File Name: E141.DOC

SLS-1 Final Report Experiment 141

**Regulation of Blood Volume During Spaceflight**

**Principal Investigator: Dr. Clarence P. Alfrey**

**Summary Abstract**

This experiment was designed to determine the effect of the microgravity
environment of space flight on the regulation of blood volume in the
rat. This effect was quantitated by assessing changes in red blood cell
mass (RBCM), plasma volume (PV), red blood cell (RBC) survival and iron
economy. The objective was to evaluate whether the rat was a suitable
animal model for further research on the elucidation of the control
mechanism responsible for the RBCM loss that had been observed in humans
after return from space flight.

Radioactive tracers were used to measure PV, RBCM and RBC survival and
iron kinetics. RBCM and PV were measured pre-flight (L-8), on landing
day (R+0) and eight days after landing (R+8). 59Fe was injected on R+0
and incorporation into RBCs was followed over the next eight days. 51Cr
RBC survival studies were from L-7 to R+0 and R+1 to R+8.

Upon landing, the mean RBCM of flight rats was significantly less than
that of synchronous ground controls, whether expressed as absolute
volume or volume normalized for body mass. PV, normalized for body mass,
was also significantly decreased on R+0 in the flight animals. The 51Cr
survival data does not implicate an increased RBC destruction rate as
the cause of the decreased RBCM. The post-flight decrease in 59Fe
incorporation into RBCs could indicate a decrease in RBC production in
response to space flight or in response to the decreased food intake and
weight gain of the flight animals during the post-flight period.

The rat, like the human, has a decrease in RBCM in response to
microgravity exposure. Neither species shows any indication that this is
due to hemolysis. It would appear that the rat is an appropriate animal
model in which to study the mechanisms involved in the control of
erythropoiesis during exposure to the microgravity environment of space
flight.

SLS-1 Final Report Experiment 141

**Regulation of Blood Volume During Spaceflight**

***Principal Investigator:***

*Clarence P. Alfrey, M.D., Ph.D.*

Baylor College of Medicine

Department of Medicine

The Methodist Hospital

6565 Fannin MS M902

Houston, Texas 77030

***Co-Investigators:***

*Mark M. Udden, M.D.*

*Ronald Nachtman, Ph.D.*

*Theda Driscoll, B.S.*

Baylor College of Medicine

Houston, Texas

OBJECTIVE

This experiment was designed to determine the effect of the microgravity
environment of space flight on the regulation of blood volume in the
rat. This effect was quantitated by assessing changes in red blood cell
mass (RBCM), plasma volume (PV), red blood cell (RBC) survival,
erythropoiesis and iron economy. The objective was to evaluate whether
the rat was a suitable animal model for further research on the
elucidation of the control mechanism responsible for the RBCM loss that
had been observed in humans after return from space flight.

INTRODUCTION

In space flights as short as seven days, an 8 to 15 percent reduction in
RBCM has been measured in astronauts upon landing. Current theories
regarding the regulation of erythropoiesis would require an increased
rate of RBC destruction to produce such changes in RBCM. However, data
from humans has indicated that RBC survival time is unchanged during
space flight. Studies in a suitable experimental animal model will help
to clarify this apparent anomaly. In the past, hematology studies have
been conducted on rats before and after microgravity exposure, but the
SLS-1 mission was the first time RBCM and PV measurements were included
in the pre- and post-flight work-up of the animals.

MATERIALS AND METHODS

The following radioactive tracer methods were used: 125I-labeled albumin
for PV, 51Cr-labeled donor RBCs for RBCM and RBC survival and
59Fe-ferrous citrate for iron kinetics. The pre-flight RBCM and PV were
measured eight days before the start of the flight (L-8), on the day of
landing (R+0) and eight days after landing (R+8). 59Fe was injected on
R+0 and incorporation into RBCs was followed over the next eight days.
51Cr RBC survival studies were from L-7 to R+0 and R+1 to R+8. Serum
ferritin levels and 51Cr spleen to liver ratios were determined on R+0
and R+9 when animals were sacrificed. These measurements were made for
fifty-nine rats pre-flight and twenty-nine rats post-flight. On L-6,
thirty additional rats were injected with labeled RBCs and 51Cr spleen
to liver ratios were determined on R+0.

This investigation was divided into two phases, the SLS-1 Flight Study
and the Delayed Flight Profile Test (DFPT). The DFPT period started ten
days after completion of the flight study. During the flight study
period, five rats were housed in each of two Animal Enclosure Modules
(AEM) and nineteen rats were housed in the individual cages of the
Research Animal Holding Facility (RAHF). The reason there were nineteen
rather than twenty was that the water delivery system in one RAHF cage
did not work and the animal was removed prior to flight. There were two
principal animal groups for which RBCM and PV were determined: flight
(n=14) and synchronous ground control (n=15). The synchronous ground
controls were housed at KSC until launch and then they were flown to the
Dryden landing site during the in-flight period for post-flight
evaluation. In the DFPT study period, the same two principal group
distinctions were employed (flight, n=15; ground control, n=15). The
animal housing conditions were the same, but all the DFPT animals
remained at 1g and at KSC Hanger L throughout the period that they were
studied.

Samples were obtained and analyzed for both serum and plasma ferritin
levels, but only the serum values are reported. Serum was available on
sacrifice day and 8 to 10 μl plasma samples were recovered after every
hematocrit. When the plasma ferritin assays were done the sequential
sample variance was outside acceptable limits. In 20 to 30% of the
samples the values were 3 to 5 times the expected value. There was no
pattern to these greatly elevated values; therefore, due to lack of
confidence in the samples, all the plasma data was considered
unreliable.

Statistical analysis, Repeated Measures ANOVA and Dunnett\'s Test, of
the DFPT data showed no statistical difference that could be attributed
to the different housing conditions. Therefore, the RAHF and AEM animals
were combined into one flight animal group and the single- and
group-housed vivarium controls were combined into one ground control
animal group.

RESULTS AND DISCUSSION

The flight study and DFPT data are presented in tabular form in Appendix
A. Missing values are a result of technical problems with the blood
sample, injection dose or injection process. The flight study data is
discussed in a paper that has been prepared for publication in
combination with the E012 investigators. The paper entitled \"Blood
Volume and Erythropoiesis in the Rat During Space Flight\" (submitted to
*Aviation, Space and Environmental Medicine)* is Appendix B. The data
that was not covered in the paper are addressed below.

Hematocrit Data

No flight related changes were found. The pre-flight mean values were
less than the post-flight mean values for all groups.

Echinocyte Data

Blood samples fixed with 0.5% glutaraldehyde were coded and the
proportion of cells that had echinocytic morphology were determined in a
blinded fashion. For all groups, flight study and DFPT, the number of
echinocytes decreased on R+0 and R+1. There was no change that could be
related to the effects of Space flight.

51Cr spleen to liver ratio in animals sacrificed on R+0

There was no change in the spleen to liver ratio that could be
attributed to microgravity exposure. On R+0, elevated values were
observed in numerous flight and synchronous ground control animals. When
there was an elevated ratio, the percentage of 51Cr remaining in the
blood was decreased proportionally. These findings could indicate a
minor incompatibility between the donor blood and the recipient rat, but
extensive testing has failed to show any incompatibility in the strain
of rats that were used for this experiment.

For the 118 spleen to liver ratios reported here, eight different 51Cr
labeled RBC pools were prepared (two to three donors per pool) and 30 to
60 injection doses were made from a given pool. The indication of RBC
incompatibility between donor and recipient involved a single pool and
only a few of the animals that were injected from that pool.

Body Mass Data

During the pre-flight period there was no difference in the growth rate
among the groups of animals. Thereafter, the flight study animal groups
grew at a slower rate than the DFPT animal groups. While there was no
difference in the landing day weights of flight animals and synchronous
ground control animals, there was a difference in growth rates during
the post-flight period. It was difficult to know how to analyze the RBCM
and PV data of these growing animals so that the effects of space flight
could be separated from the growth-dependent volume changes. A
significant decrease in PV relative to space flight was found only after
the PV was normalized for the body weight of the animal and when the
flight animals where compared to synchronous ground controls. When the
DFPT controls were included in the analysis the normalized RBCM decrease
was significant but the PV decrease was not. Because the growth rate of
the DFPT animals was different from the flight and synchronous ground
control animals the decision was made not to include the DFPT animals in
our analysis for flight-related differences.

CONCLUSION

Upon landing, the mean RBCM of flight rats was significantly less than
that of synchronous ground controls, whether expressed as absolute
volume or volume normalized for body mass. PV, normalized for body mass,
was also significantly decreased on R+0 in the flight animals. The 51Cr
survival data does not implicate an increased RBC destruction rate as
the cause of the decreased RBCM. The post-flight decrease in 59Fe
incorporation into RBCs could indicate a decrease in RBC production in
response to space flight or in response to the decreased food intake and
weight gain of the flight animals during the post-flight period.

The rat, like the human, has a decrease in RBCM in response to
microgravity exposure. Neither species shows any indication that this is
due to hemolysis. It would appear that the rat is an appropriate animal
model in which to study the mechanisms involved in the control of
erythropoiesis during exposure to the microgravity environment of space
flight.

**APPENDIX B**

**Journal Article Submitted to the**

***Aviation, Space and Environmental Medicine***

Blood Volume and Erythropoiesis in the Rat During Space Flight

Mark M. Udden, M.D.1

Theda B. Driscoll 1

Linda A. Gibson 2

Cynthia S. Patton 1

Mark H. Pickett 1

J. B. Jones, D.V.M. 2

Ronald Nachtman, Ph.D 1

Zuhair Allebban, B.V.M.S., Ph.D. 2

Albert T. Ichiki, Ph.D. 1

Robert D. Lange, M.D. 2

Clarence P. Alfrey, M.D., Ph.D. 1

1 Baylor College of Medicine

Houston, Texas, USA

and

2 The University of Tennessee Medical Center

at Knoxville, Tennessee, USA

Running head: Rat Erythropoiesis in Space Flight

Address correspondence to: Mark Udden, M.D., Medicine/Hematology, One
Baylor Plaza, Houston, TX, 77030, USA. Telephone number 713/798-6105.

Key Words: Space flight, microgravity, erythropoiesis, red blood cell
mass, plasma volume, iron utilization, erythropoietin, erythroid
progenitors

ABSTRACT

A decreased red blood cell mass (RBCM) and plasma volume (PV) have been
consistently found in humans after return from space flight. Rats flown
on the Spacelab Life Sciences - 1 mission were studied to assess changes
in RBCM, PV, erythropoiesis, and iron economy. The RBCM and PV increased
in both ground control and flight animals as expected for growing rats.
However on landing day, both the RBCM and PV, when normalized for body
mass, were significantly decreased in the space flight animals. During
an 8 day post-flight observation period, iron incorporation into
circulating red blood cells was diminished in the flight animals. During
the first four days post-flight increases in reticulocyte counts were
significantly smaller in the flight than the control animals. Fewer
erythropoietin-responsive progenitor cells were recovered from the bone
marrow of flight animals after landing than control rats. Serum
erythropoietin (EPO) levels were the same in both groups. Thus, rats
subjected to a 9 day space flight had less increase in RBCM than
controls and diminished erythropoiesis during an 8 day post-space flight
observation period.

INTRODUCTION

A consistent finding in humans after space flight is a decrease in the
red blood cell mass (RBCM) and plasma volume (PV) (3,6,7). These
changes, found in American astronauts and Russian cosmonauts, are
thought to represent an adaptation to weightlessness. Hematological
studies carried out in rats subjected to space travel during Cosmos
missions and Spacelab 3 have produced conflicting results
(4,5,12,13,14,21,22, 23). Because measurements of RBCM and PV were not
obtained in these early American and Russian studies, it was not
possible to determine from the hematocrit or hemoglobin alone whether or
not the rat, like the human exposed to microgravity, decreased its RBCM.

We report here the results of two complementary studies carried out on
rats that accompanied the human space travelers for 9 days on the NASA
space shuttle Columbia mission, Spacelab Life Sciences-1 (SLS-1/STS-40).
Our objectives were to assess RBCM, PV, reticulocyte counts, marrow
erythropoietin-responsive progenitor cell number, erythropoietin levels,
iron utilization and storage, RBC survival and RBC shape. These studies
provide the first comprehensive examination of erythropoiesis and blood
volume regulation in rats subjected to space travel.

METHODS

[Animals]{.underline}. Male, specific pathogen-free, Sprague-Dawley rats
were supplied by Taconic (Indianapolis, Indiana). These studies were
approved in advance by the animal care and use committees of Baylor
College of Medicine, the University of Tennessee Medical Center at
Knoxville and the NASA Ames Research Center. The animals were handled in
a humane manner under the supervision of a veterinarian and in
accordance with national and international standards. The animals were
housed in the Kennedy Space Center (KSC) vivarium facility for four
weeks prior to launch. There were 29 animals in the flight group and 30
ground controls. In each group, 10 rats were caged 5 to a single cage.
The remainder were caged individually. During space flight, animals
single-housed were maintained in the Research Animal Holding Facility
(RAHF). Control and flight animals group-housed were supported in Animal
Enclosure Modules (AEM). The RAHF and AEM are animal housing facilities
specifically designed for space flight. The day after launch, the ground
control animals were flown to the landing site at Edward\'s AFB, CA, and
housed in a vivarium at the Dryden Payload Receiving Facility.

On the day of landing or recovery day (R+0), 19 flight and 20 control
animals were killed. Blood samples were taken from these animals for
ferritin and EPO determinations, RBC count, morphology and indices and
reticulocyte count. Bone marrow was obtained for progenitor cell culture
and for morphologic evaluation. The remaining twenty rats underwent
determinations of RBCM and PV. Red blood cell 59Fe-incorporation studies
were initiated on R+0 within 3-6 hours after landing. These animals were
killed on R+9 and blood and bone marrow were taken for the same
measurements made on the animals killed at landing.

[Red blood cell mass and plasma volume determinations]{.underline}. RBCM
was determined using 51Cr and PV with 125I as previously described (18).
Tail veins were used for the injections and to obtain blood samples
using commercial over-the needle catheters to reduce the number of
venipunctures (17). The volume of blood withdrawn was less than 0.3 ml
for both the background and the post-injection sample.

[Blood cell counts]{.underline}. Automated blood cell counts were
performed in duplicate using the TOA (Kobe, Japan) Sysmex F-800 micro
cell counter. The determinations included: RBC count, hematocrit (Hct),
hemoglobin (Hgb), mean cell volume (MCV), mean cell hemoglobin (MCH),
and mean cell hemoglobin concentration (MCHC). A drop of blood was added
to 1.0 ml of 0.5 % glutaraldehyde in phosphate buffered normal saline
containing 1.13 mM calcium and 1.0 mM magnesium chloride buffer. Later
these were mounted wet on glass slides under cover slips and examined
under plane microscopy at 1000x magnification. One thousand cells were
counted to determine the number of normal discocytes and spiculated
cells.

[Reticulocyte counts]{.underline}. Samples of blood were shipped under
refrigeration to Knoxville where they were brought to room temperature.
Blood (5μl) was pipetted into 1.0 ml of thiazole orange stain, mixed and
incubated in the dark for 30 minutes. The samples were analyzed on a
Becton-Dickinson FAC Scan flow cytometer. The reticulocyte count was
determined after analysis of 10,000 events with a software program,
ReticCount (Becton Dickinson, San Jose, CA.).

[59-Iron incorporation into circulating red blood cells]{.underline}. On
landing day, three μCi of 59Fe-ferrous citrate in a volume of 0.3 ml
were injected intravenously. Samples were obtained 24 hours and 8 days
later and RBC 59Fe radioactivity measured. The RBCM determined by 51Cr
on R+0 and R+8 was used to calculate the respective 59Fe incorporation.

[Erythroid Colony Assay]{.underline}. One femur was removed from each of
the flight and control rats and was returned to the Knoxville laboratory
under refrigeration. The bone marrow was flushed into a 15 ml conical
tube and the cultures were carried out according to the method described
by Ogawa (19), with some modifications. The culture mixture (1 ml)
contained 3.7 x 105 total bone marrow cells suspended in
methyl-cellulose supplemented with Iscoves Modified Dulbecco\'s Medium
(Terry Fox Laboratories, Vancouver British Columbia), and 10% mouse
spleen-conditioned medium without EPO or with either one of three
concentrations (0.25, 0.5, 1.0 U/ml) of recombinant human EPO (Amgen,
Thousand Oaks, CA). DAF (2,7-diaminofluorene) stain was used to
enumerate CFU-e\'s and BFU-e\'s on days 3 and 7, respectively.

[Bone marrow differential]{.underline}. The bone marrow smears were
prepared from the head of the femur (1), fixed in absolute methanol for
10 minutes, and then stained for 10 minutes with Wright\'s stain. After
rinsing, the slides were stained for 10 minutes with freshly prepared
and filtered Giemsa stain, rinsed and dried. Differential counts of 200
cells were performed to determine the percent of cells that were
immature (pronormoblasts and basophilic normoblasts) and mature
(polychromatophilic and acidophilic normoblasts) erythroid cells.

[Erythropoietin and ferritin determinations]{.underline}. Serum was
obtained from 3.0 ml of tail vein blood and immediately frozen to -70
oC. EPO levels were measured using a commercial radio immunoassay kit
(Diagnostic Systems Laboratories, Inc. Houston, TX). Ferritin was
determined by radio immunoassay (RAMCO, Houston).

[Statistical methods]{.underline}. There were no significant differences
between single-housed and group-housed animals as determined by repeated
measurements ANOVA. Therefore, group- and single-housed animals were
analyzed together for flight and control animals and comparisons made
using the repeated measurements ANOVA, Student-Newman-Keuls Multiple
Comparison test, student t and Wilcoxon Z tests.

RESULTS

There was no difference in weight of flight and control rats at the end
of the inflight period (Figure 1). However, the flight animals grew
slower during the post-flight period of the study (Figure 1). The total
RBCM increased in the control and flight animals as they grew during the
period of study (Table I). On the day of landing and 8 days later, the
total RBCM in the flight animals was significantly less than that in the
control rats. On landing day, the RBCM adjusted for body mass was
significantly lower in the flight animals than controls. However, the
weight adjusted RBCM was not significantly different at the end of the
post-flight recovery period. On landing day, the PV adjusted for body
mass was significantly lower in the flight animals than controls (Table
I). It is possible that a greater PV deficit was present at landing but
was not observed when the volume measurements were determined 3-6 hours
later.

There were no significant differences in the RBC counts or Hgb of flight
and control animals on landing day (R+0) (Figure 2). During the
post-flight period, these values increased for both groups during the
first three days. On R+1, there was a significant increase in RBCs and
Hgb in the flight animals compared to the controls. By day 9 of the
post-flight observation, there were no differences in these values.
Values obtained for the hematocrit were consistent with the RBC and Hgb
determinations (data not shown).

There were no significant differences between the flight and control
animal MCV values (data not shown) and no significant changes
post-flight in the number of RBCs with atypical shapes. Small numbers of
spiculated cells were observed and appeared to be the most common
atypical cell present in rat blood.

The reticulocyte counts of the flight rats were not statistically
different than the values of control rats on R+0 (Figure 3), but the
increase in the reticulocyte counts of the flight animals was
significantly smaller during early recovery.

Two estimations of red blood cell survival were made. Data points for
survival curves were only collected on days when the RBCM was measured.
Since the animals were growing, this avoided errors associated with an
increasing RBCM. Because of a launch delay, the first 51Cr-labeled RBC
injection was 8 days before launch. Injections of 51Cr-labeled RBCs were
repeated on landing day and on the 8th day after landing. Thus, the
first 51Cr survival period reflects the loss of RBCs during an 8 day
pre-flight period and a 9 day flight period. The second estimate of 51Cr
survival was made from determinations on days 1 and 8 after landing.
This data is shown in Table II. There were no differences between the
two periods and thus no apparent effect of space flight on the life span
of rat erythrocytes.

Table III shows that 59Fe incorporation into RBCs was significantly
greater in control than flight animals after space flight. The serum
ferritin, which correlates with iron stores (24), was significantly
greater in the flight animals (387 [+]{.underline} 13 ng/ml;n=15) than
in controls (333 [+]{.underline} 12 ng/ml; n=15) at landing. Eight days
after landing, there were no differences in serum ferritin levels.

The effects of space flight on bone marrow erythroid progenitors are
shown in Figure 4. The flight animals had a significantly decreased
number of BFU-e\'s recovered from their marrows on landing day. A
similar but less dramatic loss of CFU-e\'s was also found. When
Wright-Giemsa stained marrows were examined, there was a decrease in the
percent of immature or mature erythroid cells in the flight animals, but
it was not statistically significant (data not shown).

There were no significant differences in serum EPO levels between
control (21.7 [+]{.underline} 0.72 mU/ml) and flight animals (22.1
[+]{.underline} 0.77 mU/ml) at landing or 9 days after landing (control
20.1 ± 0.56, flight 21.2 ± 0.67 mU/ml).

DISCUSSION

In both control and flight groups, the total RBCM increased during the
flight period of the study, as expected for growing animals. However, it
is clear that the increase of the RBCM in the animals flown in space was
smaller than the controls. The hematocrits did not differ between the
flight and control animals because the PV also decreased. During the
nine day post-flight observation period, the RBCM in the flight animals
continued to lag behind that of the control animals. However, the rate
of growth during the post-flight period was slower in the flight animals
so that the weight adjusted RBCM in the flight animals was not
significantly different than the controls eight days after landing. The
normal 51Cr-labeled RBC survival study and the absence of significant
changes in RBC morphology support the conclusion that the relative
decrease in RBCM during exposure to microgravity was not due to
hemolysis or splenic sequestration.

The absence of any significant hemolysis indicates that decreased
production of red blood cells was the cause of the relative decrease in
RBCM during space flight and recovery. The ferritin level in the flight
rats at landing was increased compared to controls. This increase is
consistent with diversion of iron from developing RBCs to iron stores as
would be expected if fewer RBCs were being produced during space flight.
The incorporation of injected 59Fe into new RBCs was also greatly
reduced in the flight animals during the post-flight observation period.
At landing, fewer BFU-e\'s and CFU-e\'s were recovered from bone marrow
cells of flight animals than controls, which is consistent with the
results of Vacek (22,23,24). Furthermore, rats subjected to a 22-day
flight on Cosmos 605 demonstrated a decrease number of bone marrow
erythroid progenitors (5). However, experiments on SL3 showed an
increase in BFU-e and CFU-e numbers which might be due to various
factors, i.e., stress, time of animal recovery post-landing and also the
use of non-recombinant EPO (13). In this study, morphologic evaluation
of the bone marrow of flight animals showed a slightly reduced number of
erythroid cells which is similar to previously reported studies (13).
There were fewer reticulocytes present in the peripheral blood collected
from flight animals during the initial days of the post-flight period.
The slow appearance of 59Fe-RBCs, the increase in iron stores, the
reduced numbers of erythroid progenitors, and the low reticulocyte
counts during the recovery period support our conclusion that
erythropoiesis was diminished in the rats subjected to space flight.

Despite the relatively decreased RBCM observed in flight animals, the
EPO level was not different than controls. Physiologically, when RBCM is
decreased, the EPO level should increase. The absence of an increased
EPO level may be due to the difficulty in discerning changes in the
normal range. An unchanged level could also mean a decrease in the
demand for more RBCs in microgravity.

Other growth factors and cytokines may participate in the regulation of
erythropoiesis in animals subjected to the stress of space flight and
landing. These include factors that stimulate erythropoiesis such as
insulin-like growth factor-I (11,20), interleukin(IL)3 (9,10), IL9 (2),
and stem cell factor (16), or inhibitory factors such as tumor necrosis
factor (8), and γ-interferon (15).

In this examination of the effects of space flight on the rat, the RBCM
and PV were decreased at landing and were accompanied by evidence for
slowed erythropoiesis. Thus, the rate of RBC production slowed, the
number of erythroid progenitor cells decreased and iron utilization was
reduced. Erythropoietin levels did not change suggesting the possibility
that the observed reduction in RBCM was appropriate for space flight or
was due to the effects of other growth factors or cytokines. During the
SLS-2 shuttle mission, crew members carried out similar experiments in
flight. We anticipate that the collection of in-flight data will better
define the effect of microgravity on these hematological parameters.

ACKNOWLEDGMENTS

The authors thank the flight crew of STS-40 and the personnel of the
Ames Research Center, the Kennedy Space Center Hangar L and the Dryden
Payload Receiving Facility. We wish to acknowledge the great efforts
made by Nina Mushell of the Ames Research Center as Experiment Support
Scientist for this project. Others whose support we appreciate include:
Joseph E. Fuhr, Ph.D., of the University of Tennessee Flow Cytometry
Facility, Frances Jones, M.D., of the Department of Pathology,
University of Tennessee; Craig Irwin, Erika Amaba, Lisa Payne and Walter
Haley for their technical assistance; and Mary Moir for preparing the
manuscript. We thank David A. Sears, M.D., for his careful review of the
manuscript. We wish to acknowledge C.D.R. Dunn, Ph.D., and the late P.C.
Johnson, M.D., who initiated this study.

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FIGURE LEGENDS

1\. Weight of flight and control animals from 28 days before launch to 9
days after recovery. The post-flight data are shown in the insert on an
expanded scale.

2\. Red blood cell count (A) and hemoglobin (B) from peripheral blood
obtained from flight and control animals before and after space flight.
L denotes launch day. R denotes recovery (landing) day. The red blood
cell count and hemoglobin in the flight animals were significantly
increased compared to the controls on R+1.

3\. Pre- and post-flight reticulocyte counts. The reticulocyte counts
from the flight animals were significantly lower than the ground
controls on days 1,3, and 4 after landing. L denotes launch day and R
denotes recovery (landing) day.

4\. Erythroid progenitors in bone marrows of flight and control animals
on landing day (R+0) and on R+9. Panel A shows BFU-e\'s on landing day
and Panel B BFU-e\'s on R+9. Panel C shows CFU-e\'s on landing day and
Panel D CFU-e\'s on R+9. Amounts of EPO added to cultures are shown on
the abscissa. The number of BFU-e\'s recovered from the flight animals
on R + 0 were significantly lower than the controls at each
concentration of EPO tested. There were no differences between flight
and control animal BFU-e\'s detected on R+9. The number of CFU-e\'s
recovered from flight animals was significantly lower than controls on
R+0 (Panel C) in the presence of 0.25, and 0.5 U/ml EPO, but not with
1.0 U/ml EPO. Differences at 9 days were not significant.

**Table I**

**RBCM and PV in Rats Flown on SLS-1**

(Mean [+]{.underline} S.E.M.)

Volume in milliliters Volume Normalized for Body Weight

(ml/100g)

8 Days Preflight Landing 8 Days Postflight 8 Days Preflight Landing 8
Days Postflight

RBCM

Flight animals (n=14) 5.75 [+]{.underline} 0.11 6.95 [+]{.underline}
0.12 \* 7.16 [+]{.underline} 0.12 \* 2.33 [+]{.underline} 0.03 2.10
[+]{.underline} 0.03 \* 2.11 [+]{.underline} 0.04

Controls (n=15) 5.73 [+]{.underline} 0.11 7.34 [+]{.underline} 0.11 7.80
[+]{.underline} 0.10 2.31 [+]{.underline} 0.04 2.24 [+]{.underline} 0.03
2.19 [+]{.underline} 0.03

PV

Flight animals (n=14) 9.85 [+]{.underline} 0.16 10.50 [+]{.underline}
0.16 10.88 [+]{.underline} 0.14 \* 3.99 [+]{.underline} 0.06 3.18
[+]{.underline} 0.05 \* 3.18 [+]{.underline} 0.06

Controls (n=15) 10.02 [+]{.underline} 0.20 10.95 [+]{.underline} 0.20
11.45 [+]{.underline} 0.21 4.04 [+]{.underline} 0.06 3.34
[+]{.underline} 0.05 3.22 [+]{.underline} 0.05

\* p [\<]{.underline} 0.05 from controls

**Table II**

**51Cr Red Blood Cell Survival in Rats Flown on SLS-1**

(Mean [+]{.underline} S.E.M.)

**T1/2 Spleen to Liver 51Cr Ratio**

Period 1 a Period 2 b

Flight animals (n=14) 18.8 [+]{.underline} 0.4 18.5 [+]{.underline} 0.5
1.3 [+]{.underline} 0.3

Controls (n=15) 18.2 [+]{.underline} 0.5 18.4 [+]{.underline} 0.5 1.0
[+]{.underline} 0.2

a From eight days pre-launch to landing day (R+0) b From day after
landing to eight days after landing

**Table III**

**Incorporation of 59Fe into Circulating RBCM of Rats Flown on SLS-1**

(Mean [+]{.underline} S.E.M.)

Percent of 59Fe Injected on Landing Day

Days Post-Injection

**1 8**

Flight animals (n=14) 30.0 [+]{.underline} 1.5 **\*** 53.9
[+]{.underline} 1.2 **\***

Controls (n=15) 43.0 [+]{.underline} 1.2 63.6 [+]{.underline} 1.2

**\*** p [\<]{.underline} 0.001 from controls

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