these coleoptiles receive no unidirectional gravitropic
stimulation during the period on the clinostat, they always show gravitropic
218 A. MERKYS and J. DARGlNAVlClENE
,t 2
Ll
0
so
p+
100 120 140 240
T i m , iiiiii
Figure 4. Retention of gravitropic induction in decapitated coleoptile. 1. Duration of
curving on clinostat (2.5 rpm); 2 . Regeneration of coleoptile tip curvature.
curvature. This means that they ‘remember’ the earlier gravitropic stimulation.
Such retention of a memory of gravitropic stimulation may last for four or more
hours (Figure. 4).
The capability of plants to retain a ‘memory’of gravitropic induction was earlier
discovered in experimentswith sunflowerseedlings employing somewhat different
methods.34The memory effect is not limited to gravitropic stimulation,but occurs
also after phototropic stimulation.Guttenberg showed in experiments with decapi-
tated coleoptiles that the memory of phototropic induction is retained in darkness
for several hours.35These coleoptiles were also capable of phototropic curvature
after physiological regeneration of their tips or after supplying them with IAA.
The memorized effect of gravitropic stimulation leads to a polarized growth of
the plant axial organ tissues. This means that the upper and lower parts of the plant
axial organ acquire different growth rates, which bring about the gravitropic
curvature. This curvature leads under normal circumstances in Earth’s gravity to
upward growth of the stem and downward growth of the root in vertical direction.
In the next section the role of the phytohormone P-indoleacetic acid (IAA) in this
polarized growth will be discussed.
111. EXPRESSION OF THE GRAVITROPIC RESPONSE

A. IAA Controls Transition from Inductionto Cravitropic Curving
After the gravitropic induction process a lateral redistribution of IAA takes place
in such a way that the IAA content in the lower part of a horizontally oriented
coleoptile or other axial plant organ increases. Gravitropic curvature can only take
place in the presence of certain levels of IAA in the tissue.” This is evident from
the following observations.Decapitated (once or twice) and gravitropically stimu-

lated coleoptilesundergo gravitropic induction and remember this stimulationuntil
IAA accumulates in the tissues, either by restoration of the endogenous IAA level
or by addition of exogenous IAA. In the absence of IAA accumulation the
expression of a gravitropic response does not take place. The optimal IAA level
required for gravitropic curving is about 100 x lower than that required for straight
growth, and it is achieved after treatment with 10-7-104 M IAA.
The question is how the physiological function of IAA may stimulategrowth that
leads to the gravitropic curvature. Cholodny and Went supposed that IAA by its
weak acid nature changes certain physical properties of the cell wall, its elasticity
and p l a s t i ~ i t y . ~ ,A~later
~ , ~ ’hypothesis was proposed by Bonner, who suggestedthat
the phytohormone interacts with nuclear histones, leading to the de-repression of
certain genes and thus the initiation of
Our idea is based on experimental evidence indicating that the principal growth-
regulating factor is not free IAA but a complex of IAA with a protein receptor. Such
complexes can express their physiological activity on different levels of protein
synthesis by inducing or increasing the synthesis of certain proteins required for
growth According to this hypothesis, bound forms of IAA are indis-
pensable for the manifestation of the gravitropic response. Experiments on artifi-
cially created lateral gradients of bound (immobile) forms of IAA in the tissue of
wheat coleoptiles provide evidence for this h y p ~ t h e s i s .Segments
~~ of wheat
coleoptiles are treated with IAA by placing agar blocks containing 14C-IAAfor 1
hour on the apical surface of decapitated coleoptiles. The coleoptile segments are
then exposed in a vertical position for 7-8 hours to agar blocks without IAA. After
this time the free IAA has moved out of the tissue (Fig. 5A). At the time indicated
by the arrow the agar blocks are replaced by agar blocks with I4C-IAAor by blocks
with unlabeled IAA.
At the same time the growth rate of the middle part (7 mm) of the segments is
determined (Fig. 5B). The length of the segments enriched with bound forms of
IAA (after cessation of diffusion of free IAA from the tissue; nr. 3 in Fig. 5B) does
not differ from that of the control segments. This part of the experiment confirms
earlier findings of Winter and Thimam1.4~However, when the experiment is
prolonged with repeated additional IAA treatment of coleoptiles enriched with
bound IAA, the growth rate of coleoptiles saturated with both bound and free IAA
is significantly increased (nr. 4 in Fig. 5B), and exceeds that of segments enriched
with free IAA only (nr. 2 in Fig. 5B). Bound IAA is the fiaction of IAA remaining
in the tissues after the initial I-hour treatment of the coleoptile segment with an
agar block, during which treatment free IAA is removed from the tissue. The
remaining IAA represents non-transportable, bound IAA, including IAA-receptor
protein complexes.
Similar data have been obtained with other test objects, such as segments of pea
and fodder bean epicotyls, and segments of kohlrabi h y p o ~ o t y l s . ~ , ~ ~
A. MERKYS and I. DARGlNAVlClENE
A
160 -
140 -
--
v)
2 120- . .
. .
!i2
0
z
(r.
100-
80 -
. '4c-w 4
-.
l 4 c - 1 ~l4c-w
E 60-
2 40 -
20 - 0 ...........O...........O...........
0
O I I I I I I I 1
0 2 4 6 8 10 12 14
4 hr
B
10 -
4
c) (r.8- 3
E
i!
$6 -
2
2
\
1
E 4 -
E
4
a
2 -
o f I I I I I I I
0 2 4 6 8 10 12 14
t, hr
figure 5. Growth of wheat coleoptile segments during basipetal IAA transport.
Additional treatment with IAA after 8 hrs (arrow). A -transport of IAA from tissue (agar
blocks with M IAA and 14C-IAA); 6 - growth of middle segment zone (7 mm);
1 - control without IAA, 2 - segments enriched with free IAA, 3 - segments enriched
with bound IAA, 4 - segments enriched with free and bound IAA.
For the investigation of the role of bound IAA in the gravitropic response the
experiment is organized in a similar way as that described in Fig. 5 . Coleoptile
segmentsare treated for 1 hour by agar blocks containingIAA(Fig. 6A). The blocks
are either placed on the entire tip of the segment (nr. 2 in Fig. 6A) or half of the tip
(nrs. 3 and 4 in Fig. 6A). On top of the control segments an IAA-free block is placed
(nr. 1 in Fig. 6A). After exposure for 1 hour all agar blocks are replaced with
IAA-free blocks, and the coleoptile segments are then incubated for 8 hours in
vertical position (Fig. 6B). After this period of incubation all free IAA has moved
out of the segments, leaving only the bound IAA. The segments are then placed in
horizontal position (Fig. 6C). When the upper part of a horizontally oriented
coleoptile segment has previously been saturated with bound IAA, a positive rather
than a negative gravitropic curvature of the coleoptile segments is produced during
the first hour of exposition (Fig. 6C, nr. 4), but after 3 hours there is a reversal from
positive to negative gravitropic curvature (Fig. 6D, nr. 4). This phenomenon must
be connected with the continuing redistribution of free IAA in the lateral direction,
leading to increased levels of free as well as bound IAA in the lower part of the
coleoptile segment.
The rate of protein synthesis,measured as incorporation of I4C-glycine,has been
studied in such coleoptile segments. The results shown in Fig. 7 indicate that the
part of the segment with a higher level of bound IAA has an increased rate of protein
synthesis. When the level of bound IAA is highest in the upper part of the segment
(Fig. 7A), protein synthesis is highest in that part (Fig. 7A, nr. 1 bars), while the
reverse is true when the immobile IAA level is highest in the lower part of the
segment (Fig. 7B, nr. 2 bars).
[~fi-4~~IH
B D
4
A C
1 3.050.4 1 28.522.2
2. r E 5 1.520.2
+ 3 &7.420.6
1 2 3 4 1 2 3 4 --4.120.G
4 4 9.5?1.5
o Control o IAA
Figure 6. Role of lateral gradient of bound IAA in gravitropic curving of wheat

coleoptile segments. A - Treatment with IAA for 1 hour for gradient development; B -
Transport of free IAA from tissue; C - Gravitropic curving in horizontal position after
1hour; D - Same, after 3 hours. 1 - control segment; 2 - segment with uniformly
distributed bound IAA; 3 - segment with excess bound IAA in lower part; 4 - segment
with excess bound IAA in upper part. Coleoptile curvature indicated in degrees;
positive gravitropic curvature of coleoptile is downward.
A B
500
-e
c
Z 400
," 300
0
g
\
200
E
3 100
0
I I1 111 IV v VI VIIVIII I I1 In IV v VIWVIII
Nr. of fraction
Figure 7. Incorporation of ''C-glycine in wheat coleoptile proteins. After 1 hr of
gravitropic curving due to lateral gradient of immobile IAA. Fractionation on a
DEAE-cellulose column. A. Bound IAA gradient towards upper part of segment; B.
Bound IAA gradient towards lower part of segment. 1. upper part of segment, 2 . lower
part of segment. I. Elution with 0.005 M phosphate buffer; I I . Elution with 0.005 M
phosphate buffer + 0.005 M NaCI; I l l . Elution with 0.005 M phosphate buffer + 0.1
M NaCI; IV. Elution with 0.005 M phosphate buffer + 0.2 M NaCI; V. Elution with
0.005 M phosphate buffer + 0.3 M NaCI; VI. Elution with 0.005 M phosphate buffer
+ 0.4 M NaCI; VII. Elution with 0.005 M phosphate buffer + 0.4 M NaCI + 0.1 M
Na2C03; VIII. Elution with 1 % NaOH, phosphate buffer pH 7.2.
These experiments show that bound (immobile) IAAcan affect the rate of protein
synthesis and thus the growth rate. However, the formation of bound IAA requires
a basipetal stream of free IAA in the tissue, as demonstrated in Fig. 5B.Thus we
can confirm the conclusion of the classical studies of Went' and C h ~ l o d n yA: ~ ~
lateral phytohormone stream in the axial plant organ leads to a gravitropic curvature
by increasing the growth rate of one part, such as, the lower part in horizontally
oriented shoots and the upper part in similarly oriented roots.
B. Role of IAA Translocation in Growth Regulation
According to the data discussed in the previous section the realization of the
gravitropic response takes place in the presence of concentration gradients of free
and bound IAA between the upper and lower halves of horizontally oriented parts
of the plant. In a large number of experiments it has been shown that the lateral IAA
gradient results from IAA transport in basipetal and lateral direction^.'^ This role
of IAA transport in the formation of an IAA gradient is beyond doubt.
Another question is: can growth regulation and, therefore, gravitropic curvature
be caused by a change in the IAA transport rate? There are no direct experimental
data to answer this question, but relations between some processes support this
assumption.
A close relation between the growth rate of cereal coleoptile segments and the
basipetal transport of free IAA has been demonstrated several time^!^"^ Triio-
dobenzoic acid, which blocks basipetal transport of IAA, inhibits The
oscillation of IAA transport along the coleoptile, followed by changes in potential
differences, leads to a wavy movement of the zone with an increased growth rate
down the ~oleoptile?~
Our experiments indicate that changes in growth rate of wheat coleoptile seg-
ments are due to changes in the rate of vectorial translocation of IAA. Basipetal
transport of I4C-IAA in coleoptile segments was measured after modification of
growth rate by means of the native hormone abscisic acid (ABA). Treatment for 1
hour with 1 pM ABA increases segment growth,while treatment for 3 hours inhibits
it. In the latter case basipetal IAA transport is found to be 30% lower than in the
former case.
The use of isolated plasmalemma vesicles and I4C-IAApermits studies of active
(energy-dependent) and passive transmembrane movements of IAA.48*49 Here
again, increased growth rate of segments coincides with increased IAA transport,
in this case both inward and outward transport of IAA across the plasmalemma
membrane is observed. The reverse has also been observed: when segment growth
is inhibited, both active and passive outward transport of IAA are considerably
decreased, but in this case entrance of IAA into the vesicles is not affected. This
experiment shows that IAA transport from vesicles, imitating secretion of phyto-
hormone from a cell, is correlated with growth rate and basipetal IAA transport
rate. These findings support the assumption that transmembrane translocation of
phytohormone plays a direct role in plant growth regulation.
This assumption is further confirmed by the following facts: (a) basipetal IAA
transport in the lower, fast growing half of the horizontally oriented segment is
more active than in the upper, slow growing half, (b) synthesis of IAA may take
place in practically every cell, but at any moment in time not all cells possess the
ability to synthesize IAA, so the site of its action may shift. On the basis of findings
of other investigators and those obtained in our laboratory, we must assume that
both linear and gravitropically induced growth is not regulated by passive cata-
phoretic translocation of IAA, as earlier proposed, but by active IAA transport.
While discussing the role of IAA in growth activation, one cannot disregard the
assumption that IAA activates the proton pump and thus leads to acidification of
the cell wall area and to its e l ~ n g a t i o n . ~If,
~ *however,
~’ auxin function in the
development of gravitropic curvature would be to produce “acid growth,” then
gravi-inducing segments on a centrifuge at 7 G for 5 minutes and transferringthese
segments to an acid medium should produce gravitropic curvature. However, this
is not the case, and gravitropic curvature occurs only after addition of IAA to the
224 A. MERKYS and J. DARClNAVlClENE
medium.52This finding suggests that IAA does not produce gravitropic curvature
by acidification of the cell wall area of the axial organ, as had been assumed by
Cholodny and Went. Moreover, such acidification is likely to occur only after the
initial phase of the action of IAA. What then is the primary phase of IAA action
during the gravitropic response? The answer to this question requires discussion of
the lateral polarization of the IAA-receptor system responsible for regulation of
growth by elongation.
C. lateral Polarization of the IAA Receptor System
Analysis of the available data has led us to assume that the growth regulating
action of IAA in plant cells is performed through an IAA-protein receptor complex.
Such a complex will have to satisfy two basic criteria: (a) it binds IAA specifically,
and (b) it expresses the characteristic primary growth regulating action of IAA in
plants. Specific binding of IAA in plasmalemma and cytosol preparations of wheat
coleoptile cells has been determined according to the method of He~tel.'~ The
physiological activity of the isolated IAA-protein complex is determined by its
activation of RNA-polymerases I and I1 in isolated wheat coleoptile cell nuclei.
Methods for the isolation of specific IAA binding proteins of plasmalemma and
cytosol and their purification have been
We have found three types of highly specific IAA binding sites in wheat
coleoptile cells by analysis of the binding characteristics of IAA with proteins from
different subcellular fractions. The physiological activity of the complexes formed
in vitro by these proteins with IAA has also been determined. Two of these sites are
located in the plasmalemma, the third one in the cytosol: (a) Site I. This is the
IAA-protein complex formed in the fraction of plasmalemma isolated from wheat
coleoptile cells with optimum binding at pH 7.2. It has a K, z 3.104 M, and n N
2. lo-' mo1.mg-' protein. The complex is present in a plasmalemma protein fraction
with molecular mass 80-90 kDa, possesses a high ligand specificity, and is capable
of increasing the activity of RNA-polymerases I and I1 of isolated nuclei of the
same cells; (b) Site 11. This IAA-protein complex of plasmalemma has a binding
optimum at pH 5.5, a K, = 1.1.104 M, n = 5.4.10-lo mol.mg-' protein, and a
molecular mass of about 20 kDa. It possesses high ligand specificity, but it has no
effect on the nuclear RNA-polymerase activity. Similarly to the IAA-binding
complex from corn coleoptiles, identified by Veni~,~' it can affect in vitro trans-
membrane cation translocation; (c) Site 111. This IAA-protein complex, isolated
from the cytosol, has a binding optimum at pH z 8, K, z 1.7.104 M, n = 0.36
vavopoh.py-' protein, a molecular mass of about 40 kDa, a high ligand specificity
for IAA, and is capable of activating RNA-polymerases I and I1 of isolated nuclei.
It resembles previously described cytosolic IAA-protein complexes from tobacco,
pea and bean cells.56
Specific binding of IAA to these three preparations from upper and lower parts
of horizontally oriented wheat coleoptiles has been determined. The results shown
18 r T
6
16
5 14
-
.-
C
0 4 -9,
.E 12
e,M 10
.
E
=
3
2
2
3
8
6
8 8
4
1
2
n 0
A B A B
figure 8. Specific binding of 3H-IAA to proteins of wheat coleoptile. Left panel:

Plasmalemma protein, site 11, pH 5.5; Right panel: Cytosolic protein, site 111, pH 8.0.
A - upper part of horizontally oriented coleoptile, B - lower part of same.
in the left panel of Fig. 8 indicate that there is no significant difference in binding
of IAA to the plasmalemmal site I1 proteins with binding optimum at pH 5.5. The
same is true for the site I protein with binding optimum pH 7.2 (not shown). The
right panel of Fig. 8 shows a much greater binding of IAA to cytosolic site 111protein
from the lower part than with that from the upper part of the wheat coleoptile. In
Fig. 9 it is shown that the activity of RNA-polymerase I1 is significantly increased
by IAA-protein complex from plasmalemma (PH 7.2) and from cytosol (PH 8.0)
derived from the lower part of horizontally oriented wheat coleoptile segments.
These findings appear to present evidence for a lateral polarization of specific
IAA binding to protein and for a functional activity of the resulting IAA-protein
complexes, which can explain the polarized growth in the horizontally oriented
coleoptile leading to gravitropic curvature.
n I T
40 40
5
Y 30 5 30
E i%
YM 20 TM 2 0
5 I
E eE
e 10 1.0
0 00
RP-I RP-I1 RP-I RP-11
Figure 9. Physiological activity of IAA-protein complexes from different halves of

horizontally oriented wheat coleoptiles. I - plasmalemma protein, site I, pH 7,2; I1 -
cytosolic protein, site 111, pH 8,O. RP-I - activity of RNA-polymerase I; RP-II - activity
of RNA - polymerase II. A - upper part of horizontally oriented coleoptile, B - lower
part of same.
226 A, MERKYS and J. DARGlNAVlClENE
IV. CONClUSlONS
A review of data in the literature and of data obtained in our laboratory allows us
to present a general view of the mechanism of the plant gravitropic response, which
is schematically shown in Fig. 10. The initial reactions-gravitropic stimulation
and its perception, and especially gravitropic signal transduction and transmission
processes-have so far been insufficiently investigated. For many years our insight
in the gravitropic stimulation process has been determined by the theory of
Nemec-Haberlandt. This theory invokes mobile particles with a density exceeding
that of the cytoplasm, the statoliths. The amyloplasts present in coleoptile cells are
believed to act as statoliths. Under the influence of gravity the amyloplasts tend to
sediment. During the last decade it was assumed that during sedimentation these
particles would interact with the endoplasmic reticulum of the cell. More recently,
actin-like proteins of the cytoskeleton have also been considered in this connection.
6
GRAVIINDUCnON
I
I Polarizationof
conipetitivity of in IAA transport system
I system directivity I
I I
t
REALIZATION
IAA-receptor protein
interartion
Changes in tanscrip- Polarization of IAA

tional level content
1 Lateral growth polarizalion [

I~~
GravitrooirC U W R ~ U ~ IC
~ ~
Figure 10. Key processes in gravitropic response.

In recent years a prominent role of calcium ions in various cellular stimulation

processes has been noticed. Evidence has been obtained for the release of free
calcium ions into the cytoplasm after gravitropic stimulation. These calcium ions
may derive from a calcium store in the endoplasmic reticulum, where they are
accumulated by means of an ATP-dependent uptake process mediated by the Ca2+,
Mg2+-ATPasesystem located in the membrane of this organelle. A sudden increase
of the free calcium level may modify the activity of certain cytoplasmic enzymes
or receptor proteins.
The gravitropic induction phase is completed when lateral polarization takes
place in tissues, which contain information about the changed direction of the IAA
transport system and about the competition of the IAA-receptor system with the
phytohormone. This information could be in 'memory' until the right conditions
for its expression are achieved.
IAA is one of the main factors in the expression of gravitropic curvature. The
Cholodny-Went theory is correct in assigning a key role in the gravitropic response
to the phytohonnone P-indoleaceticacid (IAA). However, it is wrong in postulating
that the asymmetric redistribution of IAA in gravitropically stimulated axial organs
is due to electroosmosis. The diagram in Fig. 10 shows that the main action of IAA
is related to the post-inductive phase of the gravitropic response. At that point the
basipetal IAA transport is deflected to a lateral direction. This leads to a lateral
gradient of free IAA with the formation of IAA-protein complexes. The resulting
lateral polarization of these complexes implies that not only free IAA, but also these
complexes may be a growth-limiting factor. Both of these may cause an alteration
in the growth rate in different tissues of the gravitropically stimulated axial organ.
Calcium ions, moving in the direction opposite to that of IAA, may also play a role
in the polarized growth process.
V. SUMMARY
The gravitropic response of plants to a change in the gravity vector may be divided
in the phases of induction and expression. During the induction phase the amy-
loplasts, due to their greater density than the cytoplasmicdensity,shift their position
in less than a minute. During this shift there is an interaction with the endoplasmic
reticulum, although a role of actin-like proteins of the cytoskeleton may also be
involved in this process. The endoplasmatic reticulum maintains a store of seques-
tered calcium through the action of an ATP-dependent calcium uptake mediated by
the Ca2+,Mg2+-ATPasesystem present in the membrane of this organelle. The
interaction of the amyloplast with the endoplasmic reticulum leads to the release
of free calcium ions from the endoplasmic store. The increased free Ca2+level in
the cytoplasm may modify the activities of certain enzymes and receptor proteins.
The gravitropic induction phase is completed when the lateral polarization of the
tissues has taken place. Thesetissues contain information about changes in direction
of the IAA transport system and in competition of the IAA-receptor system for the
228 A. MERKYS and j. DARGlNAVlClENE
phytohormone. This information is fixed in “memory” and its expression is

achieved when the lateral gradient of IAA concentration and of the activity of the
IAA-receptor protein complexes is formed in the horizontally oriented plant organ.
Flows of IAA and calcium ions in opposite directions may lead to the expression
of laterally differentiated growth.
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36. Cholodny, N.G. Phytohormones.Izdatelstvo Akademii Nauk Ukrain SSR, Kiev, 1939 (in Russian).
37. Burstrom, H. Influence of the tonic effect of gravitation and auxin on cell elongation and polarity
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38. Bonner, J. Molecular Biology ofDevelopment. Mir, Moskva. 1967 (in Russian).
39. Merkys, A.J. State of question related with gravitropic reaction realization. Common Principles
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40. Merkys, A. Influence ofAuxin on the Process of Growth of Higher Plants. Stimulators of Growth
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41. Merkys, A.J. Auxin and Plant Growth. Mokslas, Vilnius, 1982 (in Russian).
42. Merkys, A.J., Darginaviciene, J.V., Marciukaitis, AS., Jurevicius, J.V. Growth activity of free and
bound (immobile) 0-indoleacetic acid. Physiology and Biochemistry of Cultural Plants,
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44. Merkys, A., Darginaviciene, J. Bound indoleacetic acid and growth by elongation. Plant Growth
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45. Merkys, A.J., Darginaviciene, J.V. About participation of bound P-indoleacetic acid in processes
of growth and geotropism. Doklady Akademii Nauk SSSR,234(3):72&723, 1977 (in Russian).
46. Goldsmith, M.H.M. The polartransport ofauxin. AnnualReviewofPlantPhysiology,28:43!M48,
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47. Newman, LA. Electric potentials and auxin translocation in avena. AustralianJournal of Biologi-
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48. Darginaviciene, J.V., Merkys, A.J., Uleviciene, R.R.,Zemenas, J.A., Maximov, G.B. IAA-Binding
properties of the plasmalemmae from wheat coleoptiles. Plant Physiology (Russia). 39(2):249-
258, 1992 (in Russian and English).
49. Darginaviciene, J.V., Merkys, A.J., Krupovnickiene, A.L., Maximov, G.B. Investigations ofvector
transport of IAA on vesiculars of wheat coleoptile plasmalemma. Doklady Akudemii Nauk SU,
318(5):127%1279, 1991 (in Russian and English).
50. Hager, A., Menzel, H., Krauss, A. Versuche und Hypothese zur Primanvirkung des Auxin beim
Streckungswachstum. Planta, 100:47-75, 197 I .
5 1. Polevoy, V.V., Salamatova, T.S.About the mechanism of auxin action on proton transmembrane
transport. Plant Physiology (Soviet Union), 22(3):5 19-552, 1975 (in Russian and English).
52. Merkys, A,, Darginaviciene, J. Changes in physical and chemical properties ofcell wall under the
influence of indoleacetic acid in response to stimulation by gravity force. Organisms and Gruviry
Force. Mintis, Vilnius, 1976 (in Russian).
53. Hertel, R. Auxin Binding Sites: Subcellular fractionation and specific binding assays. Plant
Organelles. Methodological Surveys Biochemistry, Vol. 9 (E. Reid, Ed.), pp. 173-183. Ellis
Horwood, Chichester, England. 1979.
54. Darginaviciene, J.V.. Romanov, G.A., Zemenas, J.A., Merkys, A.J. Auxin-binding protein from
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and English).
55. Napier, R.M., Venis, M.A., Bolton, M.A., Richardson, L.I., Bucher, G.W. Preparation and
characterisation of monoclonal and p o l y ~ l antibodies
~~l to maize membrane auxin-binding
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Receptors and Cellular Interactions in Plants. Cambridge University Press, Cambridge, 1986.
Chapter 10
HUMAN LIFE SUPPORT FOR

ADVANCED SPACE EXPLORATION
Steven H. Schwartzkopf
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
I1. Human Life Support Requirements . . . . . . . . . . . . . . . . . . . . . . . 232
111. Life Support Functions and Technology Selection . . . . . . . . . . . . . . . 233
A . Atmosphere Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . 234
B . Water Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
C . Waste Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
D. FoodProduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
E. Food Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
IV. System Design of a Controlled Ecological Life Support System . . . . . . . . 238
A . American Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
B . Russian Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
C. Concept for a Controlled Ecological Life Support System . . . . . . . . 239
V. Conceptual Design o f a Life Support System for a Lunar Base . . . . . . . . 240
A . FoodProduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
B . FoodProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

.
ISBN:0-7623-0147-3
23 1
232 STEVEN H. SCHWARTZKOPF
C. Atmospheric Regeneration . . . . . . . . . . . . . . . . . . . . . . . . .245

D. WaterPurification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,245
E. Waste Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246
VI. In Situ Resource Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
VII. Design Characteristics and Breakeven Analysis . . . . . . . . . . . . . . . . .248
VIII. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 51
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .252
1. INTRODUCTION
Whether the human race will choose to remain an exclusively terrestrial species or
expand permanently into space by establishing colonies on the Moon, Mars or
elsewhere, will ultimately be influenced by a multitude of factors. One of the most
important of these factors will be the availability of technology to economically
support human life in extraterrestrial environments.On prior space missions, U.S.
astronauts have been sustained almost exclusively by open-loop, non-recycling
systems utilizing physicochemical life support technologies. In these systems,
consumables such as food, drinking water, and oxygen were stored in the spacecraft,
while waste materials were simplyjettisoned overboard or stored for disposal upon
the return to Earth. The simplicity, effectiveness, and reliability of this approach
successfully met the challenge of demonstrating the feasibility of human short term
spaceflight.
For activities such as the establishment of permanent human settlements on the
Moon and Mars, the open-loop, non-recycling systems used previously will not be
adequate. Designers of life support systems for such endeavors are faced with a
much different challenge. They must take into account factors such as the necessity
of providing a familiar, Earth-like living environment to promote human produc-
tivity and psychological well-being, and the need to increase the self-sufficiency
and decrease the operating costs of the life support system. To meet these design
challenges,a new approach to the support ofhuman life must be implemented. This
paper describes such an approach.
11. HUMAN LIFE SUPPORT REQUIREMENTS

Human beings require substantial amounts of consumable materials to maintain life
(Table 1). Without recycling, over 8,000 pounds of oxygen, food, and water (for
food preparation, showers, personal hygiene, and clothes washing) are needed to
sustain a person for one year. These estimates are nominal amounts for a person
with a body mass of approximately 155 pounds (70 kg). The actual amounts
required can increase substantially with higher body mass or with physiological
changes such as an elevation in the level of physical exertion.' In addition, these
mass estimates would increase even firther when the packaging materials and the
Human Life Support 233
Table 1. Nominal Life Support Consumables

Required for Humans
Consumable Mass in kgyr
Food 408
Oxygen 304
Water, food preparation 263
Water, drinking 676
Water, hand/face wash 662
Water, shower -
1325
Total 3638
structural support(s) required to contain the consumablesduring a launch into space

are included.
At the current cost of launching a Space Shuttle into low Earth orbit, estimated
to be $1 1,000 per pound,* the average annual cost of supporting a single crew
member in low Earth orbit would be over $88 million. Naturally, due to the greater
distances involved, the cost of supplying life support materials to a human crew at
a base on the Moon or Mars will be even higher. As a consequence,the development
of technologies which can produce the necessary life support consumables in the
spacecraft or at a base site is economically essential if advanced manned space
missions are to become reality. Production of consumables on site can only be
accomplished by regenerative life support system technologies which recycle
organic wastes or by technologies which use available in situ resources (such as
lunar or Martian regolith) as raw input materials.
I l l . LIFE SUPPORT FUNCTIONS AND TECHNOLOGY

SELECTlON
The primary functions required for the direct support of human life include:
atmosphere regeneration, water purification, waste processing, food production,
and food processing. There are two types of technology available to provide these
functions: physicochemical and bioregenerative. Physicochemical life support
technologies include a broad range of physical and chemical reactors, devices
such as incinerators, electrolyzers, distillation devices, and selective absorbent
filters. Bioregenerative technologies utilize living organisms (such as bacteria,
algae, higher plants, or animals) as the “reactors” to provide life support
functions. Each primary life support function is described below, accompanied
by a discussion of the types of technology available to provide the required life
support.
A. Atmosphere Regeneration
The major operations performed in atmosphere regeneration are the removal of

CO,, water vapor, and trace contaminants (both gaseous and particulate), and the
generation of 0,. Traditionally, physicochemical technologies have been used to
provide these function^.^ The most common method of removing CO, on U.S.
space missions, for example, has been by absorbing it onto lithium hydroxide
(LiOH). The adsorption reaction is irreversible, however, and requires periodic
replacement of the LiOH with fresh material to maintain operation. For short
duration missions, this replacement is feasible, but as mission durations lengthen,
the cost of resupplying LiOH becomes prohibitive. It was for this reason that the
longer duration Spacelab mission employed a system in which CO, was removed
by a regenerable molecular sieve.4This absorbent traps CO, in microscopic pores
on its surface, and it can be regenerated by exposure to vacuum and heat.
For long duration missions, once the CO, has been removed, it can be further
processed by reduction to recover the 0,.3This can be accomplished by reacting
the CO, with H, at high temperature in the presence of a catalyst (e.g., Bosch or
Sabatier reactors) or by electrochemical separation. Alternatively, the CO, can be
supplied to plants, which through the photosynthetic process will convert it to 0,.
Water vapor is emitted into the spacecraft atmosphere through crew respiration
and perspiration, as well as through activities such as eating, food preparation,
showering, and hand and face washing. On long duration missions, water vapor
will also be emitted by washing and drying of clothes. This water is generally
removed from the atmosphereby a condensingheat exchanger and liquid collection
system. In some applications, such as space suits or short duration missions,
regenerable absorbents have been used for water vapor removal. Once the water
vapor has been collected and returned to liquid form, it must be processed by a
water purification system (discussed below) before it is recycled. This purification
step is necessary because many water-soluble atmospheric trace contaminantswill
dissolve in the water as it condenses.
In the closed atmosphere of a spacecraft or habitat, gaseous or particulate trace
contaminants emitted by materials, machinery, and the crew or any other living
organisms can accumulate to levels which may be health-threatening. For the
International Space Station, particulate filters are combined with a gaseous trace
contaminant control system which uses activated carbon and catalytic oxidation to
remove contaminants. The particulate filters in this system also remove any
airborne bacteria from the spacecraft atmosphere.
On prior U.S. space missions, oxygen for the crew has been supplied in pressur-
ized cylinders stored aboard the spacecraft. For longer duration missions, this
method is inadequate, and a means of producing 0, must be provided. Physico-
chemical technologies for the production of 0, include water electrolysis, water
vapor electrolysis, and the reduction of CO,. As noted above, plants can also be
used to provide 0, to the crew through photosynthesis.
B. Water Purification
For most U.S. space missions, potable water has been provided from storage
tanks on the spacecraft. Both because water is heavy and because a significant
amount of water is required to support human life, onboard storage is not feasible
for long-duration missions. For this reason, the Space Shuttle provides potable
water as a by-product from its fuel cells, which combine H, and 0, to produce
electrical power and H,O.For vehicles which do not use fuel cells, water purifica-
tion must be provided to enable recycling.
The water availablefor recycling comes from three primary sources: atmospheric
humidity condensate, wash water (from food preparation, hand and face washing,
showering, and tooth brushing), and urine. A variety of physicochemical technolo-
gies have been developed for water recycling. These technologies include simple
distillation, filtration (e.g., reverse osmosis, multifiltration) and phase change
processes (e.g., Vapor Compression Distillation’ or freeze crystallization).
Higher plants can also supply essentially pure water through the process of
transpiration. Typically, plants transpire between 200 and 1000 liters of water per
kilogram of dry biomass per year.6 Water processing with higher plants involves
supplying the water to the plants through irrigation or in the form of hydroponic
nutrient solution. This water is absorbed by the plants and transpired as water vapor,
which is collected on a condensing heat exchanger and then purified for human use
through exposure to ultraviolet radiation (UV) or ozone to remove bacteria and
trace organic contaminants.
C. Waste Processing
Historically, the development of waste processing technology has had low

priority due to the short duration of previous missions. On most flights, feces and
solid wastes are simply stored for return to Earth, while urine is often just vented
overboard. Several physicochemical technologies have been investigated for proc-
essing and recycling solid wastes. They include dry oxidation (incineration), wet
oxidation, and supercritical wet oxidation. These high energy methods generally
convert organic waste materials into inorganic salts, water, and gases.
Bioregenerative technologies for waste processing include bacterial reactors and
combinationsof higher plant and bacterial systems.Bacterial reactors, both aerobic
and anaerobic, have an extensive history of application in domestic sewage treat-
ment plants. Aerobic systems typically require higher energy inputs to maintain
oxygenation (e.g., aerating pumps, mixers). Anaerobic systems require very little
energy, but have very slow process rates, and the anaerobic bacteria are more
susceptibleto changes in environmental condition^.^ Combining higher plants with
anaerobic bacterial systems provides several distinct advantages. The most signifi-
cant of these is the capability for increasing the removal of ammonia and nitrate
nitrogen at a higher rate than that obtained with bacterial systems without plant^.^
However, such systems are less efficient in removing carbonaceous compounds

than plant-free bacterial systems.
For longer missions, all solid and liquid wastes (e.g., feces, urine, food prepara-
tion waste) must be processed and converted into a usable product. One ofthe most
promising recycling technologies, low pressure wet oxidation, is typically carried
out at conditions below 230°C and below 3460 kPa (500 psi). The process breaks
down organic material through a combination of hydrolysis and oxidation. Since
low molecular weight compounds such as acetic acid tend to be refractory to the
process, low power wet oxidation processes lead to lower oxidation efficiency. The
result is a breakdown of solids and reduced oxidation demand, with a product liquor
containing a mixture of inorganic salts and soluble low molecular weight organics.*
D. Food Production
Previous missions have supplied astronauts with food through on-board storage.
For long duration missions, however, it will be necessary to produce food on board
the spacecraft. The food must be produced either by growing edible organisms
(microbes,plants, animals) or by direct conversion of waste or in situ materials into
food. Physicochemical methods of synthesizing carbohydrate, fat, and protein have
been developed and tested.'.'' Unfortunately, foods produced in this fashion are
typically not well-accepted by humans. Consumption of such synthetic foodstuffs
has produced a number of undesirable side effects including nausea and diarrhea.'
An additional problem with this method of food production is that many of the
chemical syntheses involved require the use of substrates of such high purity that
they would be difficult or impossible to obtain from life support system wastes. As
a consequence, if physicochemical food production were used in space, it would
require resupply shipments of the high purity substrates from Earth.
The earliest research on bioregenerative food production focused on the use of
unicellular algae (e.g., ChZoreZlu; see chapter 11 by Wolf in this volume) and small
vascular plants of the family Lemnaceae.' In both instances the biomass pro-
duced was physiologicallyunacceptable as a human foodstuff. Since the late 1970s,
attention has been focused on the inclusion of crop plants in life support systems.
Crop plants provide a nearly ideal solution to the problem of designing a food
production system for space use. Human crews are accustomed to consuming these
materials on Earth, and therefore no physiological or psychological barriers to their
use as foodstuffs exist. Current estimates of the amount of growing area required
to feed one person range from 20 to 30 square meters, depending on the species of
plants grown. For example, Hoff et al.,13 proposed a mixture of ten species
(soybean, peanut, wheat, rice, potato, carrot, chard, cabbage, lettuce, and tomato)
which satisfy all human nutritional requirements and require a growing area of
24 m2.
Animals can also play a part in food production systems for advanced missions.
Previously, the primary objection to their use has been the ecological inefficiency
Table 2. Efficiency Characteristics of Various Animal SDecies2’

Feed Conversion Harvest Production
Animalffroduct Efficiency Index Efficiency
Beef 17 49 8.3
Swine 40 45 18
Lamb 25 23 5.8
Rabbit 33 47 16
Broiler Chicken 50 59 30
Eggs 36 90 32
Milk 33 100 33
Shrimp 40 56 22
Prawns 50 45 22
Catfish 67 60 40
Carp 67 60 40
Tilapia 67 60 40
Notes: All figures in percent.

Feed Conversion Efficiency = (kg Biomass Gain / kg Feed) x 100.
Harvest Index = (Edible Biomass/Total Biomass) x 100.
Production Efficiency = (kg Edible Biomass/ kg Feed) x 100.
which occurs between trophic levels. As an example, only about 10% of the food
provided to cattle is converted into meat (Table 2). Thus, from an energetics
perspective, it would make more sense to feed plant materials to a human crew
directly rather than feeding them to an animal and subsequently using parts of the
animal as human food. This concept neglects the idea of feeding animals with the
plant parts that humans would not normally consume. Bacteria, molds and yeasts,
as well as several species of vertebrates (e.g., fish, chickens) can use such materials
as foodstuffs. In addition, Table 2 shows that several animal species have conver-
sion efficiencies considerably above the 17% value typical of beefcavle. Thus, with
proper speciesselection,animals can potentially play a substantial role in the overall
food production system.
E. Food Processing
Food processing technologies would make the biologically-produced materiaIs
suitable for human consumption. These technologies may be grouped into two
general categories: (1) processing of materials normally edible by humans, and (2)
processing of normally inedible materials to convert them into an edible form.
Edible materials may be eaten directly after washing, cooked for immediate
consumptionor for storageand later consumption,or processed to remove a specific
component, either to enhance digestibility or to obtain a component for specific
uses. Materials which are normally inedible, would be extracted or treated to
prepare them for human consumption or processed to produce feedstocksfor animal

consumption.
IV. SYSTEM DESIGN OF A CONTROLLED ECOLOGICAL

LIFE SUPPORT SYSTEM
A. American Experiments
Early in the American space program, research was conducted to evaluate these
physicochemical and bioregenerative technologies for human life support applica-
tions. Bioregenerative technology was viewed as being able to provide weight
savings in several of the basic functional areas, with the most notable being
atmosphere regeneration. Experiments were done to evaluate the effectiveness of
both algal ( e g , Chlorella; see chapter 11 by Wolf in this volume) find bacterial
(e.g., Hydrogenomonas) reactors for atmosphere regeneration. Studies which ap-
plied these reactors to regenerate the atmosphere of animals held in sealed chambers
showed that the concept was partially feasible. However, these studies also showed
that significant problems existed with respect to balancing respiratory gas (0, and
CO,) production and utilization, as well as in the development of control systems.
In two of these experiments,for example, atmospheric CO, concentrationsreached
over while in another experiment, 0, concentration reached over 60%.16
Neither of these results would have been acceptable for human life support
applications.
U.S. research into bioregenerative technology was largely terminated in the late
196Os, because planned manned space flight activities were all of short duration
and near-Earth. As a consequence, it was decided that life support for these missions
could be easily handled by a combination of on-board storage,dumping overboard,
and the physicochemical regeneration processes described above. In the late 1970s,
as the possibility of establishing a permanent human presence in space was again
considered, the American space program began to reevaluate bioregenerative
technologies.
B. Russian Experiments
In the Soviet space program, regenerative life support technologies had been
studied without significant interruption through a set of experiments conducted
with a series of closed, manned Bios chambers beginning in the early 1960s. The
first Bios was only 12 m3in volume. However,the final chamber (Bios 3) had grown
to a volume of 315 m3, and was composed of four equally-sized compartments,
including two phytotrons (where crop plants were grown hydroponically),an algal
culture room, and quarters for a crew of up to three. The earliest Bios chambers had
a total plant growing area of only 13 m2 per occupant. This growing area was
sufficient to provide each occupant with all of the oxygen for breathing and about
40% ofthe food he or she required. In the earliest Bios chambers, research was also
conducted on the use of a single-celled alga (Chlorellu) as an atmospheric gas
regenerator and food source. Although the algae worked well as a CO, scrubber
and 0, producer, it proved to be a poor source of human food.
Life support experiments were conducted in Bios 3 as recently as 1983,” when
a long duration experiment was conducted to simulate a round trip from Earth to
Mars with a crew of two. In Bios 3, the plant growing area had been enlarged to 30
m2 per person. The plants were grown hydroponically, and included wheat, chufa,
peas, dill, kohlrabi and many other vegetables. Although the later experimentswere
completed successfully, with full atmospheric and water recycling, they demon-
strated only partial recycling and closure of the life support system. Even in Bios
3, the occupants had to rely on a portion of their food (20%) from on-board storage.
C. Concept for a Controlled Ecological Life Support System
Conceptually, it is possible to design a life support system based exclusively on

either physicochemical or bioregenerative technology. However, both kinds of
technology have characteristicadvantages and disadvantages.For example, physi-
cochemical technologies tend to be fast-acting, but they usually perform only one
specific function, and frequently require large amounts of power. Bioregenerative
technologies are often characterized by slow reaction rates and larger volumetric
requirements, but are frequently multi-functional (i.e., plants can produce food,
recycle water, and help process waste all at the same time) and often operate with
very little electrical power. Thus, by carefully selectingand combiningtechnologies
with offsetting advantages and disadvantages it is possible to develop a hybrid life
support system design which can offer significant improvement of performance
over systems which are purely based upon physicochemical or bioregenerative
technologies.
An important method of integrating the two kinds of life support technology is
through the design and development of a Controlled Ecological Life Support
System, further abbreviated as CELSS. This system combinesbiological functions,
such as photosynthesis (primarily for food and oxygen production, and CO,
removal) with physicochemical functions (such as gas separation,and water vapor
condensation), and attempts to mimic some of the basic behaviors of the Earth’s
biosphere. The fundamental idea behind this kind ofhybrid design is that the system
incorporatesthe process control and recycling capabilities found in natural ecosys-
tems to provide an increased stability in the entire life support system.
A generalized top-level schematic of a CELSS is presented in Figure 1. This
figure illustrates the fundamental flows of life support materials through the system.
In this example, crop plants produce food for the crew. As additional benefits of
the food production subsystem, the plants take up CO, produced by the crew,
produce 0, for crew respiration and for use in oxidizing waste materials, and
produce water vapor which can be condensed and collected to supply the crew’s
figure 1. Generic diagram of a CELSS illustrating primary mass flows between

subsystems.
drinking and hygiene water. In the food processing subsystem, the products of the
crop plants are processed into forms which are palatable to the crew. The crew’s
metabolic waste materials (urine and feces), miscellaneous solid wastes (tissues,
wipes, writing paper, and so on), and inedible plant biomass from the food
production and processing subsystems are all supplied to the waste processing
subsystem. These wastes are oxidized, and used as a supply of inorganic nutrients
and CO, for the crop plants Pure water produced by the waste and waste water
processors is resupplied to the crew for use, or recirculated through the waste
processing subsystem.
V. C O N C E P T U A L D E S I G N OF A LIFE SUPPORT SYSTEM

FOR A L U N A R BASE
In late 1989,Lockheed initiated a NASA-funded case study to develop a conceptual
design for a lunar base life support system which could accommodate a crew that
would grow from an initial size of 4 to 100 people at base maturity. The lunar base
was chosen as a focal point because prevailing opinion supported the idea that the
Moon, and not Mars, would be our next home. During the initial portion of the
Lunar Base CELSS study, several different design concepts were evaluated. Based
upon a series of detailed analyses and trade-off studies,one ofthese design concepts
(Figure 2) was selected for the development of a detailed conceptual design.18The
five major subsystems of this design are described below.
figure 2. Functional block diagram of the Lunar Base CELSS conceptual design.
Figure 3. Cross sectional diagrams and design parameters of the Lunar Base CELSS
plant growth unit designs.
A. Food Production
For the Lunar Base CELSS project, the design rapidly focused on the selection
and development of the food production system, as it turned out to be both the
largest and the highest power consumer. Two systems were selected to produce
food: a crop growth unit and an aquaculture unit for the freshwater fish nlapia.
Based on an analysis of human nutritional requirements, the plant growth unit was
designed to include wheat, soybean, peanut, lettuce, tomato, and carrot. The use of
nlapia was incorporated as a means of producing a small amount of animal protein
for crew consumption. With this minimum set of plant species, supplemented by
about 50 gm per person per day of nlapia meat and some multiple vitamins, a
nutritionally adequate diet can be produced.
To accommodate an increase in crew size from 4 to 100, a series of three plant
growth unit designs was developed. These three designs span a range from a small
system which is ready to run upon landing (tumkey system), to a large system which
requires installation,inflation,and construction before it can begin operation.Cross
sections and fundamental design parameters of these three units are presented in
Figure 3. All three designs incorporate hydroponic plant growth techniques. The
first design uses a metal pressure hull based on the initial design and dimensions
developed for a Space Station module, and provides about 100 m2of growing area.
An artist’s concept of this unit is provided in Figure 4. The artist’s concept illustrates
both an artificial lighting system and a sunlight collection and distribution system
to supply photosynthetically active radiation.
Figure 4. Artist's concept of a Space Station module-based plant growth unit on the
lunar surface.
The second design is a hybrid which includes an inflatable shell attached to a

rigid backbone, providing about 224 m2 of growing area. This design employs an
aluminum backbone and airlock, incorporates premounted utilities, The shell
consists of a tough, polyurethane-coated nylon material. This second design com-
bines a moderate degree of assembly with partial turnkey operation, and thus
occupies an intermediateposition between the turnkey concept presented in Figure
4, and the full assembly version described below.
This third design is a large inflatable unit with approximately 528 m2 of plant
growing area (Figure 5). The shell, excluding the airlocks,would be fabricated fiom
the same polyurethane-coated nylon material used in the second design. To begin
operations, both inflatable concepts require the addition of atmosphere after they
are positioned on the moon. Plants growing in these units would make use of direct
sunlight during the lunar day, either through a sunlight collection system like the
one pictured in Figure 4, or by direct illumination through the wall. In this way
these concepts decrease the total power requirement of the life support system.
This type of inflatable technology provides a significantreduction in launch mass
(over a hard shell), and is feasible using advanced materials and materials technolo-
Figure 5. Artist’s concept of an inflatable plant growth unit on the lunar surface.
gies available today. In addition, although the concept of an inflatable greenhouse

on the surface of the Moon may seem unsafe, this is not the case. Another study
has shown that the dangers posed to plants from galactic cosmic radiation, solar
flares, and meteorite strikes are statistically very low, even over an assumed 20-year
lifetime of the fa~i1ity.I~ The primary drawback of this concept is that the lunar
night lasts two Earth weeks. As a consequence, it is necessary to provide artificial
lighting for the plants during the lunar night in order to maintain optimal growth
and productivity.
To handle the growth in crew size, different combinations of the three plant
growth unit designs would have to be installed as the base evolves. For a crew of
four, one of the Space Station-based modules would be sufficient. As the crew
grows to 30, another module and three ofthe hybrid designs would have to be added.
Finally, to meet the life support needs of a crew of 100,addition of three of the large
inflatable units would be required.
One of the primary drawbacks to the use of higher plants in food production
involves the need for lighting to support photosynthesis. The minimum acceptable
photosynthetically active radiation level is 300 to 600 pmol/m*/sec, when metal
halide or high pressure lamps are used. If artificial lighting is used for the plants,
0.5 to 1.O kW of electrical energy per square meter of growing area will be required
to produce this level. However, the electrical power required for illumination can
be reduced by using natural sunlight during the lunar day and providing low-inten-
sity artificial lighting (200 to 300 pmol/m2/sec) supplemented by higher atmos-
pheric CO, concentrations during the lunar night. The Lunar Base CELSS study
has identified two feasible methods for using sunlight, one using translucent,
greenhouse-like structures for plant growth areas and the other using sunlight
collectors and light conduits as distributors in opaque-walled plant growth units.
B. Food Processing
In the Lunar Base CELSS study, food processing hardware is minimized to

decrease launch mass. The selected food processing hardware would support
grinding of grains and beans to produce flours as well as general grinding of plant
biomass. Manual operations are assumed to be used for preparation of grain for
milling or fish meat for cooking.Human-inedibleplant material would be processed
by feeding it to the EZapia, either directly or after drying and grinding it into smaller
pieces.
C. Atmospheric Regeneration
The atmospheric revitalization subsystem in the Lunar Base CELSS design

utilizes higher plants for all CO, reduction and 0, production. The atmospheres of
the crew, plant, and animal chambers are isolated from one another by separate
physicochemical CO, and 0, removal systems (liquid-based scrubber/concentra-
tor). This atmospheric isolation provides for independent control of the respiratory
gas concentrations in the different chambers and helps prevent the spread of
contaminantsand microorganisms between the chambers. Temperatureand humid-
ity control are handled by standard condensing heat exchangers. Trace contaminant
removal is performed by a modified Space Station trace contaminant control system
design, which uses activated carbon adsorbent and a catalytic oxidizer. The acti-
vated carbon beds adsorb most of the contaminants, and the catalytic oxidizer
oxidizes any remaining contaminants at high temperature. The trace contaminant
control system adsorbent must be regenerated periodically by applying heat and
vacuum to the adsorbent beds. The effluent material can be captured and stored as
waste, or it can be degraded by the waste processing system and recycled.
D. Water Purification
Water reclamation by higher plants has been chosen as the primary method of
purification for the Lunar Base CELSS design. Water for drinking and food
preparation is obtained by treating condensate, collected directly from the crew
cabin, to remove trace contaminants. However, the volume of condensate water is
not suficient to fill the crew’s need for drinking and food preparation. Therefore,
the Lunar Base CELSS design proposes to make up for this deficit by recovering
condensate from the plant growth chamber and purifying it with the same systems.
Water for hygiene and clothes washing is taken directly from the plant condensate
collection and treated by UV light to remove bacteria and to degrade trace organic
compounds.The remainder of the condensate from the plant chamber and aquacul-
ture unit is recycled by returning it to the hydroponic nutrient solution, or by adding
it to the aquaculture system to make up for evaporative losses.
E. Waste Processing
The waste processor selected for the Lunar Base CELSS design is a low-pressure
wet oxidation system. This system is selected because it provides the most complete
processing at the lowest energy expenditure. The wet oxidation unit receives all
solid waste materials not fed to the aquaculture unit. These solid wastes includes
metabolic wastes produced by crew, animals and plants, and the non-metabolic
waste materials derived from packaging materials, daily activities,and so forth. The
wet oxidation unit will degrade these materials and then supply the effluent to the
plant growth chamber for addition to the hydroponic nutrient solution. The effluent
materials will then be further processed by the plants and bacteria.
VI. //V S/TU RESOURCE UTILIZATION

Utilization of material from the Moon could significantly affect the ultimate design
and operation of a lunar base life support system. A significant design advantage
in establishing a base on the lunar surface rather than an orbiting station is the
availability of in situ resources. Table 3 summarizes the elemental composition of
the loose surface material (regolith) of the Moon, determined in samples obtained
from the Apollo and Lunar missions.Table 4 summarizesthe elemental composition
by percent of a typical plant (corn) and the human body. It appears that over 95%
of plant biomass and over 87% of human biomass is composed of oxygen, carbon,
hydrogen and nitrogen. Thus, on a mass basis these four elements are the most
important for Lunar Base CELSS implementation. Of the four elements, only
oxygen is present in Lunar regolith in large amounts. As a consequence, from a life
support perspective the extraction of oxygen from regolith must be the initial target
for the technological development of in situ resource utilization as well as the
primary focus for interfacing with the Lunar Base CELSS. Although trace amounts
of carbon, hydrogen and nitrogen might be extracted from lunar regolith, these
elements will have to be supplied from Earth, at least initially. However, as the
capability for resource extraction develops, there will be less need to rely on
supplying even these elements from the Earth.
The Lunar Base CELSS design includes two methods by which oxygen can be
added to the support system. First, oxygen can be directly added to the crew
Table 3. Elemental Composition of Lunar Regolith22

Mare Mare Mare Mare Mare Basin Basin Basin
High Ti High Ti Low Ti LowTi Low Ti Ejecta Ejecta Ejecta
Element A-11 A-17 A-12 A-15 L-16 A-14 A-15 A-17
A1 7.29 5.80 7.25 5.46 8.21 9.21 9.28 10.9
Ca 8.66 7.59 7.54 6.96 8.63 7.71 6.27 9.1 9
Cr 0.21 0.31 0.24 0.36 0.20 0.15 0.19 0.18
Fe 12.2 13.6 12.0 15.3 12.7 10.3 9.00 6.68
K 0.1 2 0.06 0.22 0.08 0.08 0.46 0.1 4 0.1 3
Mg 4.93 5.80 5.98 6.81 5.30 5.71 6.28 6.21
Mn 0.1 6 0.19 0.1 7 0.19 0.1 6 0.11 0.1 2 0.08
Na 0.33 0.26 0.36 0.23 0.27 0.52 0.31 0.30
0 41.6 39.7 42.3 41.3 41.6 43.8 43.8 42.2
P 0.05 0.03 0.14 0.05 0.06 0.22 0.07 0.06
S 0.1 2 0.13 0.10 0.06 0.21 0.08 0.08 0.06
Si 19.8 18.6 21.6 21.5 20.5 22.4 21.7 21 .o
Ti 4.60 5.65 1.84 2.11 2.11 1.02 0.79 0.97
Table 4. Elemental Composition of Plant and

Human Tissues23
Element Plant fZea mais) Human
0 44.44 14.62
C 43.57 55.99
H 6.24 7.46
N 1.46 9.33
Si 1.17 ,005
K 0.92 1.09
Ca 0.23 4.67
P 0.20 3.11
Mg 0.1 8 0.16
S 0.1 7 0.78
CI 0.1 4 0.47
Al 0.1 1 -
Fe 0.08 0.01 2
Mn 0.04
Na - 0.47
Zn - 0.01
Rb - 0.005
atmosphere on an as required basis. Second, oxygen from in situ resource utilization

could be added to the oxygen storage buffer included in the atmospheric control
subsystem. The conceptual design assumes that in worst case the oxygen could be
isolated by the same type of installation that is to be used for the isolation of oxygen
from the plant growth unit. At best, the oxygen stream from the in situ resource
utilization plant would be filtered to remove particulates and then added to the crew
chamber or buffer. Thus, it appears that both interfaces are simple and direct, and
neither requires any unique or specific hardware.
Carbon, hydrogen, and nitrogen are also available in regolith, but at much lower
concentrations. Accordingly, the implementation of in situ resource utilization
technology for their extraction is a lower priority than that of oxygen. The addition
of nitrogen to the Lunar Base CELSS would be as straightforward as the addition
of oxygen, and should not require any unique hardware. Carbon and hydrogen
addition would be easiest in the form of CO, and water, respectively. Specific
hardware would be required to oxidize both elements prior to their addition to the
support system, but the actual addition of their oxidized forms will present no
problems, since storage buffers for both H,O and CO, exist in the conceptual
design.
Still another form of in situ resource utilization would involve the recovery of
plant macro- and micro-nutrient elements from regolith. At this point, the interfac-
ing requirements for this type of technology are difficult to establish, as the
chemical form in which the elements would be extracted determines the method of
addition to the support system.
VII. DESIGN CHARACTERISTICS AND BREAKEVEN

ANALY S IS
The estimated masses of the Lunar Base CELSS conceptual design to support the
three crew sizes of 4, 30, and 100 persons are presented in Table 5. It is clear that
the plant growth unit constitutes by far the largest subsystem in all three cases. In
the 4-person crew, the plant growth unit accounts for 82% of the total life support
system mass, while for the 30- and 100-person crew sizes, these units account for
79% and 74%, respectively, of the total mass. This percentual decrease is due to the
addition of the larger, but lighter, plant growth unit designs as the base nears
maturity. The second largest subsystem is the aquaculture unit, which comprises
9-1 2% of the total life support system mass.
The food and oxygen reserves were calculated for different time intervals. For
the food production system it was assumed that it might take up to one full crop
cycle, expected to last 60-90 days, to return to equilibrium. Thus the food reserve
was set to last for a 90-day period. Interruption of the food production cycle will
also stop the biological oxygen production. However, it was assumed that an
adequate oxygen production would already resume about 30 days after starting a
new crop.
Table 5. Mass Estimates of Lunar Base CELSS Components for Different Crew
Estimated Mass by Crew Size
Subs ystem/Component 4 30 100

Plant Growth Unit(s) 12,322 78,641 209,081
Solid Waste Processing 63 273 808
Atmosphere Regeneration 271 1,169 3,016
Water Purification 31 233 778
Aquaculture (Tdapia) 1,366 10.1 693 3,695
Food Processing 26 52 122
Inflation Gas 0 1,446 12,014
90-Day Food Reserve 565 4,239 14,130
30-Day Oxygen Reserve -3% 2352 9.840
Total 15,038 99,174 283,484
Note: All figures in kilograms.
Estimates of the electrical power required to operate the life support system for
these three crew sizes are shown in Table 6. The maximum power values are
required only during the lunar night, when the entire photosynthetic active radiation
required by the plants must be provided by artificial light. The minimum operating
power values, applicable during the lunar day when this radiation can be supplied
by natural sunlight, are also presented for comparison purposes. It is clear that the
use of sunlight will dramatically decrease the total power requirement for the life
support system.
One of the most important questions addressed by the Lunar Base CELSS study
was to estimate the economic feasibility of such a system. Myers2’ has developed
a method to do this by graphically determining the mission length at which a
regenerativelife support system begins to pay off economically.In this “breakeven”
method the mass of life supportconsumablesis added to the mass of the life support
hardware required to maintain one person. Figure 6 presents the results of such an
Table 6, Power Estimates of Lunar Base CELSS for Different

Crew Sizes
Power Requirement (kW)
~~ ~~
lunar Night lunar Day

Crew Size (maximum) (minimum)
4 72 12
30 61 7 94
100 1,700 226
RESUPPLY FOOD PRODUCTION

WITH WASTE PROCESSING
\ ...”.,..
,,..**’.
....
,...I..
. .. ......‘
...a
MASS /,
...a
. _,._. .. .’ , .. ”
PER \
ATMOSPHERE
PERSON REGENERATION
0
0
MISSION DURATION
Figure 6. Graphical method for determining breakeven points (after Myers2’).
analysis, where the mass to be launched per crew member is plotted as a function
of the mission duration. In this figure the line labeled “RESUPPLY” represents a
scenario in which all consumables must be resupplied and no recycling life support
equipment is used. The y-intercept of this line is the launch mass at the time of first
launch, which is set to zero. The slope of this line is equal to the resupply mass
required to support one person per time unit expressed as a fraction of the mission
duration.
The addition of regenerative equipment increases the initial launch mass, as given
by the y-intercept. However, the need for resupply of consumables decreases, as
expressed by a decreased slope of the line. In the scenario labeled “WATER
RECYCLING the mission launch mass reflects the addition of the water recycling
hardware. The breakeven point comes relatively soon at point 1. Thereafter, the
resupply mass required to sustain one person decreases to 35% of that required
without recycling of water in this hypothetical case. This reduction equates directly
to an economic savings, as the launch cost of each kilogram of material is
approximately constant for a specific launch vehicle.
When additionallyhardware for atmospheric regeneration is included, the break-
even point comes later (point 2), but the resupply mass decreases to 6.3% in this
case, as indicated by the line labeled “ATMOSPHERE REGENERATION.” When
in addition hardware for production of food and recycling of solid waste is
incorporated, the breakeven point comes still later (point 3), but the resupply mass
falls to nearly zero, as indicated by the line labeled “FOOD PRODUCTION WITH
WASTE PROCESSING.” It is clear that increasing the regenerative capability
lengthens the time required to reach the breakeven point, but greatly decreases the
required resupply of consumables. Full recycling pays off only for longer missions.
O F0 1 2 3 4
MISSION DURATION (yr)
Figure 7. Cumulative launch mass of open loop, partially closed ( P K ) and closed-
loop life support systems (CELSS) for a 4-person lunar base as a function of mission
duration.
By means of such a breakeven analysis we have calculated the potential mass

savings produced by a Lunar Base CELSS designed as described in this chapter. It
has been performed by using the estimated life support mass required for a 4-person
crew. Figure 7 illustratesthe cumulative mass of life support materials required by
a 4-person crew as a function of time, if no recycling is used (OPEN LOOP). If
equipment to recycle air and water is added, the initial launch mass increases, but
the breakeven point relative to 100% resupply is already reached after 1 month, and
the cumulative amount of life-sustaining materials launched during the mission is
greatly reduced (P/C). When a complete recycling system for atmosphere, water,
food, and waste is transported to the Moon, the breakeven point relative to 100%
resupply comes after about 5 months and after 2.5 years relative to air and water
recycling only. Thus, in terms of cumulative launch mass savings, a complete
recycling Lunar Base CELSS makes sense only for a mission of 2.5 years duration
or longer.
VIII. CONCLUSIONS AND SUMMARY

The requirementsfor a human life support system for long-duration space missions
are reviewed. The system design of a controlled ecological life support system is
briefly described, followed by a more detailed account of the study of the concep-
tual design of a Lunar Base CELSS. The latter is to provide a safe, reliable, recycling
lunar base life support system based on a hybrid physicochemicalhiological
regenerative technology.
The most important conclusion reached by this study is that implementation of
a completely recycling CELSS approach for a lunar base is not only feasible, but
eminently practical. On a cumulative launch mass basis, a 4-person Lunar Base
CELSS would pay for itself in approximately 2.6 years relative to a physicochemi-
cal aidwater recycling system with resupply of food from the Earth. For crew sizes
of 30 and 100, the breakevenpoint would come even sooner,after 2.1 and 1.7 years,
respectively, due to the increased mass savings that can be realized with the larger
plant growth units.
Two other conclusions are particularly important with regard to the orientation
of hture research and technology development. First, the mass estimates of the
Lunar Base CELSS indicate that a primary design objective in implementing this
kind of system must be to minimize the mass and power requirement of the food
productionplant growth units, which greatly surpass those of the other air and water
recycling systems. Consequently,substantial research must be directed at identify-
ing ways to produce food more efficiently. On the other hand, detailed studies to
identify the best technology options for the other subsystemsshould not be expected
to produce dramatic reductions in either mass or power requirement of a Lunar Base
CELSS. The most crucial evaluation criterion must, therefore, be the capability for
functional integration of these technologies into the ultimate design of the system.
Secondly, this study illustrates that existing or near-term technologies are ade-
quate to implement a Lunar Base CELSS. There are no apparent “show-stoppers”
which require the development of new technologies. However, there are several
areas in which new materials and technologies could be used for a more efficient
implementation of the system, e.g., by decreasing mass or power requirement and
increasing recycling efficiency. These areas must be further addressed through
research and development.
Finally, although this study focused on the development of a Lunar Base CELSS,
the same technologiesand a nearly identical design would be appropriatefor a Mars
base. Actually, except for the distance of transportation, the implementation of a
CELSS on Mars would even be easier than it would be on the Moon. The presence
of atmospheric CO, on Mars, although in low concentration, coupled with the fact
that the dayhight cycle on Mars is very similar to that on Earth, makes the use of
light-weight, greenhouse-like structuresfor growing food plants even more feasible
than on the Moon. There are some environmental problems, which would have to
be dealt with, like dust storms and the large amount of ultraviolet radiation incident
on the planet’s surface. However, the materials and methods are largely available
today to develop such a life support system for a Mars base.
REFERENCES
I . Calloway, D.H. Basic data for planning life-support systems. In: Foundations ofspace Biology
and Medicine (Calvin, M. and Gazenko, O.G., Eds.), pp. 3-2 1. Vol. 111. National Aeronautics and
Space Administration, Washington, D.C., 1975.
2. Bozich, W.F. Presentation at TMSA Space Requirements Conference. March, 1991, Los Angeles,
CA.
3. Gary P. Noyes, Carbon Dioxide Reduction Processes for Spacecraft ECLSS: A Comprehensive
Review. Proceedings 18th Intersociety Confirence on Environmental Systems, July 1988, San
Francisco, paper 88 1042.
4. Wieland, P.O. Designingfor Human Presence in Space: An Introduction to Environmental Control

and Life Support Systems. NASA-Marshall Spaceflight Center, NASA Reference Publication no.
1324, NASA, Washington, D.C., 1994.
5. Friedman, M.A., Schwartzkopf, S.H., Styczynski, T.E., Tleimat, B., Tleimat, M. Gray Water
Recycling with a Unique Vapor Compression Distillation (VCD) Design. Proceedings 22nd
International Conference on Environmental Systems, July, 1992, Seattle, WA, paper no. 9213 18.
6. Martin, J.H., Leonard, W.H., Stamp, D.L. Principles of Field Crop Production. Macmillan
Publishing Co., New York, 1976.
7. Wolverton, B.C., McDonald, R.C., Duffer, W.R. Microrganisms and higher plants for waste water
treatment. Journal of Environmental Quality. 12(2):23&244, 1983.
8. Lamparter, R.A. Personal Communication, 1991.
9. Berman, G.. Murashige, K. (Eds.) Synthetic Carbohydrate:An Aidto Nutrition in the Future. Final
Report of the Stanford-Ames NASNASEE Summer Faculty Systems Design Workshop, NASA
Contract NGR-05-020-409, January, 1973.
10. Shapira, J. Design and evaluation of chemically synthesized food for long space missions. In: The
Closed Life-Support System, NASA-Ames Research Center, NASA SP- 134. pp. 1 7 5 1 87. NASA,
11. Miller, R.L., Ward, C.H. Algal bioregenerative systems. In: Atmosphere in Space Cabins and
Closed Environments ( K . Kammermeyer, Ed.), pp. 186-222. Appleton. New York, 1966.
12. Ward,C.H., Wilks, S.S.,Craft H.L. Useofalgaeandotherplantsin thedevelopmentoflifesupport
systems. American Biology Teacher 2 5 5 12-524, 1963.
13. Hoff, J.E., Howe, J.M., Mitchell, C.A. Nutritional and CulturalAspectsofPlant Species Selection
f o r a Controlled Ecological Life Support System. NASAContractor Report#166324,NASA-Ames
Research Center, Moffett Field, CA, 1982.
14. London, S.A., West, A. Gaseous Exchange in a Closed Ecological Life Support System. Report
from Aerospace Medicine Research Lab, Wright-Patterson A.F.B., Ohio, 1962.
15. Bowman, R.O., Thomae, F.W. An algae life support system. Aerospace Engineering, 19(12):
2-2, 1960.
16. Bowman, R.O., Thomae, F.W. Long-term non-toxic life support of animal life with algae. Science,
134:55--56, 1961.
17. Ivanov, B., Zubareva, 0.To Mars and back again on board bios. Soviet Life. April 1985, pp. 22-25.
18. Lockheed Missiles & Space Co., Inc. Lunar Base Controlled Ecological Life Support System
(LCELSS). Preliminary Conceptual Design Study: Final Report. LMSCIF280196, April 30,199 1.
19. Schwartzkopf, S. Hazard and risk assessment for surface components of a lunar base controlled
ecological life support system. Proceedings 22nd International Conference on Environmental
Systems, July 1992, paper 921285.
20. Myers, J. Space biology: ecological aspects-introductory remarks. American Biology Teacher
25:40W12, 1963.
21. Phillips, J.M., Harlan, A.D., Krumhar, K.C., Caldwell, M.S., Crowlie, C.M., Ramsbacher, L.,
Meyer, B.S. Studies of Potential Biological Components of Closed Life Support Systemsfor Large
Space Habitats: Research and Technology Development Requirements, Costs, Priorities and
Terrestrial Impacts. Final Report, Grant NSG2309. NASA-Ames Research Center, 1978.
22. Phinney, W.C., Criswell, D., Drexler, E., Garmirian, J. Lunar Resources and Their Utilization. In:
Space-Based Manufacturingfrom Non-terrestrialMaterials (G. O’Neill, Ed.), pp. xx-xx. Prince-
ton University Press, Princeton, NJ, 1977.
23. Epstein, E. Mineral Nutrition ofPlants: Principles and Perspectives. John Wiley and Sons, New
York, N.Y., 1972.
Chapter 11
B I0R EGENERATI0N WITH M ALTOSE

EXCRETING CHLORELLA: SYSTEM
CONCEPT, TECH NOLOGICAL
DEVELOPMENT, AND EXPERIMENTS
L uz i an Wo If
I. Introduction . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . 256

.. .
11. Earlier Bioreactor Designs .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 257
111. System Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
IV. Technological Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
A. Tubular Photo-Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . 259
B. GasLiquid Separator with Low Shear-Stress Pump . . . . . . . . . . . . 262
C. Gas Dehumidifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
D. Maltose Separator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
E. Liquid Storage and Transfer Unit . . . . . . . . . . . . . . . . . . . . . 265
F. Monitor and Control System . . . . . . . . . . . . . . . . . . . . . . . . 266

ISBN: 0-7623-0147-3
255
256 LUZIAN WOLF
V. Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268
A. Open Gas Loop Experiments . . . . . . . . . . . . . . . . . . . . . . . .268
B. Closed Gas Loop Experiments . . . . . . . . . . . . . . . . . . . . . . ,269
C. Closed AlgaeInsects System . . . . . . . . . . . . . . . . . . . . . . ,269
VI. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . .272
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274
1. INTRODUCTION
The perspective of long duration missions led ESA in 1984 to initiate a technical
study of a regenerative system for the support of long duration biological experi-
ments on-board spacecraft. The study concentrated on the development of a system
for the regeneration of water, oxygen, and food from the metabolic end products of
the biological experiment. The system was to be small in size, reliable, working
fully automatic for up to one year, and satisfling the needs of the biological
experiment at all times. A trade-off of several designs favored a photosynthetic
bioreactor housing a controlled culture of the maltose excreting green micro alga
Chlorellu (strain 241.80, Gottingen).'.2 The idea was to link this photosynthetic
bioreactor to a biological experiment to form a partially closed two-compartment
artificial ecosystem (Figure 1). The algae assimilate CO, and water produced by
the biological experiment, and convert these compounds with the help of light
energy to oxygen and carbohydrates,primarily maltose, which are excreted into the
culture medium. The organisms in the biological experiment may then utilize the
carbohydratesas a source of carbon and energy under formation of CO, and water,
which can again be assimilated by the algae.
The requirement of a small size led us to study algal cultures with a high biomass
concentration. Such cultures have the obvious advantage that the culture volume
NTIENTS NUTRIENTS
I
, MALTOSE ,
02 c
LIGHT,
4
PHOTOSYN.
c02
PRODUCER 4 CONSUMER
Figure 1. Partially closed artificial ecosystem consisting of consumer and photosyn-

thetic producer compartments.
Bioregeneration with Chlorella 257
required to achieve target gas conversion rates can be relatively small, which in
turn keeps down the mass and volume of the system.
II. EARLIER BIOREACTOR D E S I G N S

High density cultures require a suitably designed bioreactor, which will supply the
substrates required for photosynthesis at sufficient rates. The two substrates are
light and CO,. The availability of sufficient light is a limiting factor in the growth
of high density cultures. Such cultures absorb much ofthe incident light in the outer
layer of the culture. In a Chlorellu culture with a biomass concentration of 4.0 g
dry weight per liter 90% of the incident light is absorbed in the outer 4-mm layer
of the culture. All algae beneath this 4-mm layer will thus receive only 10% of the
incident light. This calls for effective mechanisms to supply sufficient light to all
cells in the culture. Only then will a high-density culture be efficient.
The problem of light supply has been successfully approached with tubular
bioreactor concepts. Pirt et al.4 built and tested a tubular photobioreactor that
consists of 52 glass tubes (each 100 cm long and 1 cm inner diameter), connected
to U-bends by silicone rubber tubing. Gas exchange and pumping is provided by
an external airlift stage. Biomass concentrations of more than 20 g.1-I dry weight
were achieved with CO, concentrations of 5% in the aerating gas. Oguchi et al.5
used a semi-transparent flexible Teflon tube with 6 mm inner diameter, coiled
around a fluorescent tube. They succeeded in batch-culturing Spirulina to a biomass
concentration of 4.5 g.T’ dry weight. Gas exchange was provided by an external
hollow fiber membrane module. At the STI Company, Japan, photo-bioreactor
prototypes have been developed that use an array of light-diffusing fibers to
introduce light into the reactor vessel.
The other substrate that can limit the growth ofhigh density algal cultures is CO,.
One gram dry weight of Chlorellu can fix 2.5 ml CO, per minute and produce about
the same volume of oxygen under optimal conditions for photo~ynthesis.~ Aculture
with a biomass concentration of 4.0 g dry weight per liter may thus require an
exchange of 10 ml CO, and 0, per minute between the liquid and the gas phase. It
is difficult to achieve this gas exchange rate, when the bioreactor is aerated with
vent gas from the consumer compartment of a typical life support application,since
the CO, concentration in the respiration gas may not exceed 0.5%.67’Every liter of
culture suspension must be brought in close contact with at least 2 liters of gas per
minute to support the above mentioned rates of photosynthesis.This is a very high
aeration rate, even for terrestrial applications. The way to solve this problem is to
stage the CO, concentration, but this increases the complexity of the system.
Gas exchange techniques on the ground rely on rising bubbles or large free
gadliquid interfaces. These techniques are not feasible in spacecraft due to the lack
of buoyancy forces in weightlessness. In microgravity bubbles do not rise, and gas
and liquid do not easily separate.
258 LUZIAN WOLF
One approach to aerate algal cultures in weightlessness, while keeping the gas
and liquid phases separate,is to use artificial gravity.Abioreactor designed to utilize
this approach consists of a static cylindricaltank with a slowly rotating (< 100 rpm)
axial paddle that generates an annular fluid layer of culture on the inner wall of the
tarkg Illumination is provided through the transparent wall of the tank by a
cylindncal array of fluorescent tubes. Gas exchange occurs over the free gas/liquid
surface, and can be augmented by bubbling air through gas outlets in the paddle.
The paddle is magnetically driven, removing the need for rotary seals that are
frequently a source of contamination. A prototype of this bioreactor has been built
and tested on the ground. However, proper development of this concept will require
extensive testing in weightlessness, which so far has not been carried out.
Another approach employs permeable membranes, which have been tried in
various arrangements to provide gas exchange for algal cultures. Hollow fiber
membrane modules have been used as gas exchanger in cultures of Spirulina’ and
Chlorella.6 The algal suspension is pumped through the module casing, and gas
through the fibers. Although gas exchange was achieved, it was noticed that culture
liquid occasionally enters the hollow fibers,thus blocking an increasing number of
fibers with time. The fibers cannot be blown free and thus cannot be made suitable
for gas exchange again. Algae also become attached to the hollow fibers, thus
reducing the overall gas exchange efficiency with time. Permeable membranes add
an additional diffusion barrier that decreases gas exchange rates. Sterilization
presents another problem. Hot steam can often not be used because of a limited
temperature rating of the membranes, while sterilization with chemical agents like
hydrogen peroxide may alter the membrane properties.
111. SYSTEM CONCEPT

In this section the system concept of a photosynthetic producer compartment for
high-density algal cultures is described. Some technological developments that
make the system suitable for operation in conditions of weightlessness are detailed
in Section IV. Initial experiments conducted with a prototype system are reported
in Section V.
The system concept of the photosynthetic producer compartment encompasses
all processes necessary to maintain a high-density continuous culture of Chlorella
241.80 over long periods of time, to operate it as a CO, to 0, converter, to generate
maltose and to separate it from the medium, and to control the culture by tuning
the photosynthetic metabolism to match at all times the needs of the consumer
(Figure 2).
The core component is a dedicated photo-bioreactor for cultivation of algae with
an associated gadliquid phase separator and a circulation pump. This core is
completed by the following subsystems: (1) an intensity controlled illumination
subsystem to provide light energy for photosynthesis, (2) a temperature control
subsystem, (3) a CO,/O, gas analyzer to monitor the gas exchange requirementsof
Bioregeneration with Chlorel la 259
/I I
Figure 2. Diagram of the Chlorella bioregenerative life support system
the consumer and the metabolic state of the photosynthetic producer, (4) a liquid
storage and transfer system to supply the producer with nutrients and various
additives when required, ( 5 ) a pH and a photometer flow-throughcell for monitor-
ing culture pH and biomass concentration, (6) a dehumidifier to remove excess
water vapor from the gas recycled to the consumer,(7) a maltose separator to harvest
photosyntheticallyproduced maltose from the culture medium, (8) a cell separator
to remove excessbiomass, (9) some other storage vessels, and (1 0) aprocess control
system with a user interface.
IV. TECHNOLOGICAL DEVELOPMENT

A. Tubular Photo-Bioreactor
The tubular photo-bioreactor has been designed to culture micro algae in weight-
lessness at high biomass concentrations. The particular design chosen solves the
problems of providing an efficient gas supply and sufficient light to a high density
culture. The design is shown schematically in Figure 3.
The bioreactor consists of40 transparent Pyrex glass tubes with an inner diameter
of 4 mm and a length of 2.5 m each. The tubes are folded four times to reduce the
260 LUZIAN WOLF
Figure 3. Principle of the tubular photo-bioreactor.
geometric dimensions ofthe bioreactor. They are inserted in a base block in parallel
arrangement, and sealed with O-rings between the base block and the top plate. The
base block contains numerous ducts and elements to route culture liquid and gas to
and from the tubes.
Culture liquid and gas enter the base block through separate inlet ports, and are
routed to 40 T-junction gadliquid mixers which are located in the base block just
before the inlet of each transparent tube. The gashquid mixers are equipped with
flow restrictors (10 x 0.9 mm for culture liquid, and 10 x 0.15 mm for gas) to ensure
an equal distribution of gas and liquid flow in all tubes. The continuous injection
of gas into the liquid stream leads to the formation of a train of gas and liquid
cylinders, which move through the transparent tubes (Figure 4). Gas exchange can
thus take place over a free gadliquid interface, and at the same time the culture
liquid can be illuminated very effectively through the transparent tubes. The gas
and liquid cylinders from all tubes are collected in a collection duct, and leave the
base block through the outlet port to an external gadliquid separator and circulation
pump (described in the next section).
The prototype bioreactor is 60 cm high, 40 cm wide, 4 cm deep and has an internal
volume of 1.3 liters. At equal flow rates of gas and liquid (a liquid to gas ratio of
figure 4. Train of culture liquid and gas cylinders in tubular photo-bioreactor.
1:1) the bioreactor contains 0.65 1 culture liquid and 0.65 1gas. When the bioreactor
is illuminated on both sides, the total illuminated transparent surface is 0.45 m2,
resulting in a specific illumination area of 7 cm2.ml-' culture fluid. The average
hold-up time of gas in the liquid is about 20 seconds.The bioreactor is manufactured
from biocompatible material and can be sterilized in an autoclave or with hot
hydrogen peroxide solution. The tubular design is expected to facilitate emptying,
rinsing, cleaning, and sterilization of the bioreactor in microgravity conditions.
262 LUZIAN WOLF
A pressure gradient of approximately 50 hPa between inlet and outlet ports causes
culture liquid and gas to flow through the bioreactor at a nominal flow rate of 1.5
1.min-I. The pattern of movement of culture liquid and gas cylinders through the
narrow tubes is nearly independent of the position in which the bioreactor is
mounted, even if it is mounted upside down. We consider this as an indication that
the design should operate also in weightlessness. The length of the liquid and gas
cylinders depends on the flow rate through the bioreactor, and on the rheological
properties of the culture liquid. When the reactor is operated with distilled water,
the cylinder length is about 20 mm, whereas an average length of 5 mm is observed
for culture liquids with algal biomass concentrations from 2 to 9 g.1-' dry weight.
The cylinder movement inside a tube causes a current inside the liquid cylinder that
circulates the liquid from the inside to the outside, and vice versa.
Gas transfer rates for CO, have been measured under operational conditions. The
bioreactor was filled with 0.1 M NaOH and repetitively aerated with air and with
air + 0.5% CO, at intervals of 10 minutes. Gas transfer rates for CO,, calculated
from the amount of CO, dissolved and released in the bioreactor in one aeration
cycle, reached 5.0 ml.min-', or about 1.0 ml.min-' Pa-'.
Light energy for photosynthesis is provided by an array of 3 x 6 spot lamps
(Wotan Decostar 51, 36", 20W). These are connected to a dimmer, which allows
the light intensity at the bioreactor surface to be adjusted between 0 and 300
pE.rn-,s-l.
B. Gas/Liquid Separator with l o w Shear-Stress Pump

The tubular photo bioreactor requires a pressure gradient of approximately 50
hPa between the inlet and outlet ports in order to maintain a nominal flow rate of
1.5 1.min-l culture liquid and 1.5 1.min-' gas through the tubes. An external phase
separator is required to separate culture liquid and gas at the nominal flow rate.
Initial experiments with centrifugal and membrane pumps showed that the vitality
of the suspended algal cells is strongly affected by vigorous pumping. This means
that low shear-stress pumping is req~ired.~ The two functions of gadliquid separa-
tion and low shear-stress pumping have been implementedwith two slowly rotating
centrifugal pumps, which are operated at the same time as pneumatic pumps
(Figure 5).
The combined gadliquid separator and centrifugal pump consists of a stainless
steel chamber containing a rotor with radial vanes. The rotor is rotated by a dc
motor/gearbox/tacho-generator combination mounted on top of the chamber. The
rotor has a diameter of 130 mm, which for a nominal rotational speed of 150 rpm
provides a peripheral centrifugal force equivalent to 1.5 G and a linear velocity of
1 d s . Torque is transmitted to the rotor via a magnetic coupling, which obviates
the need for rotating seals. A window is provided to allow visual inspection of rotor
movement and liquid levels.
Bioregeneration with Chlorel la 263
gas out phase 1 phase 2
Figure 5. Microgravity-compatibleconcept for gashquid separation and low shear

stress pumping.
The gadliquid separator and the low shear stress pump work as follows (Figure
6): Bubbles are separated from the liquid by two centrifuges C1 and C2 which are
arranged in parallel. Pumping is achieved in two phases by alternatively pressuriz-
ing and venting C1 and C2 with pump P and solenoid valves V1 and V2. In phase
1 C 1 is pressurized and emptied through valve V4, while C2 is vented and filled
through V5. Valves V3 and V6 are kept closed by the pressure difference between
C1 and C2. In phase 2 C2 is pressurized and emptied through valve V6, while C1
is vented and filled through V3. Valves V4 and V5 are kept closed by the pressure
difference between C 1 and C2. A level detection system automaticallyswitchesthe
solenoid valves V1 and V2.
This prototype was tested during the 18th ESA Parabolic Flight Campaign in
March 1994. It separated gas and liquid at liquid flow rates between 65 and 550
ml.min-' and gas flow rates between 110 and 1000ml.min-'. Other flow rates were
not tested due to lack of experiment time.
C. Gas Dehumidifier
Air leaving the gadliquid separation centrifuges is saturated with water vapor.
Hence the humidity must be reduced before returning the air to the consumer
compartment. Dehumidification is performed by cooling the air below the dew
point and removing the resulting condensate.
The prototype dehumidifier, schematically presented in Figure 6, consists of a
cylindrical chamber with a volume of 400 ml. A mesh of woven stainless steel with
a pore size of 15 pm and a bubble point pressure of about 100 hPa is mounted close
264 LUZIAN WOLF
gas in gas out
Figure 6. Diagram of a microgravity:compatible dehumidifier.
to the bottom of the chamber. The mesh is cooled by an external heat exchanger.
Input air impinges on the mesh and is cooled below the dew point. Condensate
forms on the mesh and is drawn by a pressure gradient of 20 hPa into the narrow
gap between chamber bottom and mesh. Air does not penetrate the mesh, provided
the pressure gradient does not exceed the bubble point of the pores. A miniature
piston pump removes condensate from the gap in small batches of 50 pl. Test runs
with the dehumidifier prototype show that the relative humidity of air of 25OC can
be reduced from 99% to 4 5 7 0 % at flow rates of 3-8 1.min-' (Figure 7).
80
70 --
6o --
-
1 , 85 llmin
I/min
3 llmin
50 --
40 -- I I
1
I
30 -- T
i
20 --
l o -- i
0 I
I
4
I
I I I
I
I
I
Bioregenerafion with Chlorella 265
figure 8. Design of reverse osmosis maltose separator. 1. top plate; 2. viton-dia-

phragm; 3. spiral plate; 4. central screen; 5 . support screen and reverse osmosis
membrane; 7. drainage plate; 8. O-ring.
D. Maltose Separator
This component serves to separate maltose produced by the algae from the culture
liquid and to concentrate it 10-15-fold to permit its utilization for food purposes.
This function can be implemented by reverse osmosis. The design of the apparatus
is shown in Figure 8.
A batch of 100 ml cell-free culture liquid is pumped into the space between the
diaphragm and a spiral plate. The space above the diaphragm is pressurized to 14
bar. Culture liquid and electrolytes permeate through the reverse osmosis mem-
brane (DDS Filtron HC50) and return to the culture. Maltose is retained on the
pressure side of the membrane. An external pump circulates the culture liquid in a
spiral path above the high pressure side of the membrane with a cross flow speed
Of 5 m.s-l.
The maltose separator prototype has been tested with culture liquid containing
7.5 g.1-I maltose and 3.3 g.1-I KNO,. The maltose concentration in the permeate is
decreased to between 2 and 3 g.l-', while it is increased to between 80 and 100 g.T'
in the concentrate. The KNO, concentration in the concentrate was increased to 5
g.T' while it was slightly decreased in the permeate.
E. Liquid Storage and Transfer Unit

The liquid storage and transfer unit adds water, acid, base, anti-foam compound,
and macro- and micro-nutrients to the algal culture as required (Figure 9). Liquids
in the prototype assembly are stored in glass bottles, but will later be stored in elastic
bags in order to permit operation in microgravity. Liquids are transferred in 50 yl
batches by means of 7 miniature solenoid pumps (LEE LPLA1220050L). The
solenoid pumps are mounted on a base plate and connected through a manifold to
one common conduit. Liquid batches that have been pumped into the manifold are
266 LUZIAN WOLF
control
Figure 9. Design of liquid storage and transfer system.
transportedby a continuous flow of gas through the common conduit to the bioreactor.
Since solenoid pumps cannot be autoclaved without damage, the liquid storage and
transfer unit is sterilized by treatment with 70% iso-propylic alcohol for 12 hours.
F. Monitor and Control System
Parameters Monitored and Controlled
The following parameters of the algal culture are monitored and controlled: pH,
temperature, culture volume, biomass concentration,CO, and 0, concentration in
the bioreactor vent gas, flow of inlet gas, bioreactor pressure, light intensity (by
CO, concentration in vent gas), and nutrient addition (by the photosyntheticactivity
of the culture). In hture matching of the algal photosynthetic quotient to the
respiratory quotient of the experimental animals is also to be controlled.
Sensor Bypass for pH and Biomass Measurement
A bypass between bioreactor inlet and outlet guides a fraction ofthe culture liquid
through a pH flow-through cell and a small flow-through photometer for biomass
measurement. The 50 Ma pressure difference between bioreactor inlet and outlet
at nominal flow rates maintains the fluid flow through the bypass without additional
pumping.
The pH flow-through cell consists of a pH electrode (INGOLD 465-35-T-Kg) in
a custom-built cell. The pH electrode is pressurized at 300 hPa to compensate for
the positive pressure inside the bioreactor.
The photometer consists of an infrared light-emitting diode (IR-LED, 940 nm),
a photo transistor, a flow-through cell with a 2-mm light path (Hellma 138-0s)and
control electronics. The voltage output of the photometer is converted to the
biomass concentration by means of a polynomial calibration function. The pho-
tometer can be adjusted to measure biomass concentrationsfrom 0.1 to 10 g.1-I dry
weight by controlling the current of the IR-LED.
Bioregeneration with Ch lorella 267
Measurement of Other Parameters
The CO, concentration is measured in the inlet and vent gas of the bioreactor by
routing the gas to a gas analyzer assembly. The latter consists of a solenoid valve
manifold, a zirconia solid electrolyte oxygen sensor (Fujikura FCX-U-A-ST), an
infrared light absorption CO, analyzer (Servomex 'Gascard' 1370), and a pressure
sensor (Honeywell 185PC15AT). The temperature ofthe culture liquid is measured
by a temperature sensor (Honeywell TD5A) attached to the baseblock of the
bioreactor. The culture volume is estimated from the average switching time of the
debubbling centrifuges.
Control of Parameters
The algal cultivation is continuously controlled by a PC, which interfaces

(RS485) to digital and analog VO units (Mistic 200). The pH of the culture liquid
is maintained at pH 6.5 through the addition of acid or base. This pH value is optimal
for maltose production while maintaining cell g r ~ w t hThe
. ~ culture temperature is
currently maintained at 20°C, but further experiments are required to determine the
optimal temperature value.
The light intensity is controlled by the CO, concentration in the bioreactor vent
gas. It is decreased when the CO, concentration drops below 0.2%, while it is
increased when the latter rises above 0.2%.
The culture volume is controlled by the addition of water. The biomass concen-
tration is currently controlled by bleeding some of it into a waste reservoir when it
exceeds the desired value, and replacing it by the addition of an equal volume of
water. A cell harvesting system, based on cross-flow filtration, is being developed,
and will later be implemented in the prototype system.
Nutrient addition is controlled by the photosynthetic activity of the algal culture
at defined reference conditions. Once every 6 hours, the bioreactor is disconnected
from any external gas supply (or consumer compartment), the bioreactor gas inlet
and outlet are connected to each other, light is switched off and the pH control is
disabled. The bioreactor is then operated in this state until the CO, concentration
in the closed gas loop has increased to 0.4% due to respiration of the algae. At this
point, light with an intensity of 100 pE.mP2s-' is switched on, and the time is
measured until the CO, concentration has decreased to 0.2%. This time is used as
a measure for the photosynthetic activity of the culture. A long time indicates a low
concentration of photosynthesizing cells and triggers the addition of nutrients.
The photosynthetic quotient (mol 0, produced per mol CO, assimilated) of the
algal culture must be matched to the respiratory quotient (mol CO, produced per
mol 0, consumed) of the consumer compartment. A control to match these two
quotients has not yet been implemented, but experiments are in progress to achieve
this control by means of two nutrient solutions, one containing nitrate as a nitrogen
source, and the other containing urea.
268 LUZIAN WOLF
V. EXPERIMENTS
A. Open Gas loop Experiments
Description
Chlorella 241.80 was grown in the bioreactor to a biomass concentration of 0.6

to 4.5 g.1-I dry weight. The bioreactor was supplied with air + 0.5% CO, at gas flow
rates between 1.2 and 1.5 1.min-l and a liquid flow rate of 2.0 l.min-l. Temperature
was set to 20°C, the pH to 6.5, Light intensity was varied between 0 and 300
pE.rn-,s-' in steps of 50 pE.rn-,s-l lasting 30 minutes each. CO, and 0, concen-
trations at the bioreactor outlet were measured and recorded. The specific CO,
uptake and 0, production rates (in ml.min-'g-I) are calculated with the equations:
AcCO, .&as Aco2 .&a$

and ro2 =
rc02 = xv x.v
where Acco, is the difference of CO, concentrations between outlet and inlet and
Aco2 the difference of 0, concentration between the same,f,, the gas flow rate, X
the biomass concentration and V the total volume of the culture liquid.
spec.
1
0.8 0
0.6 9 t
8 QI
0.4 8
0.2
0a
-0.2
-0.4
..
O
8
* * #
I I
ii 0
I
0 8
-0.6 0 4)
4)
-0.8
-1
0 50 100 150 200 250 300
spec. C02 consumption rate [ml/min.g] light intensity [pE/m2.s]
Figure 10. Specific C 0 2 uptake rates and 0 2 production rates as a function of light
intensity in Chlorella culture in photo-bioreactor in open gas-loop mode. Biomass
concentration 4.5 g.1-l dry weight.
Results
The specific CO, uptake rates and 0, production rates in cultures with a biomass
concentration of 4.5 g.1-' dry weight reached a value of 0.6 ml.g-'min-' at a light
intensity of 300 pE.m-2s-' (Figure 10). In cultures with a biomass concentration of
0.6 g.1-' these specific rates reached a value of 1.5 m1.g-'mid at the same light
intensity. The lower value at the high biomass concentration may be explained by
auto shading of algae. The results in Figure 10 suggest that a light intensity of 300
pE.mP2s-' was not sufficient to reach saturation of the photosynthetic process.
B. Closed Gas loop Experiments
Description
ChIoreIIa 241.80 was grown in the bioreactor to a biomass concentration of 1.2

g.T' dry weight. The gas flow rate was set to 1.5 l.min-', the liquid flow rate to 2.0
l.min-', the temperature to 2OoC,the pH to 6.5 and light intensity to 200 pE.m-2s-'.
The bioreactor was supplied with air + 1.2% CO, until the CO, concentration at
the bioreactor outlet reached 1.1%. The bioreactor gas inlet and outlet were then
connected to close the gas loop, and the CO, and 0, concentrations in the closed
loop were measured and recorded. The specific CO, uptake and 0, production rates
were calculated with the equations:
where d.co2
81
is the slope of the CO, concentration trend, 2 the slope of the 0,
concentration trend, Vgasthe total gas volume, X the biomass concentration and V
the total volume of the culture liquid.
Results
The specific 0, production rate in cultures with a biomass concentration of 1.2

g.1-I dry weight reached about 1.3 ml.g-'min-' at a light intensity of 200 pE.rnp2s-',
as shown in Figure 11. The rate was somewhat lower for partial CO, concentrations
of 0.1 to 0.5%, indicating a limitation ofphotosynthesis through the availability of
the substrate CO,. Photosynthesis was close to saturation at a light intensity of 200
pE.mP2s-'.
C. Closed Algae-Insects System
Description
The feasibility of supporting the gas requirements of a cockroach colony

(Periplaneta americana) by a Chlorella culture was tested in the following experi-
2 70 LUZlAN WOLF
spec. 0 2 production rate [mUmin.g]
0 50 100 150 200

light intensity [pUmS.s]
Figure 11. Specific C02 uptake rates and 0 2 production rates as a function of light
intensity in Chlorella culture in photo-bioreactor in closed gas-loop mode.
ment. A 250-ml air-lift bioreactor, serving as the photosynthetic producer compart-

ment, was connected in a closed loop to a 1150-ml glass vessel, serving as the
consumer compartment. The bioreactor was filled with 200 ml Chlorella 241.80
culture (obtained from an axenic stock culture) with a biomass concentration of 1.6
g.1-' dry weight, pH 6.25. The bioreactor was maintained at 20°C with a thermo-
stated glass waterjacket. Two spot lamps (Wotan Decostar 5 1,36", 20W) provided
an illumination intensity of 0 to 300 pE.rn-'s-' at the culture surface. The illumi-
nation intensity could be regulated through the lamp current. In the consumer
compartment eight cockroaches with a total mass of 6.2 g were placed. The animals
were maintained at 30°C with a thermostated plastic water jacket surrounding the
glass vessel. The vent gas from the consumer compartment was dried, then passed
through a CO, gas analyzer (ADC MKIII), and finally filter-sterilized (0.2 pm)
before introducing it into the bioreactor at a flow rate of 300 ml.min-I. The vent
gas from the Chlorella bioreactor was directly led to the cockroach consumer
compartment.
The lamps were switched on or off by a threshold controller. When the measured
CO, concentration exceeded a threshold value of 0.2%, the lamps were switched
on, and below this value they were switched off. The CO, concentration and the
lamp status were continuously recorded. The light duty cycle was calculated from
the train of light-on-off cycles as
Light duty cycle = ton/ (ton+ toff)
where the light was on for ton minutes and off for toff minutes. The rates of
increaseldecrease in CO, concentration in the closed volume caused by the light-
o d o f fcycles were used to estimate the combined CO, production of insects and
algae (light-off) and the combined CO, consumption of algae minus the CO,
production of insects (light-on). The CO, production rate of insects alone was
calculated by subtracting from the combined CO, production rate of insects and
algae the CO, production rate of algae in darkness, measured before the start of the
experiment.
Results
The experiment was conducted for 100 hours. Figure 12 shows four periods
(periods 1 to 4) with a duration of 20, 19, 18, and 30 hours in which the system
remained completely closed. In the time intervals in between, the system was
opened to maintain the consumer compartment (feed the cockroaches),to measure
the CO, uptake rate ofthe photosynthetic producer compartment at a reference light
intensity of 43 pE.rn-,s-', and to measure the biomass concentration of the algae.
Before starting the experiment, the CO, production rate of the photosynthetic
producer compartment was measured at 0.8 ml/h CO,.
Biomass concentration (1.6 g.1-I dry weight) and CO, uptake rate (3.3-2.6 ml/h
CO,) of the photosynthetic producer compartment remained approximately un-
changed throughout the course of the experiment.
During periods 1 and 2 the CO, concentrations stayed within an interval o f f 250
ppm from the set point of 2000 ppm, oscillating with a period of 20 to 30 min. The
light duty cycle varied considerably within these periods. Assuming a near constant
CO, production rate of algae in light-on conditions, these variations must reflect
varying CO, production rates of the consumer. The CO, production rate of the
insects varied between 0.5 and 5.0 ml/h CO,. It exhibited a pronounced diurnal
rhythm during the first two days of the experiment. The insect CO, production rate
correlates well with the light duty cycle.
During period 3 the illumination intensity was set to 47 pE.m-'s-'. After 2.5 hours
a quasi equilibrium with a CO, concentration of 2400 ppm was established for 2
hours, but later the CO, concentrations exceeded the measurement range (2500
ppm) of the gas analyzer. Illumination was continuously on, and the photosynthetic
compartment could not compensate the CO, production of the consumer.
During period 4 the CO, concentration fluctuates very regularly within an
interval o f f 250 ppm from the set point of 2000 ppm, oscillating with a period of
20 to 30 min. The light duty cycle fluctuated around 0.5.
In the course of this 100-hour experiment the insects produced approximately
250 ml CO, and consumed an equivalent amount of 0,, which were removedhp-
plied by the algal culture. This algal CO, consumption should have produced 0.32
g maltose, but due to the insensitivity of the assay method this could not be detected
in the algal culture medium. All eight cockroaches survived the experiment,
indicating that the Chlorella air-revitalization system functioned adequately.
2 72 LUZIAN WOLF
period 1 period 2 period 3 period 4

C02 uptake rate of algae at ILight = 43 pE/m2/s and cC02 = 2000 ppm:
3.3 mllh 2.9 mllh 2.8 ml/h 2.6 ml/h
light intensity lpE/m2/sl
200- . ..
.......................................
.i 4 ......................................... ....................................... i..........
150-__ .................................. 4.. .................................... i....................................... 1 ..................i ..........
100....................................... i....................................... I
t....................................... _ ............
, .
50 ........................................ :..................................... . . . . . . . . . . . . . . /__._____________.____._ .......................
!
0 24 40 72 96 h
C02 production of insects [ml/h C021
0 24 40 72 96 h
C02 consumption of algae minus C02 production of insects Iml/h C021
m
0 24 40 72 96 h
Figure 12. Gas exchange in closed algae-insects system. Explanation in text.
ESA has been studying a small-scale bioregenerative system to support long-term

biological experiments on-board spacecraft with oxygen, water and food. Core
component of this system is a special photo-bioreactor in which a maltose-produc-
ing strain of the green micro alga Chlorellu is cultivated. A number of auxiliary
system components have been developed and are finctioning on the ground
according to the design specifications, among them a gadliquid phase separator
operating at the same time as a low shear-stress pneumatic pump, a dehumidifier,
a maltose separator, and a liquid transfer system. All components have been
designed so that-in principle-they will operate in weightlessness, though this
has so far only been verified for the gadliquid phase separator.
The bioreactor and some of the auxiliary components have been integrated in a
prototype system, which has been subjected to preliminary testing. The prototype
has been sterilized successfblly by autoclaving. except for the liquid transfer unit
which is disinfected with isopropyl alcohol. Chlorella 241.80 has been cultured
several times under controlled conditions for up to 8 weeks. Algal growth to a
biomass concentration of 9 g.T' dry weight and maltose production to a concentra-
tion of 17 8.1-I have been achieved. The low shear-stress pneumatic pump works
reliably without the mechanical cell damage produced by other types of pumps.
Contamination of the algal cultures by other micro-organisms has been avoided in
most of the experiment runs. The maximum oxygen production rate observed was
2 ml.min-', when the culture was aerated with air + 0.5% CO,. This production rate
is well below the CO, gas transfer rate of 5 ml.min-I under these conditions. It can
probably be doubled by increasing the maximum light intensity of the illumination
unit (currently 300 pE.m-'s-'). In a preliminary closed gas loop experiment with
Periplaneta as consumer, the possibility of controlling the Chlorella culture so as
to match the needs of the consumer colony has been established.
A maltose excreting ChloreIla strain has been selected as the photosynthetic
producer, because the technique for automatic culturing of this organism and
harvesting its products was expected to be much less complex than that required
for culturing higher plants. Although the prototype system developed in our
laboratory has reached a high level of sophistication, there remain still a number of
technical and biological problems to be solved before the feasibility of this concept
is definitively demonstrated.
The major problem is maintaining sterility, and eventually automatic cleaning
and resterilization when contamination occurs during operation. The culture me-
dium, which contains minerals, cell fragments and considerableamounts of sugars,
is an ideal substrate for many other microorganisms.
Another problem is long term operation. The prototype system contains many
tubes and ducts which are perfused with culture medium. These may clog, which
may lead to loss of sensor information essential for controlling the culture.
Even when we succeed in demonstrating the feasibility of this concept, it will be
a difficult task to demonstrate convincingly that the expected advantages of a
bioregenerative system can outweigh the simplicity and reliability of a non-regen-
erative stored resource system in terms of volume, mass and amount of consu-
mables required over the operational time.
ACKNOWLEDGMENTS
The author is grateful to Alan Dowson, Jutta Meier, Eva-Maria Osthof, Annette Pfeiffer, and
Jorg Rossler for their efficient support in the performance of the experimentsdescribed here.
2 74 LUZIAN WOLF
He wishes t o thank the staff of the Brunel Institute of Bioengineering, London, for their
efforts in developing the gadliquid separator, the dehumidifier and the maltose separator
prototype.
REFERENCES
1. Ziesseniss, E. Symbiose-spezijsche Synthese und Excretion von Maltose durch Chlorella spec.
aus Paramecium bursaria. Dissertation, Georg-August-Universittit zu Gottingen, F.R.G., 1982.
2. ReiOer. W., WieOner, W. Autotrophic eukaryotic freshwater symbionts. In: Encyclopedia of Plant
Physiology, N.S., vol. 17: Cellular interactions (H.F. Linskens, J. Heslop Harrison, Eds.), pp.
59-74. Springer, BerlidHeidelbergflrlew York, 1984.
3. Wolf, L., Physiological Parameters of Chlorella 241.80. ESA Technical Report, XA 931159lLW.
European Space Agency, Noordwijk, The Netherlands, 1993.
4. Pirt, S.J.et al. Atubular bioreactor for photosynthetic production ofbiomass from carbon dioxide:
Design and performance. Journal of Chemical Technology and Biotechnology, 33B:35-58, 1983.
5. Oguchi, M.,Nitta, K.. Otsubo, K., Shimada, A., Miyazaki, K., Koyano, T., Miki, K., Application
of tubular photo-bioreactor system to culture spirulina for gas exchange and food production in
CELSS. Proceedings 40th Congress of the International Astronautical Federation, paper no.
IAFIIAA-89-577, 1989.
6. Atmosphere Quality Standards in Manned Space Vehicles. ESA PSS-03-401, ESA Publication
Division, ESTEC, Noordwijk, The Netherlands, 1993.
7. Man-System Integration Standards. NASA-STD-3000. NASA, Washington, D.C., 1987.
8. Brkchignac, F. Towards bioregenerative life support systems. In: Proceedings IVth European
Symposium on Life Sciences Research in Space (V. David, Ed.), pp. 421-429. ESA SP-307, ESA
Publication Division, ESTEC. Noordwijk, The Netherlands, 1990.
9. DORNIER. Environmental LifeSupport System TechnologyStudy, Final Report. ESACR(P) 2432,
ESTEC, Noordwijk, The Netherlands, 1987.
10. Oguchi, M., Otsubo, K., Nitta, K., Shimada, A., Fujii, S., Koyano, T., Miki, K. Closed and
continuous algae cultivation system for food production and gas exchange in CELSS. Advances
in Space Research, 9(8): 169-177, 1989.
11. MacElroy, R.D. The Controlled Ecological Life Support System Research Program. Proc. of the
AIAA Space Programs and Technologies Conference, Huntsville, paper no. AIAA-90-3730,
American Institute of Aeronautics and Astronautics, Washington, D.C., 1990.
12. Gitelson, 1.1. et al. Life support system with autonomous control employing plant photosynthesis.
Acta Astronautica, 3:633450. 1976.
13. Averner, M., Karel, M., Radmer, R. Problems associated with the utilization of algae in bioregen-
erative life support systems. NASA CR 166615, NASA, Washington. D.C., 1985.
14. Radmer, R., Behrens, B., Arnett, K., Gladue, R., Cox, J., Lieberman, D. Algal Culture Studiesfor
CELSS. NASA CR 177448, NASA, Washington, D.C., 1987.
15. MacElroy, R.D. Artificial Ecological Systems: Activities in the U.S. and Japan. Proceedings
CNEYDARA Workshop on Artijkial Ecological Systems in Marseille, 1990.
16. Wolf, L., Brechignac, F. Biological Life Support System Technology for Biological Experiments
in Space. Proceedings of the International Conference on Life Support and Biosperics. University
of Alabama, Huntsville, AL, 1992.
Chapter 12
VESTIBULAR FUNCTION AND

SENSORY INTERACTION IN
ALTERED GRAVITY
L.N. Kornilova
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
I1. Perceptual Reactions during Spaceflight . . . . . . . . . . . . . . . . . . . . 278
111. Reactions during Adaptation to Microgravity . . . . . . . . . . . . . . . . . . 284
A . Spontaneous Eye Movements . . . . . . . . . . . . . . . . . . . . . . . 284
B . Target Acquisition. Fixation and Pursuit . . . . . . . . . . . . . . . . . . 285
C . Optokinetic Nystagmus . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
D. Vestibulo-Ocular Responses . . . . . . . . . . . . . . . . . . . . . . . . 287
E . Subjective Optical Vertical . . . . . . . . . . . . . . . . . . . . . . . . . 290
F. Visually Induced Vertical Self-Motion Sensation . . . . . . . . . . . . . 290
G. Time Course of Adaptation in Long-Tern Missions . . . . . . . . . . . . 291
IV. Reactions during Readaptation to Earth’s Gravity . . . . . . . . . . . . . . . 293
A. Spontaneous Eye Movements . . . . . . . . . . . . . . . . . . . . . . . 294
B. Nystagmus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

.
ISBN: 0-7623-0147-3
275
2 76 L.N. KORNILOVA
C. Saccades and Smooth Tracking . . . . . . . . . . . . . . . . . . . . . . .296

D. Otolith Reflex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .297
E. Reactions of Semicircular Canal System . . . . . . . . . . . . . . . . . . 299
F. Subjective Optical Vertical . . . . . . . . . . . . . . . . . . . . . . . . . 301
G . Vestibulo-OcularReactions during Head Movements . . . . . . . . . . . 301
H. Oculomotor Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . .303
I. Classification of Vestibulo-Ocular Reactions . . . . . . . . . . . . . . . . 303
V. Neurophysiology of Vestibular Adaptation . . . . . . . . . . . . . . . . . . .305
VI. Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . .308
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .309
1. INTRODUCTION
Experience from all spaceflights has shown that weightlessness significantly alters
the fimctioning of gravity-dependent sensory systems. Adaptation to weightless-
ness is associated with two parallel processes: (1) alteration of the habitual phylo-
genetically and ontogenetically developed interactions between sensory systems,
and (2) formation of new sensory patterns in the central nervous system. The
anomalous perceptual, sensory, sensory-motor, and autonomic reactions, develop-
ing during the initial period of adaptation to weightlessness, are reminiscent of the
clinical form of terrestrial motion sickness. This led many of the American and
Russian investigators of the physiological effects of weightlessness to refer to this
phenomenon as “space motion sickness” (SMS). Investigators, who consider SMS
a neurological disease, point to the similarity of SMS symptoms with the clinical
manifestations of other forms of motion sickness occurring on the ground.’-7
However, investigatorswho consider SMS a physiological process, see the anoma-
lous reactions characteristicof adaptation to weightlessness as natural responses of
the body to an external factor. They consider SMS to be a special space form of the
adaptation syndrome.&l4
According to current ideas about the general adaptation syndrome, the first stage
of its development is characterized by changes in functional parameters, which are
adaptive and reversible; the second stage of adaptation involves actual structural
transformations. If the intensity or duration of the external stimulation causing
adaptation-in this case weightlessness-increases further, the structural transfor-
mations may be incapable of maintaining physiological adaptation to the new
conditions. This leads to disorders in the operation of the vestibular system, which
are accompanied by clinical symptoms. If we approach the definition of SMS from
this standpoint, then the use of the term “space adaptation syndrome” is justified,
as it is based on the physiological theory of the general adaptation syndrome. SMS
then becomes a state in which sensory disintegration takes the form of vestibular
disturbances in the context of the space adaptation syndrome (Fig.1).
Vestibular Function and Sensory Interaction 277
-
intersensory interaction
A Adaptation period
Compensation
veriod period
Figure 1. Development of space adaptation syndrome.

Note: SMS = mace motion sickness.
It is also important to remember that-concurrently with general physiological

adaptation processes-adaptation of general and specific sensory functions is
taking place. These two types of adaptation do not belong to a single process.
General physiological adaptation involves mobilization of systems of general
adaptation at the level of the entire organism, while adaptation of sensory functions
is a local process aimed at sending the most appropriate information to particular
sensory channels so as to provide an appropriate response. Adaptive processes are
intrinsic to each hierarchical level of any individual sensory function. However,
adaptation of individual sensory systems to a changed environment may follow a
time course different from that of general adaptation. This is illustrated by the case
of a cosmonaut on flight 15, who had to wear shoes with special pressurized insoles
in order to prevent positional illusions. He needed increased loading on the
supporting surfaces of his feet. This indicates that adaptation of the tactile-proprio-
ceptive system to weightlessness occurs more rapidly than the general physiologi-
cal adaptation process.
Under conditions of normal gravity, the vestibular system plays a leading role in
the process of intersensory integration and orientation, since it has developed
throughout evolution to operate in a gravitational field and its input is an interre-
ceptor formation. Most investigators in the United States, Europe and Russia
associate the anomalous reactions observed in weightlessness (space adaptation
syndrome or space motion sickness) with changes in vestibular system function
and in all functions based on vestibular afferent i n ~ u t . ' * ' ~ - 'Changes
~ ~ ' ~ ~ in
~
vestibular system function are variously ascribed to changes in the labyrinth
internal environment due to the headward fluid shift, to otolith deafferentation, to
2 78 L.N. KORNILOVA
canal-otolith conflict, to interlabyrinth asymmetry, or to intersensory mismatch.

However, it seems artificial to distinguish between intra-labyrinth and extra-laby-
rinth factors in SMS pathogenesis in view of the extensive functional associations
of the vestibular system and of the fact that it is an obligatory participant in the
realization of integrated reactions.
Individuals with a non-functional labyrinth are known to be resistant to motion
sickness during parabolic flight and under exposure to Coriolis a~celeration.~~ This
confirms the leading role of the vestibular system in the development of anomalous
reactions in adaptation and readaptationto altered gravity conditions. These anoma-
lous reactions can be classified by their origins as: (1) perceptual, (2) percepto-mo-
tor, (3) autonomic, and (4)mixed. Each of these types of reactions corresponds to
a specific clinical picture, which will be described later. Here the primary emphasis
will be on describing the neuro-vestibular reactions in weightlessness and their
classification, on explaining the pathogenesis of the sensory mechanisms and the
source of perceptual and vestibular-motor disorders occurring in weightlessness.
Interest in this problem was stimulated (1) by the prevalence of neuro-vestibular
disturbances in weightlessness (up to 98%), and (2) by the lack of a simple
correlation between neuro-vestibular disorders and autonomic
II. PERCEPTUAL REACTIONS DURING SPACEFLIGHT

Procedures
Russian investigatorshave developed a battery of questionsand tests for the study

of optical and spatial illusions occurring during adaptation to weightlessness. The
most commonly used one is Anketu, which uses questionnaire forms and a dicta-
phone for recording the experiences of the cosmonauts before, during, and after
flight.'3,35*36Observations were made during the initial period of adaptation to
weightlessness. The dynamics in adaptive shifts during long and short-term flights
were also observed.
A total of 102 cosmonauts was examined by means of the Anketu questionnaires,
and 2 1 ofthese used a dictaphone for recording their verbal descriptions of illusions
and sensationsexperienced. Analysis of the data shows that 98%of the cosmonauts
have noticed illusions of (1) orientation (coordinateand kinetic), (2)position,and
( 3 ) motion of self or surrounding objects.
Description of Observed Illusions
Spatial illusions during space flight were very heterogeneous and differed greatly
between individuals. They arose immediately upon entering weightlessness and
gradually diminished over several hours (or even minutes). However, illusory
sensations arising upon closing the eyes continued for 14-30 days of flight in 19%
of cosmonauts, and even during the entire flight of 96-438 days in 7%. Illusions
Vestibular Function and Sensory Interaction 2 79
were generally noted in darkness or when eyes were closed (77%). In darkness or
during free floating with eyes closed, 98% of cosmonauts sometimes experienced
a state of partial or complete disorientation.
Cosmonautswith open or closed eyes had illusions that they described as: upside
down, face down, feet up, tumbling, flailing, ‘falling head first’, ‘hanging in a
position on their right side’, ‘had separated from the couch and were propelled
upward’, ‘rotated around the longitudinal (Z) axis to the right, then twisted to the
right around the vertical axis’, ‘that their head rotated backward and downward,
then twisted to the right around an axis perpendicular to the chest’, etc.
Difficulty in visually scanning surrounding objects or instruments on the cabin
console was reported by 2 1 % of the cosmonauts. Also reported was experiencing
an illusion of the ‘approach’or ‘displacement’of the instrument panel (in horizon-
tal, vertical, or more often upward direction). Illusions ofjerky motions of external
objects were reported by 32% of the cosmonauts when performing visual tasks
during passive or active motion of the head (oscillopsia symptoms).
Some cosmonauts showed a complete lack of height concept (up and down) in
weightlessness.All space inside and outside the cabin was represented as distance
and depth, but not as height. Harm3’ and K~rnilova’~ showed that 50-58% of
cosmonauts developed an internal image of space and their own position in it by
visual association (visuo-spatial type). The sight of another crew member, floating
‘upside down’ or objects in abnormal orientations (compared to Earth) caused
discomfort in these cosmonauts. With eyes closed they completely lost their sense
of orientation and perception of the surrounding space. Other cosmonauts (34%)
developed their image of space and their own position in it mainly on the basis of
internal body coordinates, especially the direction of their legs and the vertical axis
of their body (‘internal axis’ type). The remaining 8% of cosmonauts could not
clearly specify what helped them to develop an image of space and their body
position in it.
The most frequent type of illusion was that ofbeing upside down (1 6%), followed
by illusions of the motion of surrounding objects (15%), and illusions of rotational
body movement (9%), illusions of displacement and inclination of objects (8%),
and illusions of linear body motion (4%). The illusions were classified as: (1)
coordinationalillusions (inclinationof the body or surroundingobjects), (2) kinetic
illusions (rotary and linear movements of the body or surroundingobjects), and (3)
mixed illusions. In the group of 102 cosmonauts 41% reported mixed illusions,
3 1% coordinational illusions, and 28% kinetic illusions. These illusions were
described by means of the international terminology and classification system3’
shown in Figure 2.
Kinetic Illusions
According to the tape recordings and questionnaire data, the following three
types of kinetic illusions were observed by Russian cosmonauts:
280 L.N. KORNILOVA
x SURGE
Y HEAVE
Z BOB
\ . ,\\IS
Figure 2. Types of motion illusions in microgravity.
0 sensation of rotation of the body around the frontal (Y) axis in the sagittal
plane forward and downward, or more often with the head backward and
downward (pitch illusion);
sensation of rotation of the body around the frontal (Y) axis, followed by
rotation around the longitudinal (Z) axis, most frequently to the right (com-
bination of pitch and yaw illusion);
0 sensation of rotation around the sagittal (X) axis in the frontal plane, most
frequently to the right, combined with a sensation of right rotation around the
longitudinal axis (Z) of the body (combination of roll and yaw illusions).
Several times a combination of rotational illusions with linear ‘bobs’(illusions

of linear displacement up and down along the longitudinal body axis) and with
heave illusions (sensation of linear displacement to the right and left along the
frontal body axis) was noted. The most common type of kinetic illusions were pitch
illusions, both in pure form and in combination with other forms of motion.
Kinetic illusions generally developed into one of the following coordinate
illusions:
0 an illusion of being upside down (inversion illusion);

0 an illusion of the body being inclined to the left or more frequently to the right
(bank illusion);
0 an illusion of the body being inclined forward or more frequently backward
(pitch illusion).
Coordinate Illusions
The predominant coordinate illusions were illusions of inversion. According to

G r a ~ b i e lillusions
,~~ of inversion are caused by responses of the otoliths to weight-
lessness. Aside from illusions of displacement, there were reports of proprioceptive
illusions (9%) during and after spaceflight. Cosmonauts reported the following
sensations: ‘of trying to prevent the wall from falling by keeping it in place with
my hand’, ‘the floor was moving out from beneath my feet’. When the engines were
fired to correct the orbit of the spacecraft, the resulting linear acceleration was
perceived by some cosmonauts as a slow motion, but as a very rapid motion by
others. After the engines were turned off the sensation of continued motion persisted
for several seconds.
Triggeringand Suppression of Illusions
Illusions could be suppressed in one of the following ways: through visual

fixation on some object; through rigid fixation of the body trunk on the couch with
head or feet pressed against it; or through the use of autogenic feedback training
methods.
The majority of cosmonauts (72%) agreed that illusions were triggered during
the first few days of flight by increased motor activity, especially sharp movements
of the head and trunk. These were the major stress factor that provoked the
development of illusory, anomalous sensory motor, and autonomic reactions.
Transfer from the transport vehicle to the space station commonly led to an
intensification of illusory and autonomic reactions. Movements in the sagittal and
frontal planes were especially disruptive. There existed various individual differ-
ences in vestibular sensitivity to motion. Some cosmonauts noted an increase in the
sensitivity of the vestibular system, and others a marked decrease in the sensitivity
of vestibular input.
2 82 L.N. KORNILOVA
Other cosmonauts (2 1%) indicated that optokinetic stimulation was the trigger-
ing factor in the development of illusions and autonomic reactions, and also the
absence of the accustomed feeling of support and sensation of ‘up and down’. In
many cosmonauts tracking moving objects through the window significantly
intensified illusions and autonomic reactions.
Some cosmonauts (7%) reported illusions and autonomic reactions also when
the head and trunk were motionless. A small number of cosmonauts (9%) noted
illusory sensations with respect to orientation of various parts of the body: ‘it seems
as if my arms are pointing downward, yet they are actually pointing upward’, ‘it
seems as if I am sitting in a hunched position, but I am actually stretched out in my
sleeping bag’). In addition to these illusions, 12% of the cosmonauts also noted
symptoms of lack of coordination (e.g., missing an object when they attempted to
pick it up).
For 34% of the cosmonauts the illusions during spaceflight closely resembled
those experienced during parabolic flight. For 28% ofthe cosmonautsthe vestibular
reactions during the early days of flight were reminiscent of sensationsexperienced
on Earth during exposure to Coriolis acceleration. In the remaining 38% of
cosmonauts sharp movements of the head, especially on the first flightday, induced
unique sensations in the vestibular apparatus that were different from any sensation
occurring in response to vestibular stimulation on Earth.
All the means used to correct and ameliorate illusions and autonomic reactions
in weightlessness (muscle stress, contact with a motionless support, physical
loading, administration of negative pressure to the lower body, wearing of pneu-
matic occlusion cuffs on the legs, wearing a neck pneumatic shock absorber to
restrain head movement, drugs) improved the state of the cosmonauts and led to
attenuation of illusionsto greater or lesser extent. The cosmonauts reported that the
greatest effect came from the use of the neck pneumatic shock absorber. Perform-
ance of demanding work tasks facilitated decrease in symptoms of discomfort and
distracted the cosmonauts from the unpleasant sensations. Sleep substantially
improved the state of the cosmonauts and decreased symptoms of discomfort.
Correlation with Physical Symptoms
After experiencing reflexive perceptual motor reactions by the end of the first
day or more frequentlyon the second day, all cosmonautsfelt the sensation of blood
rush to the head, heavinessin the head, and some even developed headaches.During
this period there were sensations of nasal congestion, the eyes were bloodshot, and
facial edema increased gradually. Some cosmonauts (1 1%) associated the develop-
ment of illusions with the sensation of blood rush to the head during the acute period
of adaptation to weightlessness.
Some cosmonauts (22%), while they were experiencing the sensation of blood
rush to the head, noted the development of autonomic reactions: skin color change
(flushing more often than pallor), cold sweat, belching, a sensation of heaviness in
the epigastric region, loss of appetite, hypersalivation, nausea, and vomiting.

Vomiting, sometimes repeatedly, occurs suddenly and frequently without prodro-
ma1 nausea. Bouts of vomiting occurred at intervals of up to 3 hours. These
sensations persisted for several minutes to hours, in some cases for 6-14 days, and
in two subjects up to the 30th day of flight. Gastrointestinal symptoms occurred
most often after 30 to 48 hours of flight.
Analysis and Conclusions
In the history of Russian manned spaceflight all cosmonauts, who experienced

vestibular discomfort, were without exception ultimately able to adapt to the
conditions of weightlessness. During the final period of the adaptation process the
cosmonauts could perform a wide variety of sharp movements without provoking
any unpleasant vestibular responses. However, some cosmonauts (9 out of 47)
participating in long-term missions (85439 days) periodically developed mild
vestibular discomfort (vertigo and queasiness), especially during the increased
motor activity required in the final 10-14 days of the flight.
Analysis of the inflight Anketu data shows that illusions developed virtually
instantaneously after transition to weightlessness, while autonomic symptoms
either did not occur at all or only significantly later. This suggests that there is no
direct connection between them, which was confirmed by correlation analysis.
Although in 19%of cosmonauts illusions occur with autonomic symptoms, in most
of these cases the symptoms developed independently after disappearance of the
illusions. These findings suggest that the illusions are not primary sensoryreactions
preceding autonomic disturbance, but have an independent mechanism of develop-
ment.
On the basis of duration and severity of the reactions, we identify three types of
physiological adaptation to spaceflight conditions:
1 . Resistant40 or mild, transitory (seconds to minutes) illusions and auto-

nomic and sensory discomfort (21%);
2. Stormysevere illusions, lack of coordination, and autonomic reactions,
lasting 1-3 days (54%);
3. T o r p i h i l d illusions and symptoms of autonomic discomfort, lasting 14
days or longer (25%).
On repeated flights the first type of sensory adaptation to weightlessnessaccounted

for 61% of cases, the second for 32%, and the third for 7%.
Return to the Earth is accompanied by a renewed development of sensory, motor
and autonomic disorders. These disorders, as a rule, were more intense than during
flight and occurred in the same individuals who, according to their verbal assess-
ment, had tolerated flight well.
284 L.N. KORNILOVA
Thus, the Anketu data indicate that spatial disorders are not restricted to a few
cosmonauts, but are a common response of a majority of persons exposed to
microgravity. The reactions are somewhat individualized with respect to severity,
nature of symptoms and their duration, and may occur even if the subject feels well
and experiences no anomalous autonomic reactions. The nature of spatial illusions
was determined by the role and relative contribution of various types of sensory
input to spatial orientation.
111. REACTIONS DURING ADAPTATION T O

MICROG RAVlTY
Research conducted in the US and Russian space programs has revealed changes
in vestibular system operation, as well as in all functions based on vestibular
afferent input. However, a problem is that the results of vestibular function tests
administered during flight are highly variable and frequently even contradictory.
This variability is not surprising, but is rather the predictable consequence of at
least the following three factors: (1) individual differences in adaptive physiologi-
cal changes, (2) abnormal experimental conditions, and (3) lack of an integrated
approach.
In order to obtain unambiguous conclusions it seemed essential to continue study
of vestibular system functioning and the mechanisms of intersensory interaction
during short and long-term space flights by means of a single, integrated approach.
For this purpose a special battery of tests has been developed: Optokinez,12914*28*40,41
and O p t ~ v e r t . A
’ ~total
,~~ of~2 1 cosmonauts has performed
these tests, 8 of them during short-term flights (7-1 8 days) and 13 during long-term
flights ( 7 5 4 3 9 days). The tests were carried out on flight days 2-3, 5-6,28-30,
then once a month or once every two months until the end of the flight, as well as
preflight and postflight. The Anketu questionnaires are listed under “subjective
observations” (top row).
A. Spontaneous Eye Movements
Study of spontaneous oculomotor activity without additional sensory stimulation

showed certain changes of its magnitude and pattern, especially in the early flight
stages. 12.l4&44
On mission days 2-3 crew members, with eyes closed or wearing dark goggles,
showed increased involuntary eye movements (nystagmus). This spontaneous
oculomotor activity consisted of floating and saccadic movements (square wave
jerks). Of 2 1 cosmoauts, 16 showed an appearance of spontaneous vertical down-
beating nystagmus (amplitude 3,9 f O,SO/sec; velocity was low, 8,9 f 1,8 O/sec); 7
of them had also horizontal spontaneous nystagmus (left-beating or right-beating),
resulting in an oblique nystagmus.
On mission days 5-6, the spontaneous oculomotor activity decreased signifi-

cantly, and the spontaneous nystagmus disappeared. Reoccurrence of eye destabi-
lization and vertical downbeating nystagmus appeared after 50 days of flight in 9
out of 16 cosmonauts. One cosmonaut began experiencing spontaneous reactions
after 30 days of flight. In general, vestibular system changes appear to be of a
transitory nature during long-term exposure to weightlessness.
B. Target Acquisition, Fixation and Pursuit
Target Acquisition
On mission days 2-3, eye movements on a sound cue showed changes of

magnitude and pattern in the extreme horizontal and extreme vertical directions,
especially vertical movements of closed eyes. The amplitude of the sound-driven
eye movements with opened eyes did not change significantly.With closed eyes or
eyes covered with dark goggles, the amplitude increased significantly. Horizontal
eye movements increased from 22,7 f 3,1° to 28,5 f 4,4" (p = 0,05); vertical eye
movements from 16,2 f 4,1° to 45,7 f 6,1° (p = 0,Ol). After 50 days of flight the
eye movements showed a tendency to decrease.
Changes in the velocity of sound-driven eye movements were observed at the
beginning of the flight. Without head movement, the velocities decreased from
60-70 O/sec to 2 5 4 0 O/sec (p = 0,Ol). The duration of eye movements increased
from 4 1,O f 2,2 ms to 72,OIf:4,O ms (p = 0,05). Electro-oculograms(EOG) presented
a smooth sine curve, rather than saccadic movements.4244
Fixation
Visual stimuluswas a light spot of approximately 1mm in diameter,which moved

on the screen discretely in the horizontal and vertical directions at the angle of
1&15".
At early flight stages the fixation capability revealed a decrease in the amplitude
of gaze fixation (head fixed) in 8 cosmonauts. The largest changes were shown in
vertical oculomotor reactions: amplitude of vertical gaze fixation decreased from
10,7 f l , 1O to 7,5 +0,4O (p = 0,05). The corrective saccades were recorded.
After extensive exposure to weightlessness (flight days 145410) a significant
decrease in the vertical amplitude of the eye movements was recorded. During
fixation of random vertical movements of the stimulus, the saccades were per-
formed incorrectly in a number of cases: the eye made diagonal rather than strictly
vertical movements, when EOG deviations were simultaneouslyrecorded both at
the vertical and diagonal leads.
After active head movements, horizontal gaze fixation (yaw and roll rotation)
improved, but vertical gaze fixation remained unchanged.12,14*28*40
286 L.N. KORNILOVA
Pursuii
The pursuit function was measured during linear movements of the visual
stimuluspoint in horizontal,vertical, and diagonal directions and in circularmotion.
The light spot moved across the screen in random fashion at constant velocity, with
a frequency of 1 Hz. When the target described a diagonal or circular motion, the
pursuit reflex degraded (1 1 cosmonauts), whereas saccades remained essentially
unaltered. (Fig. 3).
The largest changes were observed when the target moved in vertical direction.
Early in flight (days 2-3), the pursuit reflex almost disappeared in 5 out of 11
cosmonauts, when the target moved downwards. In this case, vestibular stimulation
failed to improve the pursuit f ~ n c t i o n . ' ~ , ' ~
Threshold sensitivity of oculomotor function to optokinetic stimulation velocity
was investigated with a series of 20 black and white bands that moved in horizontal,
--
vertical and diagonal directions at linear velocities increasing from 1 to 20 O/sec.
Before flight
B
F W
Inflight
Figure 3. Pursuit in microgravity. A - EOG of tracking eye movements for circumfer-

ential stimulus. B - EOG of tracking eye movements for diagonal stimulus. C - EOG
of tracking eye movements for vertical stimulus.V - vertical EOG, H - horizontal EOG.
Arrows show direction of stimulus movement.
Note: Calibration: 1 On, 1 s.
There was a marked decrease of the lower and upper thresholds of the optokinetic
reaction at the beginning of the flight. Before flight the lower and upper thresholds
of the optokinetic nystagmus were 5-6 '1s and 12-19 'Is, respectively, while on
mission days 2-3 these values had decreased to 2-3 ' I s and 8-10 ' I s , respec-
tively.28,40,41
C. Optokinetic Nystagmus
Before flight, the horizontal optokinetic nystagmus (OKN) gain was 0,6-0,7 in
all cosmonauts, and was symmetrical as a rule. With regard to the vertical OKN
gain, the cosmonauts could be divided in two groups. One group (1 1 cosmonauts)
had an upward gain of 0.7 f0.1 and a downward gain of 0.5 f 0.08 (gain up > gain
down); the other group (1 0 cosmonauts) had OKN gain symmetry of 0,6 0,05in *
both directions.
In weightlessness the horizontal OKN gain showed considerable individual
variability. During early adaptation to weightlessness (3-5 days) the vertical OKN
gain decreased significantly to 0.2 (p < 0,05), and the OKN gain asymmetry
disappeared (Fig. 4A). If the preflight vertical OKN was rhythmic with stable
amplitude and without dicrotic spikes for fast as well as slow OKN constituents,
then nearly all cosmonauts had dicrotic spikes with the slow phase of up and down
OKN at all flight stages.
During long-term flights the OKN dynamics showed considerable individual
variability, and had an undulatory character. In one group of cosmonauts (13
cosmonauts) inflight OKN was increased (Fig. 4B), sometimes decreasing gradu-
ally to preflight values in the course of the flight. In the other group (8 cosmonauts)
the OKN gain was decreased during the entire mission (Fig. 4A), but occasionally
it increased to preflight values. The vertical OKN gain asymmetry was periodically
significantly increased (up to 25%), but it reversed in the course of a long-term
spaceflight (Fig. 4A and 4B).
D. Vestibulo-Ocular Responses
Eye movement reactions were also studied during voluntary head movements.
These were sinusoidal yaw movements at a frequency of 0,5 Hz, controlled by a
metronome. Before flight, vestibular stimulation by active rotatory head yaw
movements did not modify the sinusoidal curve of single nystagmus. However,
during flight on mission days 2-3 there was an appearance of strong nystagmus
upon vestibular stimulation by active rotatory head movements, in other words
there was an increased vestibulo-oculomotor response. The nystagmus, superim-
posed on the compensatory eye movements during rotatory head movements, was
consistentwith the results of an earlier study that showed a decrease in the vestibular
nystagmus threshold.
Early in flight the vestibulo-oculomotor response gain increased (p = 0.01) and
an asymmetry appeared. After 168 days of flight during this gain was significantly
288 L.N. KORNILOVA
BF , 25 53 104 146 208 279 292 332 398 2 4 6 8 I5

Days
0.9
0.8
0,7
0.6
0,s
0.4
0,3
0,2
01
I
0
Inflight I1 Postflight
BF 3 6 30 85 168 200 2 5
DAYS
O K S U P - - r - OKSDOWNI
Figure 4. Gain of vertical optokinetic nystagmus before, during, and after flight. Top -
first type; bottom - second type. Gain is measured as slow phase velocity of vertical
optokinetic nystagmys divided by stimulus velocity; BF = before flight; OKS =
optokinetic stimulus; p < 0.05.
BF 3 5 28 168
DAYS O F FLIGHT
-~
r - -
!TURN RIGHT OTURN LEFT!
Figure 5. Gain of vestibulo-ocular reflex. Yaw gain before and during flight; head
yaw rotation 0.5 Hz, eyes closed. Gain is measured as ratio of velocities of eye
movement and head movement.
Note. BF-before flight; p < 0.05
decreased compared to the preflight values or it was absent, as were also the
accompanying nystagmus responses (Fig. 5).
Opto-vestibular Reaction
The largest individual changes were observed with combined optokinetic and
vestibular stimulation during flight. Before flight, most cosmonauts showed reac-
tions of visual origin in response to combined opto-vestibular stimulation. At the
beginning of the flight (on day 3) optokinetic reaction was undetectable, while
vestibular responses increased (Fig. 6 ) .
During head movements and simultaneous optokinetic stimulation, vestibular
nystagmus or vestibulo-optokinetic reactions were revealed, which depended on
concordance or discordance of the two stimuli. On mission day 5, decreased
vestibulo-oculomotor reactions of vestibular origin and increased optokineticreac-
tions were observed. This may suggest that at the beginning of adaptation to
weightlessness the vestibular input played a dominant role, while at the end of
adaptation the visual input prevailed.
Decreased oculomotor responses to both separate and combined stimulations
were observed in some cases as late as the 50th day of the mission. The oculomotor
changes recorded during flight developed in the absence of vegetative (SMS)
reactions (as indicated by questionnairedata).
290 L.N. KORNILOVA
1 Before flight 2
3day H- M
timonth- H
- -
L?&
L- c
1 0 ° L
IS
Figure 6. Oculomotor responses to opto-vestibular stimulation. H = horizontal EOC

recorded during yaw head rotation. 1. with eyes closed, 2. with head movements
during optokinetic stimulation. Arrows show direction of head movements.
E. Subjective Optical Vertical
The subjective optical vertical (SOV) was investigated in 10 cosmonauts during

flight. The accuracy of SOV perception was evaluated in total darkness with a
background of a pattern of diffuse black and white spots, which did not move or
moved either upward or downward. Testing of SOV perception was hampered by
the simultaneous occurrence of illusions, as was clear from the recorded voice data.
The preflight error deviation in SOV perception was for all cosmonauts within
the physiological range (0.1-2"). In 8 cosmonauts the SOV error was either
predominantly to the right or to the left, with the asymmetry reaching up to 30%.
During adaptation to microgravity, the SOV error increased beyond the normal
range, reaching 8'-12', and asymmetry also increased (Fig. 7). During a long-term
mission SOV remained significantly elevated, while error dynamics had an undu-
lating character.
F. Visually Induced Vertical Self-Motion Sensation
Vertical linear vection (transference), induced by optokinetic stimulation with

linear and sinus velocity profile, was investigated in 10 cosmonauts. In 2 subjects
BF 3 6 25 53 104 146
Days of flight
I m Dark, bGore
-~ stirn 0 Dark,
__ after stlrn 0 Picture OTFUP m OKS DOWN1
~
figure 7. Subjective optical vertical. Errors of subjective optical vertical in sitting

position before flight (BF)and during flight. Error in degrees plotted against days of
flight.
Nore: OKS = optokinetic stimulation; * p c 0 01
this was done in the initial stage of microgravity (days 3 and 6 of flight) and in 9
subjects after 30 days of long-term flights (once a month or every two months to
the end of the flight)!u*
In preflight tests there were very stable vection responses with a definite fre-
quency maximum and steady phase response of about 180”. However, during the
initial flight period vection frequency,both maximal and irregular phase reactions,
was absent. Temporary directional inversion of the subjective vertical motion led
to “an inverted vection,” while an upward stimulus motion led to an upward vection
and vice versa. After the initial period of adaptation during long-term flights the
perceptual responses were unstable, presumably because the process of adaptation
alternated with periods of de-adaptation (Fig 8).
C. Time Course of Adaptation in long-Term Missions
In weightlessness,information from the otoliths reflects the position of the body

in space and its unfamiliar mode of motion. During the initial flight period the
cerebral cortex cannot properly interpret this information, since it contradicts the
information coming from the visual system. This situation is a result of disruption
in polymodal convergence mechanisms, especially between the vestibular, visual
and proprioceptiveafferent inputs.50J’In order to perform motor acts correctly and
achieve appropriate spatial orientation, the central nervous system blocks out the
anomalous vestibular signals, and information coming through the visual channel
becomes predominant.I2-I4
292 L.N. KORNILOVA
Figure 8. Vestibulo-visual interaction in weightlessness. Picture shows example of

vection induced by optokinetic stimulation on Earth and in weightlessness. Inverted
vection follows after normal vection response. Arrows show direction of movements
of stimulus and illusion. Stimulation at constant velocity of 50 “/s.
The anomalous perceptual and sensory-motor reactions that occur at the

beginning of a flight attenuate over time as a result of adaptive changes in the
central nervous system. Adaptation to weightlessness presupposes formation of
a “neural model” of sensory information in the central nervous system, which
supports appropriate representation of the altered sensory afferent information
and the anomalous environmental conditions in space. However, research on
long-term exposure to weightlessness has demonstrated that this neural model
is relatively unstable and is easily disrupted under the influence of additional
factor^.'^
After 50 days of exposure (in one cosmonaut after 30 days) illusory and
anomalous spontaneousand induced eye movement reactions began to occur. This
suggests that the changes in the interaction between the sensory systems during
long-term exposure to weightlessness are transitory, i.e., during flight periods
of dominance of adaptation alternate with periods in which de-adaptation
dominates.
Data on the alternation of periods of adaptation and de-adaptation are of funda-
mental importance for the diagnosis and prediction of the status of the sensory
systems, of the effects of the duration of exposure to weightlessness, and of the
capacity of crews to perform critical operational tasks while in orbit.
IV. REACTIONS DURING READAPTATION T O EARTH'S

GRAVITY
Results ofpostflight physiological examinations are discussed.Vestibular reactions
have been observed in cosmonauts during readaptation to conditions of normal
gravity. These are similar in nature to reactions occurring during adaptation to
weightlessness, but in a number of instances they are more severe. The author has
developed a special test battery, providing separate and combined stimulation of
sensory input channels during passive postural stimulation for the study of mecha-
nisms, time course and postflight vestibular readaptation (Table 1).'7,25*27,32,33,35
Table 1. Pre- and Postflight Tests of Vestibular Function
Number of Subjects
~
Short Long
Parameters Tested Method Used Flights Flights Total
Subjective observations SAS (SMS)questionnaire 56 46 102
Spontaneous oculomotor EOG, central & max. divergence 18 31 49
activity (nystagmus) gaze, eyes open/closed. Position:
vertical (0");supine (90");head
down tilt (120").
Gaze fixation ability EOG, gaze fixation at target. Posi- 18 31 49
tion: vertical (0");supine (90");
head down tilt (1 20").
Pursuit reflex EOG, pendular tracking eye move- 18 31 49
ment. Position: vertical (0"); su-
pine (90");head down tilt (1 20").
Vestibulo-oculomotor EOG; head rotations. Position: verti- 18 31 49
reflexes cal (0");supine (90");head down
tilt (1 20").
Tactile-proprioceptive EOG during muscle strain. Position: 18 31 49
oculornotor reactions vertical (0");supine (90");head
down tilt (1 20").
Cerebral-oculomotor EOG; digitonasal test. Position: ver- 18 31 49
reactions tical (0'); supine (90");head
down tilt (1 20").
Postural oculomotor EOG, after changing position 18 31 49
reactions oo-+1200-too
Otholith reflex After-image method, ocular 28 20 48
torsional counterrolling
Subjective optical vertical Angle between subjective and 19 20 39
gravitational verticals
Optokinetic nystagmus EOC, rotating drum 9 9 18
Vestibulo-oculomotor EOG, rotating chair 0 18 18
reflexes
Canal/otolith interaction EOG, rotating chair, stop stimulus 19 10 29
w/wo head inclinations
Vestibulo-oculomotor EOG, rotating chair w/wo target 10 8 18
suppressing fixation
Caloric responses EOG, air thermal calorization 0 9 9
294 L.N. KORNILOVA
Some or all ofthe tests were performed before and after flight by 102 cosmonauts,
56 of whom completed short-term flights (7-18 days) and 46 long-term flights
(75-439 days). Of the 102 cosmonauts 60 had flown once, 29 'mice, and 13 from
3 to 5 times. Subjects were tested 45 and 30 days before launch, and on days 0-1,
3-4,5-6,8--10 after return (in some instances 14-15 and 75 days postflight).
On days 0-1 postflight and sometimes also on days 3-4,96% of cosmonauts
returning from long-term flights and 27% of those returning from short-term flights
exhibited symptoms that could be designated as 'clinical vestibular dysfunction'.
These symptoms of varying severity consisted of illusions (e.g., dizziness,illusions
of movement of self or surround), motor reactions (e.g., pointing errors) and
vestibular reactions (e.g., nystagmus, which was central or sometimes peripheral
in nature).
On the first day postflight, all cosmonauts complained of instability when
standing and of 'swaying' from side to side while walking. Active and passive
movements (e.g., transfer of the cosmonauts to stretchers)caused stomach discom-
fort and nausea, and provoked intense dizziness and vomiting.
A. Spontaneous Eye Movements
Postflight cosmonauts exhibited spontaneous dysmetric and dysrhythmic nys-

tagmus, which was generally tonic, but sometimesjerky. This occurred both at rest
in a sitting position and during active and passive shifts of body position with eyes
either closed or covered by dark glasses. Nystagmus was directed downward and
to the right in 44% of the cosmonauts and upward and to the left in 56%. In most
cosmonauts (82%) the direction of spontaneous nystagmus was not altered by
changes in body position, but the intensity was increased in the head-up position
and decreased in the head-down position (Fig. 9).
Spontaneousnystagmus was either constant or occurred in bursts. Gaze fixation
of a point was impossible, as the eyes continued to oscillate due to the nystagmus.
In some cases the typical clonic (jerky) form of nystagmus changed to an atypical
form of square wave jerks, which either remained atypical or reverted to the clonic
form during subsequent sessions. Change in spontaneous nystagmus occurred in
response to all tests in 33% of the cosmonauts, while in 35% it was induced only
by opto-vestibular and proprioceptive stimulation and by tests of cerebellar coor-
dination (e.g., touching the nose generated a square wave nystagmus). In 2 cosmo-
nauts the spontaneous nystagmus, recorded after their first flight, reoccurred in the
same form and with the same characteristics after the second flight.
In addition to spontaneousnystagmus, cosmonauts also exhibited the phenome-
non of "wandering eyes," i.e., inability to fix the gaze on an object. In these
cosmonauts, the eyes continually moved involuntarily upward or to the side. The
amplitude of the eye movements increased after vestibular stimulation. This phe-
nomenon was of vestibular-reticular (not collicular) origin and could not be
attributed to sleepiness. While it usually disappeared after 7 or 8 days, it remained
Figure 9. Spontaneous eye movements after return from long-term spaceflight.

Electro-oculograms were recorded on 1 st day postflight after 237-day spaceflight, in
standing, lying and antiorthostatic positions.
Nofe: H = horizontal EOC, V = vertical EOC. Calibration: 109 Is.
in 3 cosmonauts for years along with spontaneous nystagmus. Several cosmonauts

exhibited rhythmic smooth sinusoidal eye movements in the EOG (vertical lead).
These interfered with their performance of tests involving fixational rotation of the
eyes. To these cosmonauts moving objects appeared indistinct and floated out of
the field of view.
B. Nystagmus
Positional Nystagmus
Marked positional nystagmus was observed in 9 cosmonauts during the first 3

days after return from long-term flights (176, 310, 365, and 439 days). Intense
dizziness and queasiness were experienced upon tipping the head back and when
lying down. Only in a sitting position with head held straight did they feel alright.
Any head movement, especially backward inclination of the head, caused intense
dizziness and nausea. On the day of landing and the day after, rotary positional
nystagmus could be visually detected when they were lying down or tipped their
head back. The EOG showed positional nystagmus with a fixed direction, either
upward or to the left. The eyes could not fixate a point, but wandered and underwent
nystagmus. Positional nystagmus had a latent period, and was subject to adaptation
and fatigue. Two cosmonauts, who participated in two flights, exhibited identical
responses after each flight.33
296 L.N. KORNILOVA
It is assumed that the directionally-fixed nystagmus, occurring only in certain

critical head positions, is due to increased static excitability of vestibular function,
in this case of the utricular portion of the otolith.
Gaze or End-Position Nystagmus
After long-term flights movement of the eyes to an extreme position (right, left,
up, or down) was generally accompanied by gaze nystagmus. In 37% of the cases
this was a reverse spontaneous nystagmus, i.e., nystagmus occurred when the eyes
were moved to the extreme position, and reversed direction when the gaze returned
to the center of the field.
Optokinetic Nystagmus
Changes in eye movement responses to optokinetic stimulation, relative to

preflight tests, were noted for several days after landing. This occurred in 32% of
cosmonauts returning from short-term flights and in 26% after long-term flights.
Changes were noted in the following parameters: frequency and amplitude of
horizontal and especially vertical optokineticnystagmus (OKN), and the magnitude
and asymmetry of OKN gain.
After short-term flights, the amplitude and tracking frequency of OKN decreased,
especially for vertical OKN. The amplitude of the spontaneous nystagmus was
increased,when both types of nystagmus were in the same direction, and decreased
when they were in opposite directions. The direction of the asymmetry of OKN
amplitude postflight did not correspond with that observed preflight. The gain of
vertical OKN showed a pronounced and reliable asymmetry, and its direction
differed from that before flight (reversal of asymmetry: gain down > gain up).
After long-term flights, cosmonauts exhibited a decrease in the amplitude of
OKN, especially in vertical, and preflight asymmetry was maintained. Magnitude
and direction of the asymmetry of OKN gain were virtually identical to those before
flight. Cosmonauts with changed OKN also showed a decrease in optokinetic
threshold and significant changes in vestibular function, like negative otolith reflex
and multiple spontaneous nystagmus. OKN parameters generally returned to
preflight levels by 5-7 days after short-term flights and 10 days after long-term
flights.
C. Saccades and Smooth Tracking
Changes in tracking of a sinusoidal (1 Hz), horizontally or vertically moving

stimulus in both the vertical and 30" head-up positions were exhibited by 18% of
cosmonauts after short-term flights and by 83% after long-term flights. These
changes were more pronounced with the vertically-moving stimulus. They in-
volved loss of smoothness of slow tracking and occurrence of jerky, stepwise
figure 10. Gaze fixation and pursuit reflex after return from long-term spaceflight.
Electro-oculograms (EOG) were recorded on 1st day postflight after 186-day space-
flight, in standing and antiorthostatic position. Arrows show direction of eye move-
ments.
Note: H = horizontal EOC, V = vertical EOC. Calibration: lo", 1s.
movements. In half of the cases, the tracking process improved in the 30 O

head-down position (Fig. 10).
The gaze fixation response (accuracy of fixational rotations) was changed in 9%
of cosmonauts returning from short-term missions and in 7 1% of those returning
from long-term flights. These changes were characterized by saccade dysmetry
(range of variance 4 O ) , occurrence of corrective microsaccades, and appearance of
gaze nystagmus in some cases.
D. Otolith Reflex
Otolith function was assessed by means of the torsional ocular response (OCR).
This parameter was measured by means of an after-image method.5' A box with a
vertical slit is placed in front of the seated subject, who is then exposed to a
lightflash to establish an after-image. Any torsion occurring while the after-image
is still visible is observed as a tilt of the after-image relative to an objectivereference
298 L.N. KORNILOVA
line. The subject, keeping head and trunk aligned, is then tilted 90" to the right or
left. After 20 seconds in the tilted position, a clock face with a white arrow is placed
in the line of vision. The subject, with eyes open, rotates the arrow on the clock to
match the after-image.This procedure is repeated 5 to 9 times for both tilt directions.
Changes in OCR were observed on days 0-1 and 3 4 after landing in 92% of
cosmonauts returning from short-term missions, and in all cosmonauts returning
from long-term flights. Results are shown in Fig. 11. OCR hyperreflexia (bi- or
unilateral) was displayed by 48% of cosmonauts returning from short-term flights,
but not by any of those returning from long-term flights. Hyporeflexia (bi- or
unilateral) was recorded in only 28% of cosmonauts returning from short-term
flights, but was the predominant response (62%) in those returning from long-term
flights.
During the first hours after landing 24% of cosmonauts after short-term flights
and 38% of cosmonauts after long-term flights exhibited a paradoxical reaction: a
negative or reverse otolith reflex (rotation of the eyes in the direction of inclination)
for one or both tilt directions. Subjects were tested 5 times, and they displayed this
reaction each time. In most subjects the paradoxical otolith reaction had disap-
peared by day 3-4 postflight, but in some cosmonauts it persisted for several days
postflight.
Another change observed in the OCR was the postflight appearance of asymme-
try (Fig. 12). While this was before flight no more than 3", it increased up to 14"
30
25
20
15
m
10
-5
-10 ' 28 62 12 18 12 20 48 0
x
;Short fl nght OShort fl left iLong fl nght OLong fl left
figure 11. Torsional counterrolling of the eye after spaceflight. Counterrolling in

degrees plotted against percentage of cosmonauts before flight and on 1st day
postflight. Body positioned on right or left side, conditions indicated by shade of bars,
for types see Fig. 15, Fisher method used. Short flights 7-1 8 days, long flights 75-439
days.
Note: *p c 0.05;** p = 0.01.
A I l n .plm fibm
Figure 12. Change in otolith reflex asymmetry after spaceflight. Asymmetry in
degrees plotted against days postflight for 48 cosmonauts returning from long-term
flights.
after landing. Asymmetry of the reflex was observed in all individuals completing
long-term flights and in 22% of those returning from short-term flights. Changes
in the direction of asymmetry were found in 10% of all cosmonauts. These
parameters returned to normal on days 3-4 after short-term flights and on days 8-9
after long-term flights, but 12%of the subjects did not return to baseline levels until
one month after landing.
E. Reactions of Semicircular Canal System

Reactivity of the cupulo-endolymph system was examined by means of cupu-
lometry, in which the duration of post-rotational nystagmus and illusions of
counter-rotation were measured after rotation at 30, 45, 60, and 90 "Is in both
clockwise and counterclockwise directions. On days 3-4 postflight 42% of cosmo-
nauts returning from long-term missions, who had displayed normal preflight
responses, exhibited a decrease in the duration of post-rotational nystagmus and
illusions, as well as development of asymmetry. Cosmonauts, who displayed
nystagmus and illusions of short duration in preflight testing, continued to exhibit
a quick extinction of these responses after landing. On days 7-14 postflight, there
was an increase in nystagmus duration and illusions in half of the cosmonauts, who
displayed vestibular recruitment, i.e., addition of spontaneous nystagmus to the
response. In most cases rotation was accompanied by sweating and nausea.
Visual fixation on apoint attachedto the head (rotating with the subject)had little
effect on the intensity of rotational and post-rotational nystagmus in 41% of
cosmonauts returning from long-term missions. There was an attenuation of the
fixational reflex, with the magnitude of the fixational index (ratio of response with
fixation to that without fixation) increasing from 0.2-0.5 before flight to 0.6-0.8
after landing.
300 L.N. KORNILOVA
Otolith-canal lnferacfion
Interaction between otolith organs and semicircular canals was studied before
and after flight by measuring intensity and duration of post-rotational nystagmus.
In these tests the subject is rotated in a chair at a velocity of 180 Ohec for 3 to 4
rotations (6 to 8 sec), after which the chair is brought to a stop in 0.5 seconds, and
the head is tilted (nystagmus dumping). Before flight a reciprocal canal-otolith
relationship (decreasing time constant of post-rotational nystagmus) was exhibited
by 86%of the cosmonauts, 8%exhibited a synergisticrelationship (increasingtime
constant), and in 6% otolith stimulation did not affect the intensity of the semicir-
cular canal response (unchanged time constant).
On days 3-4 after long-term flights, the distribution of the subjects over these
groups was altered: 39% displayed the reciprocal canal-otolithrelationship, 7% the
synergistic relationship, and 54% the absence of otolith inhibition of the canal
response. The percentages were also altered after short-termflights:35% of subjects
exhibited the reciprocal relationship, 26% the synergisticrelationship, and in 39%
no change was elicited. By day 8 postflight, the preflight pattern of otolith-canal
interactionswas restored in cosmonauts returning from short-term flights, while by
day 14 postflight only 49% of the long-term flyers exhibitedresponses as exhibited
before flight.
~- ~~ __ .__
__ __.
.short fl right Oshort fl left .long fl. nght along fl left1
Figure 13. Errors of subjective optical vertical after spaceflight. Tests on 1st day
postflight, body positioned on right or left side. Error in degrees plotted against
percentage of cosmonauts, short flights 7-18 days (20 subjects), long flights 75-439
days (20 subjects), conditions indicated by shade of bars, for types see Fig. 15.
Note: * p < 0.05.
F. Subjective Optical Vertical
Spatial orientation was studied by having the cosmonauts look through a 60 cm

long tube and set either a horizontal or a vertical line at the end of the tube.52This
task was performed with the cosmonauts seated upright or rolled laterally to the
right or left side. On days 0-1 postflight virtually all cosmonauts returning from
long-term flights displayed a decreased accuracy of perception of the subjective
optical vertical with the variability and magnitude of errors increased for both the
sitting and lateral positions.
In the lateral positions, the magnitude of asymmetry of errors increased sharply
(Fig. 13), and in the majority of cases its direction altered, compared with that
observed preflight. In a number of cosmonauts (10% after the short-term and 18%
after the long-term missions), the previously occurring Aubert phenomenon gave
way to the the Muller phenomenon. Recovery of the preflight accuracy of percep-
tion of the subjective optical vertical occurred by days 10-14 postflight. The
accuracy of subjective perception of the position of the vertical body axis was
virtually unchanged after
G. Vestibulo-Ocular Reactions during Head Movements
Eye movement reactions during volunrary sinusoidal pitch, roll or yaw head
movements at a frequency of 0.125 Hz were studied in the sitting, supine, 30"
head-down and 30" head-up positions with eyes either open or closed. The
vestibulo-ocular reaction gain was decreased (relative to preflight) in most cosmo-
nauts on days 0-1 after long-term flights, when head movements were performed
with eyes closed. Vestibulo-ocular reactions were virtually absent in 39% of
returning cosmonauts; and there were no accompanying nystagmus responses.
Active head movements with eyes closed did not evoke the appropriate vestibulo-
ocular reaction in 54% of cosmonauts upon yaw rotation and in 60% upon pitch
rotation, but the reaction was reduced or missing (Fig. 14A and 14B).
An increase in horizontal vestibulo-ocular reaction gain (relative to preflight)
was noted in 17% of cosmonauts, while the gain in roll and pitch rotation remained
near preflight levels in these subjects. With eyes open and without gaze fixation,
62% of the cosmonauts exhibited an increase in the amplitude of eye movements
in a direction opposite to that of the head and the appearance of nystagmic cycles.
The vestibulo-ocular reaction gain with eyes open was increased (relative to
preflight) for head movements in all planes.
After short-term flights, the vestibulo-ocular reaction gain for head pitch rota-
tions with eyes closed on postflight days &1 was decreased (relative to preflight)
in 26% and increased in 18%of cosmonauts. On day 5 after return from a short-term
flight, the vestibulo-ocular reaction gain was virtually the same as preflight. In the
postflight period, another characteristic of the vestibulo-ocular reaction was the
appearance of a directional asymmetry in the gain of this reaction.
'
302 L.N. KORNILOVA
0.4
0,35
0.3
0.25
9 0,2
0.15
0,I
0.05
0
83 0 I7 I2 0 34 0 54
x
OSHORT FL LEFT .SHORT FL. RIGHT!LONG FL LEFT OLONG FL RlCw
0,35
0,3
0,25
0.2
0.15
0,1
0.05
0
56 12 18 0 26 28 O M )
%
Figure 14. Gain of vestibulo-ocular reflex after spaceflight. Top - during yaw rotation;
bottom - during pitch rotation. Head rotation speed 0.125 Hz. Eyes closed. Gain
plotted against percentage of cosmonauts, conditions indicated by shade of bars, for
types see Fig. 15.
Note: * = p < 0.05.
H. Oculomotor Reactions
Proprioceptive Stimulation
Muscle tension after return from spaceflight may induce oculomotor reactions.
After short-term flights muscle tension induced eye tremors in 18% of the cosmo-
nauts and proprioceptivenystagmus in 2 1%. After long-term flights,muscle tension
generally stabilized eye position and extinguished spontaneous nystagmus, but
after cessation of muscle tension nystagmus returned and was then more intense.
Effects of Cerebellar Coordination Tests
Performance of the finger-nose test, a test for cerebellar coordination, did not
significantly affect the nature of eye movement activity after short-term flights.
However, performance of this test in all positions was accompanied by occurrence
of a square wave cerebellar nystagmus in 3 1% of those returning from long-term
flights.
Effects of Postural Stimulation
In some returning cosmonauts changes in body position (passive tilting from a

30” head-up to a 30” head-down position and back) stimulated oculomotor reac-
tions. In the majority of cosmonauts returning from short-term flights there was
virtually no effect, but in 11% of cosmonauts there was an increased saccadic
activity.
After long-term flights there was an effect on the spontaneousnystagmus in 17%
of the cosmonauts:postural nystagmus was superimposed on vertical spontaneous
nystagmus. These cosmonauts also displayed orthostatic intolerance, and the two
effects were strongly correlated (r = 0.8, p = 0.05).
In most cosmonauts the vestibular reactions became normal by days 5-6 after
short-term flights, by days 1&14 after long-term flights. However, 11 cosmonauts
returning from long-term missions exhibited signs of enhanced vestibular reactivity
on day 75 postflight. In 3 other subjects, in whom spontaneous nystagmus was
recorded after their first long-term flight (in 1983 and 1985, respectively), the
phenomenon of “wandering eyes” was still evident &5 years postflight.
1. Classification of Vestibulo-Ocular Reactions
Analysis of inflight and postflight vestibular hnction has shown that there are
clear individual differences in nature and dynamics of the sensory system adapta-
tiodre-adaptation to gravity. The cosmonautsreturning from long-term flights can
be divided in four groups on the basis of eye movement responses to stimulation
(Fig. 15):
304 L.N. KORNILOVA
lTVPES OF THE NATURE OF OCULOMOTOR RESPONSES
Figure 75. Types of oculomotor responses to various sensory stimuli after spaceflight.
1. Individuals who displayed no nystagmic response to postural and vestibular

stimulation and weak responses to opto-vestibular and proprioceptive stimu-
lation and cerebellar coordination test (37%).
2. Individuals with altered spontaneous nystagmus in response to all types of
stimulation (33%). Vestibular and eye movement responses in the 30"
head-down position were closer to normal than in the 30" head-up position,
presumably because the headward shift of fluid in the head-down position
resembled that in flight. Some individuals of this group displayed an atypical
square wave nystagmus, resembling that observed in persons with cerebellar
lesions.
3. Individuals exhibiting altered spontaneous nystagmus only upon passive
postural stimulation by shifting from head-up to head-down position V.V.
(1 7%).
4. Individuals with nystagmus insensitive to changes in body position, but
sensitive to vestibular, opto-vestibular, and proprioceptive stimulation
(13%). In later postflight testing, members of group 2 demonstrated re-
sponses typical of either group 1 or 3, indicating that response to stimulation
became more selective during the postflight period.
Inflight and postflight reactions, resembling the clinical signs of vestibular

dysfunction, were transient. They reflected the normal progress of adaptatiodre-
adaptation of the sensory system to the altered gravitational environment. Re-
Table 2. Types of Sensory System Adaptation to Altered Gravity

1. Marked response to any stimulus
2. No or greatly decreased response to all stimuli
3. Selective response to certain types of stimulation only
sponses differed to some degree between individuals with regard to severity,nature,

time of development, and duration. Nonetheless, analysis of eye movement re-
sponsesto various types of stimulation, displayed by the four groups of cosmonauts,
enabled us to identify three types of sensory system adaptatiodre-adaptation to
altered gravity (Table 2). The first type of adaptation is characterized by a strong
response to any stimulus presented during the initial adaptation period. The second
type of adaptation is characterized by responses that are drastically decreased or
even absent. The third type of adaptation is distinguished by a selective response
of the sensory system only to certain types of stimulation.
The battery of tests, employing separate and combined stimulation of various
sensory inputs during postural stimulation, allowed us to identify (1) the relative
contributions of various mechanisms (hemodynamic and sensory) to the develop-
ment of anomalous vestibulo-ocular responses after flight, and (2) the pathways
and forms of adaptationhe-adaptationof vestibular function.
V. NEUROPHYSIOLOGY OF VESTIBULAR ADAPTATION

United States, European and Russian studies of neurovestibular adaptation to
altered gravity indicate that development of vestibular disorders (spatial orientation
illusion, anomalous sensory-motor and autonomic responses) during and after
flight is not limited to certain individuals, but is a general response of the sensory
system to microgravity exposure. These responses differ to some extent between
individuals in severity, nature, time of occurrence, and duration. However, the
results of our studies provide a picture of the forms, mechanisms, and periods of
adaptatiodre-adaptation of the vestibular and vestibulo-ocular systems under al-
tered gravitational conditions.
The following types of sensory disorders during adaptation to weightlessness
have been identified perceptual (67%); perceptomotor (12%); autonomic (2%);
and mixed (19%). Each type corresponds to a particular clinical picture:
0 The perceptual type is marked by spatial illusions in the form of coordinate

or kinetic illusions in various planes.
0 The perceptomotor type has illusions accompanied by motoric disturbances:
spontaneousnystagmus, lack of motor coordination, impeded visual tracking
and fixation.
306 L.N. KORNILOVA
0 The autonomic type is marked by the occurrence of sweating, pallor, or

flushing, hypersalivation, nausea, vomiting, and symptoms indicative of
cardiovascular and respiratory changes.
0 The mixed form is marked by various combinations of the symptoms de-
scribed for the other three types.
Sensory disorders may be accompanied by headache, a feeling of a rush of blood

to the head (in weightlessness only), and sleepiness. In a number of cases there is
also a sharp decrease in work capacity and job performance.
There exist some definite differences in the genesis of vestibular dysfunction, as
well as in the pathways and forms of vestibular adaptation and re-adaptation.
Adaptive changes in the sensory systems, particularly the vestibular system, begin
during the initial period of exposure to altered gravity. At that time the primary
triggering perceptual and sensory-motor disturbances operate by means of a reflex-
ive mechanism.53Changes in eye movement responses. observed after 2-3 days of
exposureto microgravity,suggest: (1) the level oftonic (static) vestibular excitabil-
ity decreases. This is confirmed by the decrease in speed and amplitude of
compensatory ocular counterrolling during roll and pitch movements of the head
(and inversion of the amplitude in some cases); by a decrease in gain of the
vestibulo-ocular response in roll, pitch, and sometimes yaw; and by a decrease in
optokinetic nystagmus gain without head movement, and (2) the dynamic excit-
ability of the vestibular and sensory inputs increases. This is suggested by the
occurrence of spontaneous nystagmus; by a decreased threshold induced by the
combined or separate application of vestibular and optokinetic stimulation; and by
increased gain of yaw vestibulo-ocular response in some cases.12,14*2127*28*54*59
After the reflexive mechanism of sensory-motor responses to microgravity has
ceased to function, a mechanism of psychosensorymotor disintegration begins to
operate in the processing of visual and kinetic information. Dissolution of the
“gravitational neural model of sensory stimuli,” the vestibulo-visual and vestibulo-
proprioceptiveassociations that were developed during evolution in a 1-G environ-
ment, disrupts the integrative mechanisms of the central nervous system, which
supportthe interaction of sensory systems. This is accompaniedby the development
of anomalous vestibular responses to the complex vestibular and visual stimuli
directed to various components and levels of the vestibular system.
Sensory deprivation is another mechanism, underlying vestibular disorders and
deterioration of the visual tracking function during this period. This is confirmed
by the observation that active head movements in microgravity’2*28and tactile-
proprioceptive effects during immersion6’ decrease spontaneous eye movement
activity and degrade the tracking function. Decreased flow of proprioceptive
stimulation, caused by loss of supporting afferent stimulation and decreased muscle
activity, alters the level of functioning of the vestibular nuclei and activates the
midbrain structures. This may be particularly relevant to the observed changes in
vestibular excitability.60*61
The decreased threshold of sensory reactions may be due
to a diminished reverse cortico-fugal flow, to a decreased cortical inhibition of

subcortical structures, and to a liberation of the sensory systems from central
control.62It should also be remembered that the properties of cerebral receptors,
neurons and fbnctions depend on the hnctional status of the brain.63@In weight-
lessness this status is determined by metabolic shifts65and by the level of cerebral
oxygen supply.66In turn,the latter are influenced by changes in cerebral microcir-
culation, fluid-electrolyte metabolism and neurohumoral regulation!’
Studies with primates have shown that changes in spontaneous and evoked eye
movement responses may result from (1) changes in afferent stimulation from the
vestibular channel (ie., by increased activity of the vestibular nerve) and (2)
changes in neuronal activity at the level of relay structures (flocculusof cerebellum
and vestibular nuclei of midbrain).68The static excitability of the floccular lobe of
the cerebellum, which has an inhibitory effect on the neurons of the vestibular
nuclei, did not change in flight. However, the dynamic excitability increased
sharply upon initial exposure to weightlessness and persisted at elevated levels
during the later days of the flight. These findings suggest that inhibition of
vestibulo-ocular transmission during sensory adaptation to altered gravity is active
in nature and has its origin in the ~ e r e b e l l u mParticipation
.~~ of the cerebellum in
vestibular responses and in the subsequent process of sensory adaptation to weight-
lessness has also been demonstrated in other ~tudies.’~.’~ Square wave jerks were
observed in a study of spontaneous eye movement activity and disrupted smooth
visual tracking. These changes may be associated with the participation of the
central nervous system at the cerebellarand brainstem levels and with the disruption
of the vestibulo-cerebellar association.
Concurrently with the process of sensory disintegration, there runs a process of
adaptation, which involves a search for the afferent stream that will facilitate
orientation under the altered gravity condition. Turning ‘ ‘ o r of some information
systems and turning “on” of others leads to a change in the relative contributions
of various sensory channels to the initiation of neuro-vestibular reactions. Russian
investigators have noted after 5-6 days of microgravity exposure: disappearance
of spontaneous nystagmus (decreased spontaneous eye movement activity); lack
of nystagmus upon active head movements with eyes closed; and occurrence of
only optokinetic responses under combined vestibulo-optokinetic stimula-
This indicates a decreased importance of vestibular afferent input in
tion.12*’4,59*70
initiating eye movement responses, which could be described as functional vestibu-
lar de-afferentation. The observed partial or complete loss of reactivity of the
vestibular system, after initial adaptation to microgravity and during re-adaptation
to Earth gravity, can be ascribed to central inhibition of transmission of vestibular
signals that are inappropriate under the new conditions. In other words, the human
brain learns to ignore information inappropriate to the environment and to accept
only useful information, i.e., visual information.
Altered contributions of the various sensory input channels in the formation of
integrated reactions are the result of the adaptation of intersensory interaction
308 L.N. KORNILOVA
processes and of sensory systems to the new sensory environment. This occurs by
two mechanisms operating in parallel: (1) selective activity of the relay and control
parts of the central nervous system (thalamic structures, reticular formation, hip-
pocampus, cerebral cortex), and (2) the central integrated mechanisms of the central
nervous system. This leads at a functional level to responses appropriate to the new
conditions of existence.
The compensatory period is marked by the recovery of the orientational h c t i o n
through the formation of new intersensoryassociations,of a new microgravitational
neural model of sensory stimuli.This new model is relativelyunstable, as suggested
by the anomalous spontaneous and evoked eye movement responses often observed
after 30 to 50 days of exposure to weightlessness. Perception disruption (illusions
ofposition and movement, disrupted spatial orientation),observed during the initial
period of adaptation to microgravity, is correlated with anomalous vestibulo-
oculomotor responses, but not with the anomalous autonomic reactions.
Morphological studies of the brains of rats after spaceflight have confirmed that
changes in the vestibulo-oculomotor systems are due to adaptive restructuring in
the central nervous system at the cellular level. Also observed was morphological
evidence of hypofunction of receptor cells in the utriculus and of diminished
vestibular impulses entering the nodulus of the vermis ~erebelli.~' At the beginning
of the flight a reorientation of the mass of dendrites of giant multipolar neurons in
the reticular formation was noted. There was a decreased orientation of dendrites
toward the vestibular nucleus and pyramidal tract and an increased orientation to
the visual nucleus.72Brains of rats after a 14-day flight revealed an increase in the
orientationof dendrites toward the vestibular nuclei,73suggesting formation of new
visual-vestibular associations in the central nervous system after long-term expo-
sure to weightlessness.

The effects of weightlessness on vestibular function have been studied since the
beginning of manned spaceflight. The results of these studies have been highly
variable and to some extent even contradictory, which makes it difficult to draw
unambiguous conclusions. This variability is probably due to at least three factors:
( 1) individual differences in the adaptive process, (2) non-standardized experimen-
tal methods and conditions, (3) a lack of integrated experiments. For this reason,
we have used a single integrated approach with a specially developed battery of
tests. The results thus obtained for 21 cosmonauts on short- and long-term flights
are reviewed here, and discussed in the light of the results obtained by others.
Changes in the operation of the vestibular system and in all functions based on
vestibular afferent input are commonly observed in spaceflight. These changes are
characteristicfor the process of adaptation and re-adaptation to altered gravity. They
occur in a high proportion of persons exposed to such conditions, although there
are individual differences with regard to severity, nature, time and duration of
occurrence, and the dynamics of the process.
Analysis of the observations in a large number of cosmonauts has permitted to
distinguish three types of adaptation of the sensory system to altered gravity. The
first type of adaptation is characterized by a strong response to any stimulus during
the initial adaptation period. The second type of adaptation is characterized by
responses that are drastically decreased or even absent. The third type of adaptation
is distinguished by the selective response of the sensory system to certain types of
stimulation only.
After long-term missions the process of re-adaption usually takes a more severe
course than the earlier process of adaptation to microgravity. Both adaptation and
re-adaptation follow an undulating course, in which adaptation and re-adaptation
are alternating. This is most conspicuous during long-term flights, and it suggests
that in the initial stage of adaptation to weightlessness the vestibular input plays a
dominant role, while at the end of the adaptation process the visual input prevails.
ACKNOWLEDGMENTS
I would like to express my deep gratitude to Professor I.Ya. Yakovleva, who was my first
guide in biospace medicine, and to Professor I.B. Kozlovskaya, who supports and inspires
me in my investigations.
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INDEX
Adrenocortotropic hormone (ACTH), Atmosphere regeneration, 233, 234,

150-153 238,245,246-248,250-252
A43 1 epidermoid cells, 46,67-68,74 Atrial natriuretic peptide, 154-155,
in hypergravity, 47, 63 159-160
in microgravity, 47-48, 50-51, 58, Atrophy, 102,110,116,174
63,65,69,75 (see also Muscular system)
Aldosterone, 131, 139, 147-149, 153,
158, 159-160 Basipetal transport, 222-225,227
Algal cultures, 233, 236, 238-239, Bed rest studies, 19,82, 101-102, 117,
256-273 118-120, 156
(see also Chlorella) Bioavailability, 109, 110-113
Allergic reactions, 94, 99 Bioregeneration, 233-252
Amyloplasts, 214,215-216,226,227 with Chlorella, 256-273
Analgesics, 98,99, 100 Blastogenesis, 11, 19,20, 27
Animal studies Blast transformation, 20,21, 23
of immune system, 18-19, 21-23, Blood pressure, 27, 115, 172,209
27,73 Blood volume, 124, 125, 126
in life support systems, 233, 236- Body temperature, 177-179
237 Body weight and mass, 125, 135, 140-
in microgravity, 172, 194,207-208 141,159,182
(see also Frogs; Mice studies; Rat Bone density, 102, 103, 112, 118, 169,
studies; Rhesus monkey 174
studies) in frogs, 208,209
Antibiotics, 98, 99, 100, 101, 103,
111-112, 117 Calcium
Antidiuretic hormone (ADH), 141- in frogs, 208
146, 158-160 in humans, 141, 145
31 5
31 6 INDEX
in plants, 214,216, 217,227,228 Cortisol, 150-153, 157, 159-160

Canal-otolith conflict, 277-278 Creatinine, 136, 140
Capillary pressure, 133-134 Cytokines, 22, 23, 35,40,46,51,75
Carbon dioxide, 142, 176, 234, 239, Cytoskeleton
256 of cells, 62,64-65,69,70-7 1,72,75
Cardiovascular system, 99, 101, 116, of plants, 216,226,227
177, 180,181, 189 Cytotoxic cells, 12, 14-15, 18,20,58
Catecholamines, 150, 155-157, 158,
160 Decongestants, 99, 100-101
Cell differentiation, 34, 35-36, 73-74 Dehydration, 125, 127, 128, 131, 135,
Cell mediated immune (CMI) system, 141
15-17 Delayed-type hypersensitivity (DTH),
Cell metabolism, 46,54-56,58-62,64, 16-17,23
69-70 Digestive disorders, 23-26, 94-95, 98,
Cell proliferation, 34-39, 43-46, 56, 99, 100,101,236,283
74-75, 172 Diuresis, 124, 135-137, 145, 158, 159
(see also Clinostat studies) DNA, 3,5,36,38,39,73
Central nervous system, 276, 293, Dopamine, 156-157
307,308 Drugs, 94-103, 108-118, 120
Centrifuge studies (see Hypergravity)
Chlorella, 236, 238,239, 256-273 Electrolytes, 72, 100, 124-160,209
Chloride, 128, 137, 138 Emergencies, medical, 94, 168, 175-
Clinostat studies 176
cell changes in, 63 Endocrine system, 141-158, 158-160,
Clinostat studies (simulated micro- 177
gravity) Endoplasmic reticulum, 216-217,226,
cell changes in,69,74-75 227
cell proliferation in, 35, 39,43,46 Environmental adaptation, 169, 171,
genetic expression in, 47-48 183
plant gravitropic response in, 217- Epidermal growth factor, 48, 49, 50-
218 5 1, 63,65,74
Closed algae-insects system, 269-27 1 Epidermoid A43 1 cells (see A43 1 epi-
Coleoptiles, 217-221, 223-226 dermoid cells)
Concanavalin A, 12-13,26,37,73 Epinephrine, 155-157
in microgravity, 34, 39-40, 50, 51- Erythrocytes, 66-69, 169, 182
53,63,65 Erythropoietic cells (see K-562 cells)
Confinement studies, 19, 21, 82-83, Eukaryotes, 35-36,70
84-85, 103, 186-188 Exercise, 20, 114, 116, 124, 127, 137,
Controlled Ecological Life Support 142, 156, 175
System (CELSS), 239-240, Extravehicular activity (EVA), 20-21,
25 1-252 172, 177-179, 188, 189
(see also Life support systems)
Corticosteroids, 150-154, 157 Fever, 94-95,99
Index 31 7
Fluid shift, 25, 173, 277-278, 303 Hypoactivity, 84, 85, 87, 89
anddrugs, 111,113,115,117,120 Hypobaric studies, 19,20-21
Fluid volume, 124-128, 133-135, 158- Hypodynamia (see Bed rest studies)
160 Hypogravity (see Clinostat studies;
and endocrine regulation, 141- 158 Microgravity)
renal function in, 135-141
Food, 124, 135, 232-233 IAA (see Indoleacetic acid)
and bioregeneration, 236-237, 242- IAA-protein receptor complex, 2 19,
245,256,272 224-225,227-228
production of, 236-238, 239-240, Illusions, 277,278-284,293,299,305,
242-245,248,250-25 1,252 308
Friend cells Immune system, 2-28,5 1-54,75
in hypergravity, 37,47,66,75 Indoleacetic acid (IAA), 214, 217-
in microgravity, 41,46,56-57,66 225,227
in simulated microgravity, 39,46 Infection, 22,23-26,28,94-95, 118
Frogs, 194-209 Injuries, 95, 118, 168, 171
Interferon, 12-13, 18,22-23,51-58,75
Gastroenteritis, 23, 24, 283 Interleukin, 12-13, 19, 20, 40, 51-56,
Gaze fixation, 286,294-295,296, 305 74,75
Genetic expression, 34-36, 58-62, 74, in animals, 18,22-23
75 Isolation studies, 82-83, 84-85, 88
in hypergravity, 46-47
in microgravity, 49-58 JTC-12 cells, 38, 63,67
in simulated microgravity, 47-48 Jurkat cells, 53-54, 64-65,67,74
Glomerular filtration, 118, 140, 160
Granulocytes, 11,73, 156 K-562 cells, 38,39,47
Gravitropic curvature, plant, 2 17,218- Kidney function, 116, 117, 118, 140-
224,225,227 141
Gravity readaptation, 293-305
Gravity receptors, 70-7 1 Labryinth internal environment, 277-
278
Hamster kidney cells, 43, 58 Lateral polarization, 215, 224-225,
HeLa cells, 36,43,74 227-228
in hypergravity, 37, 38, 46-47, 58, Leg volume, 174, 183
62-63 Leukocytes, 11, 19
Hepatic first pass effect, 109, 112-113, (see also under specific cell name)
117 Life support systems, 232-252
High density algal cultures, 257-273 failure of, 168, 176, 186-188
(see also Chlorella) Liver function, 116, 117,208,209
Hybridoma cells, 30-41,43,56,66,74 Low shear-stress pump, 262-263,272-
17-hydroxycorticosteroids, 150, 153- 273
154 L8 rat myoblast cells, 43,68,69
Hypergravity,cells in, 35,36-39,43,58 Lunar Base CELSS, 240-252
318 INDEX
(see also Life support systems) Morphology, cell, 62-69,7 1-72,75

Lung adenocarcinoma cells, 66,68 Motility, cell, 62-69, 72-73, 75
Lymphocytes, 2, 10-13,34-75 Motor system functions, 175, 283,
in animals, 18, 22-23 293-294,305
and mitogen challenge, 41,73-74 Muscular system, 102, 110, 116, 174,
and PHA, 3-10 182,189
and stress, 20, 2 1, 26-27
Natriuretic hormones, 154-155, 160
Macrophages, 12, 17,22,53 Natural killer (NK) cells, 12, 14-15,
Maltose producing algae, 256, 258, 20,23, 27
259,265,267,272,273 Nausea, 236,283,299,305
(see also Chlorella) Neuroendocrine system, 2, 12
Marangoni convection, 62,64-65,72 Neurologic system, 34, 99, 101, 177,
MC3T3-El cells, 37, 38 278,305-308
Medical monitoring, 168-189 Neutrophils, 3-4, 10-12,20,22-23,49
Mice studies, 21-23, 58,73 Newcastle disease virus, 12-13
Microgravity Norepinephrine, 155-157
adaptation to, 276-309 Nutrition (see Food)
body fluids in, 124-160, 173 Nystagmus, 295-296, 299, 301, 303,
cell changes in, 19, 34-75,70-71 304,306-307
countermeasures to, 174-175,282
drug disposition in, 94-103, 108- Ocular response (OCR), 296-299
120 Oculomotor activity, 284-293, 301,
eye movement in, 284-293 303
frog behavior in, 194-209,205-209 Optokinetic nystagmus (OKN), 287-
illusions in, 277,278-284,293,299, 289,296,306
305,308 Orthostatic intolerance, 101-102, 116,
medical monitoring in, 168-189 124-125, 127, 160, 169, 175,
perceptual reactions in, 278-284, 303
305,308 Orthostatic suspension, 19,21-23
physiological changes in, 108-120, Osmolality, 133-134, 138-139, 141,
158-160,283-284 159
vestibular system in, 284-293 Otolith-canal interaction, 299-300
Mitogen activation, 11, 51-52, 53, 74- Otoliths, 277-278,292-293,296-299
75
of glycoproteins, 50 PHA (see Phytohemagglutinin)
of lymphocytes, 4-8, 10-12, 34-37, Pharmaceutics, 35, 94-103, 108-120,
39-40,4 1 ,71,73-74 124
Monocytes, 11-12, 16-17, 26-27, 35, Photobioreactor, 258-259,272-273
5 1-52,53-54,65-66 Photosynthesis, 234, 239, 244-245,
in animals, 22-23 249,256-273
proliferation of, 39-41,46,73-75 Phytohemagglutinin (PHA), 3- 10, 19-
Monotony, 82-83, 84, 87, 89 20,21,27
Index 319
Plant gravitropic response, 213-228 Space analogs (see Simulation stud-

Plants, life support, 233,234,235-236, ies)
238,242-245,248,252 Space-induced syndromes, 173-175
Plasma volume, 124, 125-128, 134, Space motion sickness (SMS), 25-26,
141, 145, 158-160 115, 135, 141, 160, 276-279
Platelets, 49,64,68 drugs for, 94-95,98, 100-101, 103
Proprioceptive stimulation, 301, 303, in frogs, 206, 209
306 Spirulina cultures, 257, 258
Protein binding, 114-115, 117, 118 Spontaneous eye movements, 284-
Proteins, 48,49,58, 70, 74, 133-134 285,294-295
Protein synthesis, 208,209,219-222 Spontaneous nystagmus, 305, 306,
Psychological issues, 17, 27, 34, 82- 307
90,99,103,157,187 Statoliths, 215-216,226
(see also Stress) Stress, 124, 172,281,282
Psychoneurologicaldisorders,169-170 and endocrine system, 150-157
Pursuit function, 286-287 and flight duration, 26-28, 169, 187,
188
Radiation, 69-70, 82, 102, 176, 180, and immune system, 2, 19-21, 26-
244,252 27,34
Rat studies, 18,43,49, 68,69,73 simulation studies of, 19-23,27,82,
Regolith, 246-248 84
Renal function, 118, 135-141, 160 Subjective optical vertical (SOV),
Renin, 147-149, 159-160 290-291,300-301
Respiratory system, 23, 25, 94-95, Suppressor lymphocytes (see Cyto-
177,305 toxic cells
Rhesus monkeys studies, 18-19
RNA, 3-4,46-47,48 T-cells (see Lymphocytes)
Thermodymanics, 69-74
Saccades, 286,296,303 THP-1 cells, 53-54
Semicular canal system, 299-300 T-lymphocytes (see Lymphocytes)
Sensory functions, 276-309 Topical drugs, 95,99, 100-101
Signal transduction, 35,46-62,65,69, Total body water, 125, 127-128, 159
70, 74,75 Tubular photobioreactor, 259-267
Simulation studies, 19-23,82-90,101- Tumor necrosis factor, 51, 52, 55, 58,
103, 118-120, 156 75
Sleep disorders, 99, 100, 101, 187,
282,305 Urine analysis, 23, 24, 100, 124, 135-
Sodium, serum, 128-131, 133-134, 141, 137-139,182, 189
139,149,150,159-160 Urodilatin, 154-155
Sodium, urinary, 137-138, 140, 141,
148, 153,154 Vanilmandelic acid, 156-157
Space adaptation syndrome (see Space V-79 Chinese hamster lung cells, 38,
motion sickness 63
320 INDEX
Vertical self-motion sensation, 292 Water

Vestibular system, 276-309 bioregeneration of, 250, 25 1-252,
of frogs, 200-201,208 256,272
Viral infections, 22, 25, 100 for life support systems, 232, 233
Vitamins, 99,100,242 purification of, 235,240,245-246
Vomiting, 283,305 Water loading tests, 140-141, 145, 160
W138 cells, 41, 66,68,75
Waste processing, 232, 233, 239-240, Weightlessness (see Microgravity)
250-25 1

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ADVANCES IN

Editor: SJOERDL. BONTING

@) JAl PRESS INC.

/A1 PRESS LTD.

Manufactured in the United States of America

August0 Cogoli Space Biology Group

Marianne Cogoli-Greuter Space Biology Group

). Darginaviciene Institute of Botany

A.D. Eggorov Institute for Biomedical Problems

A.I. Grigoriev Institute for Biomedical Problems

A. Cue11 lnstitut de Medecine et de

G. Houin Laboratoire de PharmacocinCtiqueet

Akerni Izurni-Kurotani Space Utilization Center

Richard Jennings NASA-JohnsonSpace Center

Nick Kanas Department of Psychiatry

lrena Konstantinova Institute for Biomedical Problems

Ludmila Kornilova Institute for Biomedical Problems

lane M. Krauhs KRUG Life Sciences Inc.

Carolyn 5. Leach NASNJohnsonSpace Center

A. Merkys Institute of Botany

Yoshihiro Mogami Department of Biology

Makoto Okuno Department of Biology

A. Pavy-Le Traon lnstitut de Medecine et de

M. Pujos lnstitut Europ4en de T6IPrnCdecine

S. Saivin Laboratoire de Pharmacocinetique et

Steven H. Schwamkopf Lockheed Missiles and Space

Scott M. Smith NASA/Johnson Space Center

Gerald Sonnenfeld Carolinas Medical Center

C.Soulez-LaRiviere European Space Research and Technology

Gerald R. Taylor NASA-JohnsonSpace Center

Luzian Wolf European Space Agency

Masa m ichi Yarnashita Space Utilization Center

medical monitoring system for long-term missions. Schwartzkopf reports on the

CHANGES IN THE IMMUNE SYSTEM

Gerald R. Taylor, lrena Konstantinova,

I. Introduction ..... ... .. . . ... ....., . ... . ... .. ... .. 2

Advances in Space Biology and Medicine

II. IMMUNOLOGY STUDIES IN SPACE AND ON THE

Table 1. Summary of Apollo Results

Source: After K i m & ' 201

Response of lymphocytes to Mitogenic Challenge

In the Russian space program, alterations in the in v i m response of cosmonaut

Table 2. Summary of Skylab Immunology Results

Nofe: All numbers represent celldmm3.

after a stay of 60 to 90 days in space. Despite identical conditions, there were no

figure 3. PHA reactivity of lymphocytes in cosmonauts after prolonged spaceflights

deviations in 4 out of 10 crew members, and a moderate decrease in the other 6

lymphocyte Responses After Multiple Missions

Multiple examination of 12 cosmonauts participating in two or three long-term

figure 4. Average PHA reactivity of “T” lymphocytes in cosmonauts after (landing +

following example exemplifies these results. One cosmonaut participated in an

Changes in Humoral Blood Components

Numerical Changes in Cells of the immune System

Table 3. Summary of Postflight Changes in Shuttle Crew Peripheral Blood Cells

Source: From Taylor and Dardanoa

(CD4) lymphocyte Additional data from 11 of these crew members

Table 4. Effect of Spaceflighton Peripheral Blood Leukocytes

Source: After Meehan et al.lU

be preceded by related neuroendocrine changes. Meehan and co-workers recognize

Production of Interleukin-2, Interferon-2, and Interferon-y

In vivo T cell proliferation requires interferon-gamma (IFN-y) and probably

CyiotoxicActivity of Natural Killer Cells

Figure 6. Cytotoxic activity of natural killer (NK)cells in cosmonautsbefore and after