File Name: E012.DOC

**REGULATION OF ERYTHROPOIESIS IN RATS**

**DURING SPACE FLIGHT**

**FINAL REPORT**

Principal Investigator

Robert D. Lange, M.D.

The University of Tennessee Medical Center at Knoxville, TN 37920

Co-Investigators

A.T. Ichiki and J.B. Jones

Co-Authors

Z. Allebban, L.A. Gibson, C.E. Irwin, and C.C Congdon

Technical Assistants

L. Payne and E. Amaba

Statistical Analysis

P. Wright and E. Walker

This study was funded by NASA contract NAG 2-494, Experiment No. E012.

**TABLE OF CONTENTS**

1\. Introduction

2\. Chapter 1 - Regulation of erythropoiesis in rats during space flight

3\. Chapter 2 - Effects of space flight on rat spleen weight and
histology

4\. Chapter 3 - Effects of space flight on rat peripheral blood
leukocytes

5\. Chapter 4 - Effects of microgravity on rat spleen and peripheral
blood lymphocytes

6\. Chapter 5 - Summary, conclusions and recommendations for future
studies

7\. Bibliography

8\. Acknowledgments

9\. Appendix A - Materials and Methods

10\. Appendix B - Extra charts and tables (chapters 1-4)

11\. Appendix C - Raw data (chapters 1-4)

**[INTRODUCTION]{.underline}**

One of the old gospel hymns is entitled \"There is Power in the Blood.\"
From antiquity, this precious fluid has been vital to life and, indeed,
the early Hebrews were prohibited from eating blood (Genesis 7:4, RSV
Bible).

One of the most consistent findings observed in men exposed to orbital
space flights has been a decrease in the total circulating number of red
blood cells (red blood cell mass - RBCM) (1,2). Figure 1 shows the RBCM
changes in astronauts on SpaceLab 2,3, and 4 flights. It should be noted
that the deficit was repaired soon after the astronauts\' return to
Earth\'s gravity.

The RBCM is the product resulting from the production versus the
destruction of red blood cells. Normally functioning bone marrow has the
capacity to increase its production up to 6- to 8-fold. Originally, it
was thought that the decrease in the RBCM in astronauts was due to an
increased destruction of red blood cells due to the use of 100% O2 at a
partial pressure of 258 mm Hg. On the Skylab and Cosmos flights,
however, hyperoxia was not present but a decrease in RBCM was still
noted. It then became apparent that weightlessness was, in some manner,
leading to erythropoietic suppression. The implication of hemolysis
cannot be completely forgotten, however, since animal studies aboard
Cosmos 782 and 936 (4,5), as well as in astronauts aboard SpaceLab 1,
indicated that hemolysis may still have a role in the pathogenesis of
the anemia of space flight.

Accompanying the decrease in the RBCM is a decrease in plasma volume. As
a consequence, little change is noted for such parameters as hematocrit
(Hct), red blood cell (RBC) count, and hemoglobin (Hgb), since their
measurements are a result of their concentration in the plasma. The

decrease in these RBC parameters, seen immediately following a return
from orbit, is probably due to an increase in the plasma volume of the
astronauts.

The feedback circuit that links red blood cell production is portrayed
in Figure 2 (adapted from a diagram developed by Erslev, 6). Surrounding
the diagram are the measurements which were made on Space Life
Sciences-1 (SLS-1) to investigate particular components of
erythropoiesis. The starting point in the development of RBC\'s begins
at the top of the diagram with multipotential stem cells becoming
committed stem cells of the erythroid series.

Figure 3 portrays a model of hematopoiesis (modified from one developed
by Quesenberry and Levitt) (7) that is useful in considering stem cell
problems. Current thought is that hematopoietic tissues contain
pluripotential stem cells, which give rise to lymphoid, megakaryocytic,
erythroid, and granulocytic committed stem cells. Existing evidence
suggests that cells which form lymphoid and spleen colony cells (colony
forming unit-spleen \-- CFU-S) are derived from a common cell. In the
mouse, the erythroid and megakaryocytic cell lines apparently have a
common ancestral cell but some develop separately committed cells.
Several committed cell types can be grown *in vitro* by appropriate
culture methods. Colony forming unit-megakayrocyte (CFU-Mk) is the
cultured precursor cell of platelets. The earliest erythroid cells are
termed burst forming units-erythroid (BFU-E) and they give rise to
colony forming units-erythroid (CFU-E).

The granulocytic precursors develop into at least four separate clonal
systems: the granulocyte-monocyte (CFU-GM), granulocytic (CFU-G),
monocytic (CFU-M), and eosinophilic (CFU-Eos) colonies.

Several hormones and cytokines effect various phases of stem cell
activity. One such hormone we studied is erythropoietin (Epo), which
acts on erythroid committed cells to cause differentiation leading to
mature RBC\'s. A burst promoting activity (BPA) acts to enhance
differentiation of BFU-E. Production of platelets is thought to be
regulated by thrombopoietin. A heterogeneous group of substances
stimulate the *in vitro* solid phase colony formation of leukocytes.

Inhibitors of cell proliferation are known, e.g., chalones, lactoferrin,
and prostaglandin, and may have colony inhibiting activity. Indeed, at
least 20 inhibitors of hematopoietic growth factors have been described
(8). The influences of cell-to-cell interactions also have considerable
importance in stem cell activity. The monocyte-macrophages, in
particular, are thought to have a regulatory function because they not
only produce colony stimulating activities (CSA) but also prostaglandin
E2, which has an effect opposite that of CSA.

The committed erythroid stem cells, BFU-E and CFU-E are measured by
their ability to form colonies in methylcellulose or agar. Under the
stimulus of Epo, they go on to form pronormoblasts, normoblasts,
reticulocytes, and mature RBC. These production phases were looked at on
SLS-1 by enumerating the number of reticulocytes. The type of cells
(erythroid, myeloid, etc.) in the bone marrow and spleens of the rats
were enumerated since, in the bone marrows and spleens of the rats, the
circulating hematopoietic cells are formed. The RCM was measured by the
isotope 51Cr (E-141), as well as the RBC count. The plasma volume was
measured by the use of 125I-tagged albumin (E-141). As previously
stated, the Hgb and Hct are measures of hemoglobin concentration. All of
these factors come together to determine the total O2-carrying capacity
in

the RBCM. The availability of O2 is sensed by cells in the kidney, from
which the glycoprotein hormone Epo originates. As mentioned before, this
hormone stimulates the conversion of progenitor cells to differentiated
cells of the erythroid series. The concentration in blood serum of the
EPO hormone was determined in controls and in the rats exposed to the
weightlessness of microgravity.

At the bottom of Figure 2 are listed other essential measurements
performed on SLS-1. They include: body weights, food and water
consumption, white blood cell counts and differentials, and platelet
counts.

A summary of the hematological parameters measured on SLS-1 is given in
Table 1.

The objective of the hematology studies was to gain an understanding of
the regulatory parameters which modulate RBC production and destruction.
The original questions addressed were:

**OBJECTIVES**

1\. Determine if any changes in serum erythropoietin levels occurred in
rats exposed to microgravity.

2\. Determine if there were any changes in standard hematological
parameters: hemoglobin, hematocrit, red blood cell count, platelet
count, reticulocyte count, white blood cell count, and white blood cell
differential count.

3\. Enumerate lymphocyte subsets in peripheral blood.

4\. Determine the effect of weightlessness on the responsiveness of
erythropoietin-sensitive cells in *in vitro* cultures.

5\. Determine if the rat is an appropriate model for hematological
changes which occur in astronauts during space flight.

This report will be divided into the following chapters:

Chapter 1. Erythroid Parameters

Chapter 2. Spleen Studies

Chapter 3. Leukocytes and Platelets

Chapter 4. Lymphocyte Immunophenotyping

Chapter 5. Summary, Conclusions and Recommendations for Future Studies

Bibliography

Acknowledgments

Appendix: A. Materials and Methods

B. Tables and Figures not used in text

C. Raw Data on Individual Animals

Each chapter will contain: A. Significant Flight Results, and B.
Significant Results of the Delayed Flight Profile Test (DFPT). All
values presented are the means and standard error of the mean (SEM).

Table 1

Animal Parameters Measured in SLS1-012 Hematology Studies

A. General

1\. Body weight/mass

2\. Food consumption

3\. Fluid consumption

4\. Space craft environment

5\. RAHF & AEM environment

B. Peripheral Blood

1\. Hematocrit

2\. Complete blood count, including RBC indices, platelet count, and
leukocyte differential and lymphocyte subsets

3\. Reticulocytes

C. Erythroid Regulatory Parameters

1\. Erythropoietin

D. Medullary Parameters

1\. Spleen weight

2\. Bone marrow and spleen differential counts; spleen histology

3\. Bone marrow BFU-E and CFU-E

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**Chapter 1**

**Regulation of Erythropoiesis in Rats During Space Flight**

**[INTRODUCTION]{.underline}**

Astronauts have been found to have a reduced red blood cell mass after
exposure to microgravity (1-3). On the other hand, far fewer animal
studies have been performed with varied results. Thus, the Soviets and
their co-investigators have reported studies in rats flown on the
following Cosmos flights: 605 (4-9), 690 (10), 782 (11, 12), 936
(11-15), 1129 (16), 1514 (8,17), 1667 (8,18), 1887 (8,19), and 2044
(20,21). Rats have also been studied on two NASA flights (22-26). Mice
were flown and studied on Apollo XVII (27) and monkeys on two Cosmos
flights (28,29).

In this chapter, the results of the erythroid parameters studied will be
examined. These include:

Red blood count

Hemoglobin

Hematocrit

Mean corpuscular volume (MCV)

Mean corpuscular hemoglobin (MCH)

Mean corpuscular hemoglobin concentration (MCHC)

Reticulocytes (manual and flow cytometry)

Erythropoietin

Bone marrow differentials

Bone marrow response to EPO (CFU-E and BFU-E)

**[MATERIALS AND METHODS]{.underline}**

See Appendix A.

**[RESULTS]{.underline}**

The different housing groups were combined for the flight animals
(Animal Enclosure Module \[AEM\] flight + Rodent Animal Holding Facility
\[RAHF\] flight, AEM control + RAHF control).

This section will present the results for the erythroid parameters for
both the flight and the DFPT. Those tables and charts not included in
this chapter will be found in Appendix B and the results on individual
animals will be found in Appendix C.

**A. [FLIGHT]{.underline}:**

[Erythroid Parameters of the Complete Blood Count]{.underline}:

Pre-Flight (L-4 - L-2): No significant differences in the erythroid
parameters of the complete blood count (CBC) were seen between the
control (AEM control and RAHF control) and flight (AEM flight and RAHF
flight) animals.

Landing (R+0): Upon landing, statistical differences appeared for the
MCV and MCHC between the flight and control groups. The flight animals\'
values were lower for the MCV and higher for the MCHC. Even though the
computer determinations of these two groups are statistically,
significantly different, examination of the means and standard
deviations or the standard error of the mean showed very little
difference.

Post-Flight (R+1 - R+9): The remainder of the rats were bled postflight
on R+1, R+3, R+4, R+8, and R+9. During this postflight or \"recovery\"
period, the flight animals showed significant increases on R+1 in the
RBC, Hgb and Hct values. On R+3, the flight animals\' values again were
increased but only for the MCHC parameter. The remainder of the

parameters on phlebotomy days showed no significant differences between
control and flight rats.

[Reticulocytes]{.underline}: The reticulocytes, either counted manually
or in the flow cytometer, showed no significant differences between
control and flight rats during preflight or at landing. Postflight,
however, the flight animals showed significantly lower values than
controls using the flow cytometer method on R+1 and R+3. On R+4, both
methods indicated that the flight animals had lower values compared to
the controls. On R+8, the manual counts indicated that the flight
animals had significantly higher values.

[Erythropoietin]{.underline}: The serum Epo levels were tested on
specimens obtained upon landing (R+0) and 9 days postlanding (R+9). No
significant differences between the flight and control groups were
found.

[Bone Marrow]{.underline}: The responsiveness of the bone marrow to Epo
was tested on R+0 for one group and its control and 9 days post-landing
for the remaining group and its control. The early progenitor (BFU-E)
showed the most differences on R+0, with flight animals having lower
values than controls at the three doses of Epo (Fig. 1). The CFU-E, or
later progenitors, were more variable, with flight animals having lower
colony counts at the .25 and .50 U/ml dose levels on R+0 (Fig. 2). On
R+9 with added Epo, there were no significant differences between the
flight and control groups.

[Bone Marrow Differentials]{.underline}: The only changes seen in the
bone marrow differentials were on R+0. The flight animals were slightly
but significantly higher in numbers of immature myeloid cells and
eosinophils and in lower numbers of lymphocytes.

**B. [DFPT RESULTS]{.underline}:**

1\. R+0 Animals: The only major statistically significant differences
were found in the clonal studies:

a\. CFU-E with 0.25 U Epo added. AEM control lessthan RAHF control.

b\. CFU-E with 1.0 U EPO added. The AEM control and RAHF flight had
fewer colonies than the RAHF control group.

2\. R+9 Animals - Major differences

a\. Erythroid parameters of the CBC

1\. RBC. The AEM control rats had lower values than the RAHF controls on
R+0 and R+3 and than the AEM flight animals on L-4.

2\. Hgb. AEM control group was less than RAHF control on R+0.

3\. MCHC. On L-3, the AEM flight group had lower values than the RAHF
flight animals.

b\. Reticulocytes: By the manual determinations on L-4, RAHF flight
animals were decreased in comparison to RAHF controls.

c\. Bone marrow differentials. The mature erythroid cells of the AEM
flight animals were increased in comparison to the RAHF flight group.

d\. Clonal studies - BFU-E. The AEM control group had a greater number
of colonies than the RAHF controls in cultures with 1.0 U Epo added.

**[BODY WEIGHTS]{.underline}**:

Body weights were taken on the animals at intervals, beginning at their
arrival at Kennedy Space Center (KSC) and ending at the final phlebotomy
at the Dryden Payload Receiving Facility (DPRF).

[Flight]{.underline}: As seen in Fig. 3, all of the rats grew at a
consistent rate up through R+0. Between R+0 and the remaining postflight
period, however, the rats showed a distinct separation of growth
patterns between the control and flight animals. Both groups had an
unexplained loss in body weight from R+0 to R+1. The flight animals
continued to lose weight through R+2 before regaining weight, starting
at R+3. The control animals started regaining weight at R+2 but at a
much slower rate than preflight.

[DFPT]{.underline}: Fig. 4 depicts that all groups of the R+0 animals
showed consistent and nominal growth patterns throughout the entire
DFPT.

As shown in Fig. 5, the R+9 animals\' growth pattern flattened out after
R+0.

**[FOOD AND WATER CONSUMPTION]{.underline}**:

Food and water also were measured from the rats\' arrival to the end of
postflight activities. During the flight, only the RAHF control and
flight animals had food and water measured.

[Flight]{.underline}: As seen in Figs. 6 & 7, the food and water
consumption between control and flight animals are consistent with each
other preflight. During the flight, distinct differences were noted. The
flight animals consumed both food and water at a faster rate than the
control animals, whose consumption decreased during this time. Upon
landing through R+1, the decrease in food intake of both groups
continued. The flight animals continued to have a decrease in water
intake through R+1, while the controls resumed their normal intake of
water from L+7 through the post flight period.

[DFPT]{.underline}: R+0 and R+9 animals: As can be seen in Figs. 8-11,
the DFPT food and water data does not present a concise pattern, as did
the flight data. The reason for this lies with the fact that a lower
number of samples in each group created a greater variability.

Age and RBC: As shown in Figs. 12 and 13, the RBC increased as the
animals grew. This was true both during the flight determinations and
the DFPT.

**[DISCUSSION]{.underline}**

**[CBC Erythroid Parameters]{.underline}**: In previous studies,
variations have been found in the RBC parameters of laboratory animals
flown in microgravity. Thus, Kalandarova has written that increased
values, decreased values, and unchanged values have been reported.

The Spacelab 3 differences, depending on the age of the animals, were
reported as shown in the following table (24-26):

  ----------------------- ----------------------- -----------------------
                          Small Rats              Large Rats

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  Hct                     Increase\*              Increase

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  RBC                     Increase\*              Increase

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  Hgb                     Increase\*              Increase

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  MCV                     Decrease                Decrease

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  MCH                     Decrease                NC

  ----------------------- ----------------------- -----------------------

  ----------------------- ----------------------- -----------------------
  CHC                     Decrease                Increase

  ----------------------- ----------------------- -----------------------

\* = Sig. \<0.05

LeBlanc on Space Transport 8 (ST8) rats found a significant increase in
RBC, Hgb, Hct, and MCV, but these animals were thought to be dehydrated
(22). It is of interest that Gazenko (10) and Leon *et al.* (12) found
evidence for a hemolytic component. However, in later studies, Leon

*et al.* (14) found that when the animals were centrifuged in flight, to
produce the effect of gravity, the hemolysis was prevented, indicating
that the hemolysis probably resulted from the lack of gravity rather
than other factors in the flight environment.

On SLS-1, we found no significant differences between flight and control
animals on R+0 in their RBC, Hct and Hgb. The MCV of flight animals were
lower than control animals, but their MCHC was increased. On R+1, the
RBC, Hgb and Hct were increased and on R+3, the MCHC of flight animals
increased. However, on R+9, there were no differences in the RBC
parameters of flight and control animals.

**[Reticulocytes]{.underline}:**

Previously, investigators have reported that the number of reticulocytes
(young circulating red blood cells) in humans and animals exposed to
microgravity are reduced in number immediately postflight
(3,9,10,19,30). This has been thought to be due to a slight depression
in red blood cell formation due to exposure to microgravity. In the
present studies, the reticulocyte values of the flight rats were not
statistically different than the values of control rats on either R+0 or
R+9. Lower values for flight animals were found on R+1, 3 and 4 when
reticulocytes were counted by flow cytometry (Fig. 12). This was also
true for manual counts on R+4, but on R+9 by manual counting, the
reticulocytes of flight animals were increased compared to their
controls.

Flow cytometry has been used to enumerate reticulocytes, allowing a
large number of cells to be counted (31-33). This method eliminates the
human element. Some authors have reported good agreement between the
values obtained by flow cytometry and the standard manual method of
counting reticulocyte numbers (33). We found that, although the trends

were the same, the flow cytometric method gave distinctly higher values.
Leach *et al.* (30,34,35), in human studies, also found that their flow
cytometry values were higher than those obtained by manual methods. It
is obvious that further studies using the two methods are needed and,
perhaps, the preservation of cells will allow studies to be made
in-flight.

**[Erythropoietin]{.underline}**: There have been very few serum Epo
level determinations carried out on flight rats. On SL3, we found the
Epo levels measured by radioimmunoassay to be 15.4 [+]{.underline} 0.8
in flight animals and in the controls 19.0 [+]{.underline} 3.7. In 5
rats studied after the 2044 Cosmos (21), flight animals had a level of
26.54 [+]{.underline} 0.77 and the synchronous controls 27.12
[+]{.underline} 1.78 (23). In the present studies, at R+0 there were no
essential differences between flight and control animals.

**[DFPT]{.underline}**: On R+9, the AEM flight animals had a
statistically higher value than the AEM control animals and were also
higher than either the RAHF control or flight animals. However, this
increased level is of questionable physiological importance.

Studies in astronauts have shown the Epo levels to be decreased during
flight and to sharply increase after landing, and to remain elevated
through 14 days after landing (30,34-37). This postflight elevation was
also found in Soviet Cosmonauts (37). Kalandarova (3) also reported that
Cosmonauts in microgravity over two months had lower Epo levels than
Cosmonauts in space for shorter periods of time.

**[Bone Marrow Colonies]{.underline}:**

The earliest clonal studies on the effect of space flight measured the
CFU-S. Vacek *et al.* found a decrease in CFU-S from spleen, liver and
bone marrow cells of flight animals from Cosmos 1667 and 1514, 936, and
1887 (15-17,38). CFU-S are thought to be very early stem cells. On
Cosmos

2044, Vacek *et al.* (38) found a decrease in the number of
hematopoietic progenitor cells (Granulocyte-Monocyte-Colony Forming
Cells \[GM-CFC\], BFU-E, CFU-E, Colony Forming Unit-Fibroblast
\[CFU-F\]) in the tibial bone marrow cells in animals which had been
injured prior to flight.

On SL3, we found significantly more erythroid colonies from bone marrow
cells of flight animals in response to Epo at levels of 0.02 and 1.0U
Epo/ml; but at the 0.20U Epo/ml level, the bone marrow cells from
control animals formed more colonies (23-26). Results today may be more
meaningful because the Epo being used is produced by recombinant
technology, compared to the use of urinary Epo, which was less pure. In
studies carried out post- Cosmos 2044, we found control marrow cells had
increased BFU-E\'s both without added Epo and at the 0.25U Epo/ml level
(21). There were no differences in the CFU-E colonies in the control and
flight groups.

In the present SLS-1 studies, R+0 flight animals had a decreased number
of BFU-E and in CFU-E at Epo dose levels at 0.25 and 0.50 U/ml, but not
a 1.0 U/ml. There were no significant differences discerned on R+9. The
results of clonal studies may indicate an inhibition of the earliest
erythroid progenitor cells in the animals exposed to microgravity. By
R+9, there were no differences observed between flight and control
animals, which could be due to the readaption to 1-G.

**[Bone Marrow Differentials]{.underline}:**

The results of bone marrow examinations showed, on the Cosmos flights, a
decrease in the number of erythroid cells and lymphoid elements with a
small increase in myeloblastic cells (5,7,8,13). The Soviet
investigators also reported a decrease in the numbers of marrow cells.
However, on Flight 936, the differences were not statistically reliable.

However, on SL3, we found no significant changes in the bone marrow
differentials (23-26) and on Cosmos 2044, while some minor differences
were found, there were no essential differences between flight and
control bone marrow differential counts (21).

In mice on Apollo XII studies, Ellis *et al.* (27) found that the
hyperoxic environment depressed erythropoiesis both in the flight and
control animals. In monkeys, a decrease in bone marrow erythropoietic
activity was found in flight animals (28,29). Six days after the Cosmos
2044 flight, the Soviet investigators reported there were marked signs
of erythroid production activation.

In the present studies, on R+0, there were no differences between flight
and control animals in their bone marrow differential counts. On R+9,
the flight animals had a slight increase in the number of immature
myeloid cells and eosinophils, but a slight decrease in the number of
lymphocytes. The changes were probably not physiologically significant.

**[SUMMARY]{.underline}**

Red blood cell parameters were examined in flight and control animals
during the actual SLS-1 flight and again during DFPT. During the actual
flight, there was no difference between flight and control animals for
the following parameters:

A. Pre-Flight

Complete blood counts

Reticulocytes enumerated either manually or by flow cytometry. The
manual counts gave lower values.

B. At R+0

Red blood cell counts

Hemoglobin

Hematocrit

Mean corpuscular hemoglobin

Reticulocytes, counted either manually or by flow cytometry. Manual
counts were lower than flow.

Bone marrow differential

Erythropoietin

C. On R+9

Complete blood counts

Bone marrow clonal studies

Erythropoietin

The significant differences were found in the clonal studies. On R+0,
the flight animals had significantly fewer BFU-E than the controls at
all doses of added Epo. The CFU-E values were also lower for the flight
animals at dose levels of 0.25 and 0.50 U Epo/ml. By R+9, there were no
significant differences. These studies indicate that the earliest
erythroid

progenitor cells were probably decreased in number due to exposure to
flight and would be compatible with studies showing a decrease in red
blood cell mass in astronauts and rats exposed to microgravity. The bone
marrow cells, however, are able to respond to added erythropoietin and
by R+9 have recovered in numbers.

At DFPT, inconsistent variations were found. While some differences were
observed by severe statistical analysis, it is doubtful that they were
of physiological significance and no consistent pattern seemed to be
present.

It should be noted that there were some differences in caging at DFPT in
comparison to the flight conditions. Further, the control animals were
not flown to Dryden as were the flight controls.

Another caveat should be made. Due to the sheer number of tests
performed, some statistical differences may appear without their being
directly related to a difference due to exposure to microgravity.

Although no differences were found due to the manner of caging, the
erythroid parameters change quite definitely with the growth of the
animals.

Another difficulty arises when we attempt to compare the SLS-1 results
with previous flights due to the different strains of rats used, the age
of the animals, and the length of the flight.

**Chapter 2**

**Effects of Space Flight on Rat Spleen Weight and Histology**

**[INTRODUCTION]{.underline}**

Previous studies of lymphatic tissues in space flight experiments with
rats have shown changes. The Cosmos 605 space flight of 22 days
demonstrated significant atrophy of spleen, thymus, and inguinal lymph
nodes examined two days after the flight landed. At 27 days postflight,
recovery had occurred, indicating reversible atrophy of lymphatic
tissues (1). Leukopenia and lymphopenia were also reported with recovery
to normal values in 60 and 30 days, respectively (2). These changes in
lymphatic tissues were attributed to environmental stress associated
with the longer flight. On the shorter, 7-day flight with SL3 there was
a mild lymphopenia and spleen lymphocyte depletion (3).

The white pulp of the rat spleen is a typical example of lymphatic
tissue. The red pulp contains some blood-forming elements like those in
the bone marrow in these young animals. The purpose in examining the
spleen (weight, histology and cytology) during the SLS-1 experiment was
to look for effects of microgravity and other aspects of the flight
experiment on this organ.

**[MATERIALS AND METHODS]{.underline}**

(See Appendix A.)

**[RESULTS]{.underline}**

The spleen to body weight ratio and hematoxylin and eosin (H&E)
histology of a cross-section of the rat spleen showed no obvious
differences for flight and control groups at either R+0 or R+9 (Table 1,
Appendix B, chapter 2). An increase in body weight between R+0 and R+9
in flight animals was less than in the controls and was attributed to
reduced food

intake in flight animals compared with normal food intake in the
ground-based controls that did not need to adjust to the landing and
return to normal gravitational influence. In these same animals, brush
preparations of spleen were made to evaluate cell differentials and no
differences in flight and control groups were observed (Table 2,
Appendix B, chapter 2).

Similarly, the DFPT studies on the same variables did not provide
significant differences based on group or single animal caging (Tables 3
and 4, Appendix B, chapter 2).

**[DISCUSSION]{.underline}**

While this flight with nine days of microgravity only caused a temporary
reduction in food intake in the flight animals on landing and no
discernible effect on spleen weight, histology or cell differentials,
longer and shorter flights and duration of microgravity have produced
significant changes, as shown in a review of the literature. Serova (4)
discusses the relation of body weight changes to lymphatic tissue and
comments on the post-flight functional hypokinesia or \"physiological
reduction of motor activity\" in Cosmos biology experiments with rats.
This behavior was present in the SLS-1 rats for about 8 hours
post-flight.

The most obvious basis and explanation for lymphatic tissue changes,
where they have been noted, is possible stress of the experimental rats
during the total preflight, flight, and postflight experiences.
In-flight sampling of lymphatic tissues has the potential for separating
out the portion (i.e., launch and flight vs. the total experiment) of
the experience that contributes most to the stress reaction.

Even in SLS-1, the peripheral blood circulating lymphocytes showed some
depletion when measured after landing (see Chapter 3). Probably the
thymus, traditionally the most sensitive organ for measuring stress in

young animals, should be examined in microgravity studies. The classical
work of Selye and his general adaptation syndrome is quite relevant to
flight personnel and animals in the context of microgravity experiences.
Selye recognized adrenal hypertrophy, lymphatic tissue atrophy and
gastrointestinal ulcers as being the key events in chronic stress. These
were central features in an adaptation syndrome (5,6).

**[SUMMARY]{.underline}**:

Although there was some peripheral blood lymphocyte depression after
landing in SLS-1 flight animals, when compared to controls, this was not
detectable in the study of lymphatic tissue in the spleen. Other studies
of lymphatic tissues in space flight experiments have shown significant
atrophy of several lymphatic tissues. Fairly prompt recovery after
landing has been reported.

The reported lymphatic tissue depression is most likely related to
stress in the total experimental situation and future in-flight studies
may show which portion of the experiment produces a stress reaction. All
organ systems involved in the classical Selye stress adaptation syndrome
could be monitored for relevance to humans and animals.

**Chapter 3**

**Effects of Space Flight on Rat Peripheral Blood Leukocytes**

**[INTRODUCTION]{.underline}**

Several experiments on orbiting spacecraft during the past decade have
shown significant changes in both the number and functional capacity of
rat peripheral blood leukocytes. The basic mechanism for these changes
during spaceflight has yet to be resolved. In rats flown on Cosmos 936
and 1129 flights, a leukocytosis with lymphopenia and neutrophilia
occurred during the acute postflight period with values returning to
control levels by 3 days (1-3). Ilyin *et al.* found a leukocytosis and
eosinophilia in rats flown on Cosmos 605 (4). Rats flown on SL3
demonstrated no significant changes in the white blood cell counts but
showed a mild lymphopenia and neutrophilia (5).

It is difficult to compare the results from the different flights since
the duration of the flights differed, as did the strain of rats and the
time the animals were sampled postflight. Studies conducted on the first
41 U.S. space shuttle astronauts demonstrated that the absolute number
of lymphocytes and eosinophils in the peripheral circulation and
lymphocyte blastogenesis were decreased after flight (6,7). Conversely,
there was an almost universal doubling of the absolute neutrophil number
and, often, an increase in the helper cluster destination (CD)
T-lymphocyte population, as determined by flow cytometric analysis. Data
from space shuttle flights 41B and 41D involving 11 crew members
indicated a postflight decrease in circulating monocytes and
B-lymphocytes (7). Prolonged spaceflight resulted in hypoplasia of
lymphoid organs and alterations in mitogen-induced blastogenesis (7).
This chapter includes the study of total and

absolute leukocyte counts in both flight and control rats prior to and
after exposure to microgravity.

**[MATERIALS AND METHODS]{.underline}**

(See Appendix A)

**[RESULTS]{.underline}**

[Flight Animals]{.underline}:

[R+0 Animals]{.underline}: The mean and standard error of the mean of
total and absolute counts of peripheral blood leukocytes of control and
flight animals are presented in Table 1. At preflight, there were no
statistically significant differences in any of the parameters between
the control and flight animals. At landing, there was a significant
decrease in the number of total leukocytes and absolute counts of
lymphocytes, monocytes and eosinophils of flight animals compared with
controls. A slight decrease in the absolute neutrophil count of the
flight animals compared with the controls was also observed.

[R+9 Animals]{.underline}: On R+1 (1 day post landing) there was a
doubling of the absolute neutrophil, monocyte and eosinophil numbers
accompanied by a slight decrease in the total leukocyte and absolute
lymphocyte counts of flight animals compared with the controls (Table
1). The platelet counts of flight animals were slightly higher than the
controls. The values of all the parameters of flight animals returned to
control levels by 9 days post landing.

[DFPT Animals]{.underline}:

[R+0 Animals]{.underline}; The mean and standard error of the mean of
total and absolute peripheral blood leukocyte counts are presented in
Table 2. At L-3, the total white blood cell (WBC) counts of both AEM and
RAHF flight animals were slightly to markedly higher compared with AEM
and RAHF

control animals, respectively. The absolute lymphocyte count of RAHF
flight animals were markedly higher than the RAHF controls. At R+0,
there was a marked increase in the total WBC count of RAHF flight
animals compared with controls. There was a moderate increase in the
number of absolute lymphocytes and monocytes of RAHF flight animals,
compared with RAHF controls. Conversely, there was a marked increase in
the absolute counts of both neutrophils and monocytes of RAHF flight
animals compared with AEM flight animals.

[R+9 Animals]{.underline}: At R+0, the white blood cell count (WBC) and
absolute lymphocyte counts were markedly lower in RAHF flight animals,
compared with controls as shown in table 3. Conversely, a moderate
increase was observed in the number of neutrophils, monocytes, and
eosinophils of AEM flight animals, compared with controls. No
significant changes in the values of any of the parameters were observed
at days 1,3,4,8, and 9 post flight. No significant changes were observed
in the platelet counts of DFPT animals.

**[DISCUSSION]{.underline}**

It was previously observed that, in rats exposed to microgravity, a
leukocytosis with lymphopenia and neutrophilia occurred during the acute
postflight period with values returning to preflight levels by 3 days
(1-3). Several factors could contribute to these effects, including
microgravity, stress, radiation, and interaction with other host
biological systems. Also, it is difficult to compare the results from
the different flights, since the duration of the flights differed as did
the strain of rats and the time the animals were sampled postflight. In
the present studies, there were statistically significant decreases in
total white blood cell counts, and the absolute number of lymphocytes,
monocytes and eosinophils, and a slight

decrease in the neutrophil count at the time of landing. Twenty-four
hours postflight, the neutrophil, monocyte and eosinophil counts of
flight animals were higher than that of the control animals. The
lymphocyte count returned to normal physiological levels but was still
lower than that of the controls. The changes in the absolute number of
lymphocytes, monocytes, and eosinophils in the current study indicated a
similarity with the results reported by Taylor *et al*. (7). We also
found a slight decrease in neutrophil counts rather than an increase, as
indicated by some studies (5).

The results of peripheral blood leukocytes of DFPT animals indicated
that they are a heterogeneous population. This heterogeneity is clearly
demonstrated in the preflight period, which showed marked difference
between control and flight animals in various peripheral blood leukocyte
parameters, both among and within the groups, as presented in Tables 2
and 3. These changes and the variation in the growth rate that were
shown by these animals made it difficult to study the effect of housing
on these animals. Since the number of rats in the DFPT groups (N=5) was
much smaller compared to the actual flight number (N=19, N-20), more
variability was observed between DFPT groups. The flight animals flown
aboard the space shuttle Columbia during their preflight period showed
no differences between or among the groups in any of the studied
parameters. On the day of landing, these flight animals showed a
significant decrease in total white blood cell counts and also in the
absolute count of lymphocytes, monocytes, and eosinophils, compared with
control animals. These leukocyte values returned to the level of control
animals on day 9 postflight. This clearly demonstrated the effect of
microgravity and/or other factor(s) (i.e. stress, radiation, etc.) on
the blood values of the flight animals compared with DFPT animals.

The mechanism responsible for changes in peripheral blood leukocytes is
not well understood. The number and functional activities of these cells
are regulated by a complex network of cytokines and endocrine factors.
Thus, spaceflight could affect the generation of these cells by
affecting these regulatory mechanisms. Other studies also reported a
decrease in the function of both human lymphocyte subsets and monocytes
(7). Here again, it is possible that stress induced by spaceflight could
change hormone levels, which could alter cytokine production by
lymphocytes, monocytes, and other cytokine-generating cells. In
addition, there could be an increased production of a soluble inhibitor
which acts as negative regulators on the function and the production of
these cells. These negative regulators, such as tumor necrosis factor α
(TNF-α) and transforming growth factor β1 (TGF-β1), either have a direct
effect on the response of these cells to other cytokines or affect the
number of receptors on their surfaces. Also, it is well established that
these negative regulators have either a direct or indirect effect on
erythropoiesis, which leads to the inhibition of maturation of erythroid
progenitor cells (9,10).

Our results indicated that the platelet counts of flight animals didn\'t
differ from the ground controls either preflight or postflight. This is
comparable to results reported in human studies (11).

**[SUMMARY]{.underline}**

The effects of microgravity on peripheral blood leukocytes are as
follows: On the day of landing (R+0), there was a significant decrease
in the total white blood cell counts (p\<0.0001) of flight animals in
comparison to control animals. There was also a significant decrease in
the absolute number of lymphocytes (p\<0.0001), monocytes (p\<0.0001),
and eosinophils (p\<0.0001) in the flight animals. A marked decrease in
the number of

neutrophils of flight animals was also observed. By day 3 post flight,
the total and absolute leukocyte counts returned to control levels.
These changes in peripheral blood leukocytes may be due to a stress
reaction or physiological readaptation to microgravity.

To better understand the various factors affecting the leukocyte
parameters of flight animals, inflight testing during the upcoming Space
Life Sciences-2 (SLS-2) mission will be critical. We hypothesize that
various factors are contributing to the changes in erythropoiesis of
flight animals, either directly or indirectly. As the levels of serum
Epo, Interleukin-3 (IL-3), and Interleukin-9 (IL-9), and the negative
regulators possibly change due to microgravity, erythropoiesis will be
directly affected. An indirect effect on erythropoiesis, such as stress,
is very difficult to quantify. However, measuring serum corticosteroids
and examining the weight and histology of the thymus aids in determining
if the animals have been stressed.

**Chapter 4**

**Effects of Microgravity on Rat Spleen and Peripheral**

**Blood Lymphocytes**

**[INTRODUCTION]{.underline}**

There have been studies indicating that humans and laboratory animals
exposed to the microgravity environment of space flight become
immunodeficient (9,10). Immune cells, especially CD4 T-lymphocytes, have
a major role in the regulation of hematopoiesis by interleukins and
cytokines, which are elaborated when these cells are activated.
Specifically, activated T-cells produce IL-3 and IL-9, together with
Epo, promote the differentiation of progenitor hematopoietic cells along
the erythrocytic lineage (3). In this study, we assessed the
immunocompetence of the flight rats by immunophenotyping the peripheral
blood and spleen lymphocytes to ascertain the immunodeficiency of the
flight animals. The lymphocyte subsets were enumerated with monoclonal
antibodies directed against T helper (CD4), T suppressor (CD8), and
major histocompatibility complex (MHC) class II for B cells.

**[MATERIALS AND METHODS]{.underline}**

(See Appendix A.)

**[RESULTS]{.underline}**

[Flight]{.underline}: The absolute number of lymphocytes (see Table 1)
was compared for the flight and control animals in the groups tested on
days 3 or 4 before launch (L-3/4), on recovery (R+0), and on number of
mission days following recovery (R+9). A difference in the absolute
lymphocyte counts was discerned in the rats in the flight group on R+0
when compared

to the control group. Otherwise, there were no differences between the
two animal groups.

The percentage and absolute number of the CD4, CD8, and B cells are also
shown in Table 1. There were no differences detected between the animal
groups prior to flight on L-3/4). There were significant decreases in
the numbers of CD4, CD8, and B cells in the flight animals on the day of
recovery, R+0, and even a decrease in the percent of B cells. The
increased percentage of CD4 and CD8 cells in the flight animals on R+9
did not have a corresponding increase in number of cells.

[DFPT]{.underline}: No differences in the percent of CD4, CD8, or B
cells were noted in the rats included in the DFPT studies (see Tables 2
and 3). However, some differences in the absolute counts were noted in
the RAHF housed rats only in the groups where the lymphocyte counts were
different.

[Spleen Cell Immunophenotyping]{.underline}: The spleen cells from the
flight and control animals were compared for percent reactivity on R+0
and R+9, where no differences were discerned. In the DFPT study, there
was an increased level of B cells in the flight animal group.

**[DISCUSSION]{.underline}**

Rats flown on SLS-1 returned to earth in a transient state of
immunodeficiency because of a lowered lymphocyte count. The animals were
all bled within 8 hours of the landing of the shuttle. The percent CD4,
CD8 and B cells from the flight animals was not different from the
control animals; whereas, the number of lymphocytes in each subset was
significantly depressed. Inasmuch as an increase in the percent CD4 and
CD8 cells was noted on R+9 in the flight animals, there was no
corresponding increase in the absolute number of lymphocytes in these
subsets. We speculate that there was an overproduction of these two T

lymphocyte subsets to compensate for the lower numbers found on R+0. We
have noted the overproduction of CD4 and CD8 lymphocytes in several
kidney transplant patients made immunodeficient with the monoclonal
antibody OKT3 or antithymocyte serum therapy following the completion of
their treatment (Ichiki and Goldman, unpublished results). Further, we
believe that the immunophenotyping values obtained from peripheral blood
lymphocytes can better reflect transient immunodeficient states because
it is possible to get the total white blood count and the percent
lymphocytes, which cannot be obtained for bone marrow aspirates or
spleen cells (8,9).

Some differences in the absolute number of CD4, CD8 or B cells were
observed between the RAHF housed animals in groups where there were
increased lymphocyte counts between the control and flight groups. It is
possible that only differences discerned were found in the RAHF housed
groups because of the larger number of animals (N=10) as compared to the
AEM housed group (N=5). If housing affected these results, there would
have been a consistent difference noted, which was not. Hence, the
differences noted in the DFPT study only reflect the heterogeneity of
the rats included in the study, which had been previously noted.

The depression of the number of lymphocytes evidently is due to factors
such as microgravity, other physical stresses, physiological stresses,
and other yet to be determined factors, which can impact the number of
lymphocytes. The depressed white blood cell level on R+0 was in the
normal range the next day, R+1 (see Chapter 3); hence, this transient
state of low lymphocytes was very short lived. Blood specimens will be
obtained from the rats on SLS-2 so we can compare the results from
in-flight specimens to R+0 and R+9 samples. If there are no statistical

differences between the results from the animals bled in space and the
animals bled upon landing, we will be more confident in attributing the
immunodeficient state to microgravity and not to the return to 1 g.

It was observed that cultures of human lymphocytes were not activated by
the T cell mitogen, concanavalin A (Con A), in the cultures flown aboard
Spacelab 1, as compared to the ground controls (2). This observation, in
light of contemporary immunology, would suggest that the CD4
helper/inducer T-lymphocytes lacked the capacity to bind and endocytose
the Con A, which would result in many events leading to the production
of interleukin 2 (IL-2) and high affinity IL-2 receptors, and the
subsequent production of many interleukins, cytokines, and growth
factors. Consequently, there have been studies to determine whether the
level of these immunological factors are affected by space flight in
humans or laboratory animals (4,5,7), as well as cultured murine and
human immune cells (1). Inasmuch as activated CD4 cells produce factors
associated with immunological differentiation and proliferation, these
cells also produce interleukins, cytokines and growth factors which are
required for the differentiation and proliferation of other
hematopoietic cells. Hence, immunodeficiency resulting from microgravity
would impact hematopoiesis and, in our study, on erythropoiesis.

IL-9 and IL-3 were found to be abundant products of activated T cells
and were suggested to be the most likely major factors contributing to
the erythroid burst-promoting activity (BPA) (3). IL-11, which was
originally isolated from a primate bone marrow stromal cell line, and
the ligand for the c-kit encoded factor (stem cell factor or c-kit
ligand) stimulate highly proliferative erythroid progenitors (7).

We consider the following a working model to study the role of
interleukins, growth factors, and Epo for erythropoiesis:

Stem Cell Factor

IL-3

IL-9 IL-9

Hematopoietic Epo Epo

Progenitor - -\>- -\>- -\>- -\>- -\>- -\>BFU-E - -\>- -\>- -\>-
-\>Erythrocytes

Cells CFU-E

It is our intention to assay for IL-3, IL-9, and stem cell factor, as
well as for Epo, in the serum of the rats flown aboard SLS-2. By
assaying for these factors in serum obtained from the rats in-flight and
comparing the results post-flight on R+0 as the immunophenotyping
studies, it could be possible to demonstrate that the immunodeficiency
caused by microgravity impacts erythropoiesis.

**[SUMMARY]{.underline}**

Rats flown on SLS-1 returned to earth in an immunodeficient state
because of their depressed level of peripheral blood lymphocytes.
Although the percentage of CD4 helper/inducer T lymphocytes, CD8
suppressor/cytotoxic T lymphocytes, and B cells was not found to be
depressed on R+0, the numbers of each of these T cell subsets and B
cells were significantly lower. This state of immunodeficiency was
observed to be transient because it was not observed on R+8.

**Chapter 5**

**Summary, Conclusions and Recommendations for Future Studies**

**[SUMMARY]{.underline}**

The objective of these experiments was to aid in answering the question:
What mechanism(s) is responsible for the anemia experienced by the
astronauts? It is also to be determined if the rat is an appropriate
animal model to study these mechanisms. The E012 experiment was carried
out to make measurements of the various factors affecting erythropoiesis
in a group of rats and their appropriate controls.

The results of these hematological studies indicated that on R+0 there
was a significant decrease in the number of Epo-responsive erythroid
progenitor cells as enumerated by the BFU-E progenitor cells. Also, the
peripheral blood showed a significant decrease in the total WBC and in
the absolute number of lymphocytes, monocytes, and eosinophils.
Immunophenotyping studies of peripheral blood lymphocytes indicated a
significant decrease in the absolute number of B-cells, T-helper cells,
and T-suppressor cells. All values returned to the control levels by
R+9. No significant differences between flight and control animals were
observed in the red blood cells parameters (RBC, Hgb, Hct), serum
erythropoietin level, and reticulocyte counts.

**[CONCLUSIONS]{.underline}**

The exact mechanism(s) that lead to these observed changes during this
flight is not completely defined. While the primary cause might be the
influence of microgravity, the etiology is probably multifactorial. This
might include altered hemodynamics, changes in oxygen demand or oxygen
carrying capacity, and metabolic disturbances. In vertebrates, including
man and rat, the overall control of red cell production is

controlled by a complex network of hormones and cytokines which affect
red blood cell production.

Changes we observed in the bone marrow\'s responsiveness to
erythropoietin indicated that differentiation was either slowed or
altered in some manner. With progenitor cells always present in the bone
marrow, some mechanism(s) is preventing those cells from fully
differentiating into mature red blood cells. It was previously thought
that Epo was the major determinant in erythropoiesis. However, as
technology has developed, new cytokines are being identified as having
an important part in the regulation of erythropoiesis, either positively
or negatively. In addition to Epo, those that react positively include
IL-3, IL-9, IL-11, and stem cell factor. Negative regulatory factors
include TNF-α and TGF-β. Since we did not observe changes in the Epo
levels post flight, it might be possible that these new cytokines may be
involved, whether by increasing, suppressing, or altering the process of
erythropoiesis in some form.

As the lymphocyte population decreases, the production of several
cytokines could decrease, which could contribute to the reduction in red
blood cell production. As new information becomes available, the
enumeration of T-cell subsets, which was the initial step of our
determining the role of lymphocytes in the complex network of
hematopoiesis, may greatly contribute to the understanding of
lymphocytes and erythrocytes.

In examining previously published data by other investigating teams, it
is extremely difficult to make direct comparisons with their results and
those presented in this report. The reasons for this are differences in
length of flights, strains of rats, different post flight animal
receiving times, housing differences (food and water included), and the

general overall handling of the animals. However, it is not to say
previously reported data is not useful. It provides a baseline and is
used as a guide to the results other investigators are acquiring. With
SLS-2 forthcoming and the basic scenarios being comparable to those of
SLS-1, in addition to inflight experimentation, this data will be
corroborated.

**[RECOMMENDATIONS FOR FUTURE STUDIES]{.underline}**

1\. Expanded inflight tissue procurement

2\. Inflight Epo IP injections (E141)

3\. Measure serum regulatory factors Epo, IL-3, IL-9, IL-11 and stem
cell factor which have a direct and indirect effect on erythropoiesis.

4\. Measure negative regulators (TNF-α and TGF-β) that suppress
erythropoiesis.

5\. Continue to gather basic hematological parameters

a\. Erythroid (RBC, Hct, Hgb, MCV, MCH, MCHC)

b\. Reticulocytes

c\. RBCM, plasma volume

d\. Bone marrow differentials and cell cultures

e\. Peripheral blood differentials

f\. Peripheral blood lymphocyte subset enumeration

6\. Examine thymus (weight, cellularity) and serum corticosteroids to
determine stress levels

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**[Introduction]{.underline}**

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**[Chapter 1]{.underline}**

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36\. Leach CS, Johnson PC, Cintron NM. The endocrine system in space
flight. Acta Astronautica 1988; 17: 161-6.

37\. Legen\'kov VI, Kiselev RK, Gudim VI, Moskaleva GP: Peripheral blood
changes in the crew members of the Salyut-4 orbital space station. Kosm
Biol Aviakosm Med 1977; 11: 3-12.

38\. Vacek A, Rotkovska D, Serova LV, Michurina TV, Bartonickova A. the
effects of space flight factors on hemopoietic stem cells of rats.
Symposium Current Problems of Cosmic Biology and Medicine 1989: 271-6.

**[Chapter 2]{.underline}**

1\. Durnova GN, Kaplansky AS, Portugalov VV. Effect of a 22-day space
flight on the lymphoid organs of rats. Aviat Space Environ Med 1976; 47:
588-91.

2\. Rakova IA, Shvets VN. Morphologic study of hemopoietic organs of
rats during hypokinesia. Kosm Biol Aviakosm Med 1978; 4: 64-8.
(Russian), Space Biol Aerospace Med 1978; 12: 85-90. (English
translation of above).

3\. Lange RD, Andrews RB, Gibson LA, Congdon CC, Wright P, Dunn CDR,
Jones JB. Hematological measurements in rats flown on spacelab shuttle
SL-3. Am J Physiol 1987; 252: R216-21.

4\. Serova LV. Weightlessness effects on resistance and reactivity of
animals. Physiologist 1980; 23(Suppl 6): S22-6.

5\. Anthony CP, Kolthoff NJ. Textbook of anatomy and physiology. St.
Louis: C.V. Mosby, 1975: 534-44.

6\. Selye H. Stress without distress. New York: Signet, 1975.

**[Chapter 3]{.underline}**

1\. Gazenko OG, Genin AM, Ilyin EA, Oganov VS, Serova LV. Adaptation to
weightlessness and its physiological mechanisms (results of animal
experiments aboard biosatellites). Physiologist 1980; 23(Suppl 6):
S11-5.

2\. Kozinets GI, Korol\'kov VI, Britvan II, Bykova IA, Spitsyna NE, et
al. Morphofunctional properties of the peripheral blood and bone marrow
cells of rats following a flight on board the Kosmos-936 biosatellite.
Kosm Biol Aviakosm Med 1983; 17: 61-5.

3\. Shvets VN, Vacek A, Kozinets GI, Britvan II, Korol\'kov VI.
Hemopoietic status of rats exposed to weightlessness. Kosm Biol Aviakosm
Med 1984; 18: 12-6.

4\. Ilyin EA, Serova LV, Portugalov VV, et al. Preliminary results of
examinations of rats after a 22-day flight aboard the Cosmos-605
biosatellite. Aviat Space Environ Med 1975; 45: 319-21.

5\. Lange RD, Andrews RB, Gibson LA, Congdon CC, Wright P, Dunn CD,
Jones JB. Hematological measurements in rats flown on Spacelab shuttle.
Am J Physiol 1987; 252: R216-21.

6\. Taylor GR, Dardano JR: Human cellular immune responsiveness
following spaceflight. Aviat Space Environ Med 1983; 54: S55-9.

7\. Taylor GR, Neale LS, Dardano JR. Immunological analyses of U.S.
Space Shuttle crew members. Aviat Space Environ Med 1986; 57: 213-7.

8\. Durnova GN, Kaplansky AS, Portugalov VV. Effect of a 22-day space
flight on the lymphoid organs of rats. Aviat Space Environ Med 1976; 47:
588-91.

9\. Khan KNM, Kociba GJ, Wellman ML, Reiter JA. Effects of tumor
necrosis factor-alpha on normal feline hematipoietic progenitor cells.
Exp Hematol 1992; 20: 900-3.

10\. Miller KL, Carlino JA, Ogawa Y, Avis PD, Carroll KG. Alterations in
erythropoiesis in TGF-B1-treated mice. Exp Hematol 1992; 20: 951-6.

11\. Legen\'kov VI, Kiselev RK, Gudim VI, Moskaleva GP: Peripheral blood
changes in the crew members of the Salyut-4 orbital space station. Kosm
Biol Aviakosm Med 1977; 11: 3-12.

**[Chapter 4]{.underline}**

1\. Chapes SK, Morrison DR, Guikema JA, Lewis ML, Spooner BS. Cytokine
secretion by immune cells in space. J Leukoc Biol 1992; 52: 104-10.

2\. Cogoli A, Tschopp A, Fuchs-Bislin P. Cell sensitivity to gravity.
Science 1984; 225: 228-30.

3\. Donahue RE, Yang YC, Clark SC. Human P40 T-cell growth factor
(interleukin-9) supports erythroid colony formation. Blood 1990; 75:
2271-5.

4\. Gould CL, Lyte M, Williams J, Mandel AD, Sonnenfeld G. Inhibited
interferon-g but normal interleukin-3 production from rats flown on the
space shuttle. Aviat Space Environ Med 1987; 58: 983-6.

5\. Konstantinova IV, Sonnenfeld G, Lesnyak AT, ***et al***. Cellular
immunity and lymphokine production during spaceflights. Physiologist
1991; 34(1 Suppl): S52-6.

6\. Quesniaux VF, Clark SC, Turner K, Fagg B. Interleukin-11 stimulates
multiple phases of erythropoiesis in vitro. Blood 1992; 89: 1218-23.

7\. Sonnenfeld G, Mandel AD, Konstantinova IV, et al. Effect of space
flight on immune responses: Bone marrow cell response to colony
stimulating factor and leukocyte subsets. Physiologist 1992; 35(1
Suppl): S222-3.

8\. Sonnenfeld G, Mandel AD, Konstantinova IV, et al. Effects of
spaceflight on levels and activity of immune cells. Aviat Space Environ
Med 61:648-653, 1990.

9\. Sonnenfeld G, Mandel AD, Konstantinova IV, et al. Spaceflight alters
immune cell function and distribution. J Appl Physiol 1992; 73(2 Suppl):
191S-195S.

10\. Sonnenfeld G, Taylor GR. Effects of microgravity on the immune
system. SAE Technical Paper Series, #911515, 1991.

**APPENDIX A**

1\. Ogawa M: Methylcellulose clonal cell culture of murine and human
erythropoiesis. In: Murphy MJ, Peschle C, Gordon AS, Mirand EA, eds. In
vitro aspects of erythropoiesis. New York: Springer-Verlag, 1978: 248.

2\. Burke WT, Brotherston G, Harris C. Improved technique for obtaining
bone marrow smears from the rat. Am J Clin Pathol 1955; 25: 1226-8.

3\. Harris C. The lymphocyte-like cell in the marrow of rats. Blood
1961; 18: 691-701.

**ACKNOWLEDGMENTS**

The authors wish to thank the following for their assistance and
encouragement for which our experiments could not have been completed:

STS-40 Flight Crew

Ames Research Center Personnel

Kennedy Space Center Hangar L Personnel

Dryden Payload Receiving Facility Personnel

Nina Mushell - Experiment Support Scientist for E012

University of Tennessee Flow Cytometry Facility - Joseph E. Fuhr, Ph.D.,
Director

Hematology Laboratory, Department of Pathology, University of Tennessee
Medical Center at Knoxville, Frances Jones, M.D., Chairman

Erika Amaba and Lisa Payne for their technical assistance

Paul Wright, M.S., for all statistical work

The original E012 experiment was designed by:

P.C. Johnson, M.D.\*

C.D.R. Dunn, Ph.D.

Their many contributions are acknowledged.

\*Deceased

**APPENDIX A**

**MATERIALS AND METHODS**

[Experimental Design and Selection Process]{.underline}:

[Animals]{.underline}: 163 Sprague-Dawley rats were obtained from the
Taconic Farms (Indianapolis, IN). They were brought into the Kennedy
Space Center at approximately five weeks of age. At that time, they were
singly housed with their food and water given *ad* *libitum*. The food
and water consumption was monitored and recorded. Body weights were
taken at regular intervals in order to establish the normal growth
pattern and any of those that did not show a consistent pattern were not
chosen either for flight or as ground controls. At L-4, the rats were
randomly divided into group or individually housed animals. The first
injections and phlebotomies were performed on all of the animals on L-4
(R+9 animals) and L-3 (R+0 animals). Subsequent 24-hour blood samples
were taken on L-3 (R+9 animals) and L-2 (R+0 animals). After the last
blood sample was taken on L-2, the final flight selection was made. The
only criteria available to the #E012 investigators in order to make this
decision were the following:

Food and water consumption

Body weight

Complete blood cell count - focusing mainly on the red blood count and
white blood count

Hematocrit

The flight selection was given to Ames personnel for random placement
into the RAHF and AEM modules. Contingency plans were utilized to repeat
this procedure. Thus, a second group of animals was prepared, selected,
and placed into the modules.

Prior to launch, a malfunction of one of the RAHF water lixits prevented
one of the RAHF rats from flying. This brought the number of RAHF
animals flown to 19 from the original 20. The number of animals and
their designated housing plan is seen in Table 1. There were 10 AEM
flight animals and 19 RAHF animals flown aboard Columbia. On the ground
were matched 10 AEM controls and 20 RAHF controls.

After the launch of Columbia, all supplies, equipment, and ground
control animals were flown to the DPRF. This took place two days post
launch. The AEM ground control animals were in a nonflight AEM while
being transported to the Dryden facility. The RAHF animals were removed
from their individual vivarium cages and placed in groups in regulation
transportation boxes. Once at Dryden, the RAHF animals were then placed
back into individual vivarium cages. The light and dark cycles for both
the AEM and RAHF were kept the same as those flying on Columbia.

Figure 1 portrays the experimental plan. On R+0 or day of landing, five
of the AEM control animals had their final 3 ml blood sample taken and
were then killed to obtain other tissues. Upon arrival at the payload
facility, five of the AEM flight and 10 of the RAHF flight animals
followed the same protocol, followed by ten RAHF ground controls. The
remainder of the animals, both flight and controls, were held 9 days
post landing and then proceeded to follow the same protocol as on R+0.

[Delayed Flight Post Test (DFPT)]{.underline}:

The DFPT was performed at KSC. This test was performed to assure that
results seen on SLS-1 were really the effect of microgravity and not
housing, noise, etc. Environmental factors recorded aboard Columbia were
simulated on the ground as much as was feasible. This included the
light/dark cycle, temperature, humidity, noise, and housing. A nonflight

RAHF and AEM were used for the simulation. Timing of all procedures were
flight simulated. The only simulation not included was the
transportation of the ground controls from KSC to Dryden 2 days post
launch.

**[HEMATOLOGICAL TECHNIQUES UTILIZED]{.underline}:**

Determination of hematological parameters were performed during the
preflight period, on R+0, and during the postflight period (Table 2).
The procedures were repeated during the DFPT. The total number of
determinations on specimens collected for subsequent analysis are also
presented in Table 2. Table 3 summarizes the methods used.

[Blood Cell Counts]{.underline}: Automated blood cell counts were
performed using the TOA Sysmex F-800TM microcell counter. These
measurements were made on-site shortly after the acquisition of the
blood samples. Standard blood controls were analyzed prior to any test
samples to ensure the performance of the machines. Standard procedures
were used on 20 μl of blood, with the exception of the lysing solution
used on the white blood cell count; three drops are standard, but seven
drops were required in order to lyse all of the RBC\'s. All samples were
analyzed twice and the average computed. Any obvious anomalies were
discarded and the samples re-analyzed. The counts included: white blood
cell count, platelet count, red blood cell count, hematocrit,
hemoglobin, and red blood cell indices mean cell volume, mean cell
hemoglobin, and mean cell hemoglobin concentration.

[Reticulocyte Counts]{.underline}:

Two methods were utilized in determining reticulocyte counts:

A. Flow Cytometry: Blood was collected via the rat tail vein as part of
the regular blood sampling. Ten microliters were removed and

placed into a prelabeled, heparinized microtube and held on ice until
ready for shipment to Knoxville. The samples were shipped under
refrigeration and, when received in the Knoxville laboratory, brought to
room temperature. Each sample was thoroughly mixed. The blood sample, 5
μl, was pipetted into 1 ml of thiazole orange stain, mixed, and
immediately placed in the dark at room temperature for a minimum of 30
minutes. The samples were remixed and placed on the Becton-Dickinson
FACScan flow cytometer. A software program, ReticCountTM by Becton
Dickinson, San Jose, CA, analyzed the results of 10,000 events. The
percentage and number of reticulocytes was then calculated.

B. Manual Reticulocyte Counts: The reticulocyte slides were prepared at
the dissection site shortly after blood collection. Ten microliters of
blood were added to 10 μl of New Methylene Blue-N. This mixture was
allowed to stain for a minimum of 3 minutes, after which standard smears
were prepared. The slides were returned to Knoxville, where they were
counter-stained using a Dif-QuikTM staining procedure. A total of 2,000
red blood cells were counted by 2 different technicians. The number of
reticulocytes were noted and percentages recorded. The average between
the two values was recorded.

[Erythroid Colony Assay]{.underline}: The cultures were carried out
according to the method described by Ogawa (1978), with some
modifications. One femur was removed from each of the flight and control
rats and was returned to the Knoxville laboratory under refrigeration,
where the bone marrow was flushed into a 15 ml conical tube. The cells
were washed 3 times using Iscove\'s Modified Dulbecco\'s Medium (IMDM)
(Whittaker, Walkersville, MD) and suspended in a final concentration of
4 x 106 cells/ml. Four 15 ml conical tubes containing the culture
mixture were

prepared for each rat. One milliliter of the culture mixture contained
1.3 x 105 total bone marrow cells suspended in 0.8% methylcellulose
(Terry Fox Laboratories, Vancouver British Columbia) in IMDM, 10% rat
spleen-conditioned medium, L-glutamine, 30% fetal calf serum, 1% bovine
serum albumin, 10-4 beta-mercaptoethanol, 10,000 units/ml penicillin,
10,000 μg/ml streptomycin, and either one of 3 concentrations of Epo
(0.25, 0.5, 1.0 U/ml) or none. One milliliter from each tube was
aliquoted into one Petri dish, repeated 6 times, and incubated at 37oC
in a humidified atmosphere containing 7.5% CO2. Any remaining cells were
immediately frozen using a standard 90% fetal calf serum and 10%
dimethyl sulfoxide (DMSO) mixture and placed under liquid nitrogen. On
day 3 after culturing, three plates per each Epo dose were stained with
2,7 Diaminofluorene (DAF) and the CFU-E were enumerated. The other three
plates per EPO dose were stained with DAF on day 7 and the BFU-E
colonies were enumerated. All cultures were read using a Nikon inverted
microscope.

[Bone Marrow Differential]{.underline}: The bone marrow smears were
prepared at the Knoxville laboratory from the head of the femur. The
\"paint brush\" technique was used to make bone marrow preparations for
differential counting (2,3). Bone marrow slides were fixed in absolute
methanol for 10 minutes and then stained for 10 minutes with Wright\'s
stain, after which tap water was added for 20 minutes to neutralize the
stain. After rinsing, the slides were stained for 10 minutes with
freshly prepared and filtered Giemsa stain. After rinsing and drying,
200 cell differential counts were performed. The bone marrow cells were
classified into the following cell types:

Small lymphocytes

Large lymphocytes

Plasma cells

Reticulum cells

Mature myeloid cells (metamyelocytes, bands, segmented)

Immature myeloid cells (myeloblasts, promyelocytes, myelocytes)

Eosinophils

Tissue Basophils

Mature erythroid cells (polychromatophilic and acidophilic normoblasts)

Immature erythroid cells (pronormoblasts and basophilic normoblasts)

The myeloid to erythroid ratio was calculated in order to determine
which cell lineages were suppressed

[Spleen Cell Differentials]{.underline}: Spleen slides were prepared at
the DS using the same \"paint brush\" technique as the bone marrow. The
slides were fixed and then transported back to Knoxville, where they
underwent staining on a Miles Hema-Tek 1000TM. Two hundred spleen cells
were classified as small lymphocytes, large lymphocytes, myeloid cells,
and erythroid cells.

[Peripheral Blood Differentials]{.underline}: Peripheral blood smears
were prepared at the DS using 10 μl whole blood plus 5 μl sterile
saline. This method was utilized as a precaution in case the rats became
dehydrated, causing the peripheral blood to increase in viscosity. The
smears were fixed at the DS, transported to the home lab, and stained on
the Miles Hema-Tek 1000TM. Three personnel read separately 100 cells
from each slide for a total of 300 cells and the results averaged.

[Peripheral Blood and Spleen Lymphocyte Subsets
Enumeration]{.underline}: A method for lymphocyte subsets, developed by
Becton-Dickinson and printed in their monoclonal antibodies package, was
modified to accommodate rat blood and spleen for the SLS-1 flight. Both
the blood and spleen cells were obtained at either KSC or Dryden and
then shipped back to the Knoxville laboratory. The blood was held at
room temperature and the spleen sections under refrigeration in
RPMI-1640 medium supplemented with calf serum, L-glutamine, and
betamercaptoethanol. The cells were then prepared by performing a series
of washings using media and followed by incubation with specific amounts
of the following monoclonal antibodies: (obtained from Bioproducts for
Science, Indianapolis, IN)

43F - Leukocyte common antigen

48P - T-suppressor/cytotoxic

55F - T cells (helper) and macrophages

340 - B-lymphocytes

341 - Monocytes and macrophages

149A - Granulocytes (spleen only)

Both spleen and blood cells from each rat had a control determined
simultaneously. The cells were fixed and enumerated on the
Becton-Dickinson FACScan flow cytometer using the Consort 30 software. A
total of 2,000 events were collected for each sample. All data were
stored on discs and each rat\'s sample data individually analyzed at a
later date.

[Serum Erythropoietin Level]{.underline}: Prior to decapitation, blood
(3 ml) was obtained from the tail vein using no anticoagulant. The blood
was allowed to clot and then centrifuged to obtain the serum samples.
The serum was immediately frozen to -70oC. The samples were then
transported to

Knoxville on dry ice and stored at -70oC. Epo was measured using a
commercial radioimmunoassay kit manufactured by Diagnostic Systems
Laboratories, Inc. (Houston, TX). The samples were counted and Epo
levels calculated using the Cobra 5005 gamma counter and software by
Packard (Downers Grove, IL).

[Spleen Histology]{.underline}: Spleen samples were obtained at
dissection. Weights were taken and blocks made. The histology sample
(approximately 1/12 of the spleen) was placed in 50 ml of 10% neutral
buffered formalin and kept at room temperature. The fixed samples were
sent to Knoxville for processing. Paraffin blocks were sectioned for
Hematoxylin and Eosin staining of slides. The slides were examined by
light microscope.

[Statistical Analysis]{.underline}: Statistical analyses were performed
by the biostatistician at the University of Tennessee Medical Center.
The flight data from the AEM flight and RAHF flight animals were
combined as were the AEM control and RAHF control data. However, for the
DFPT, all groups were kept separate, include the rats killed on R+0,
which were kept separate from those killed on R+9. The statistical test
utilized varied depending on the variability of the data. The following
tests were used throughout the data analysis:

Student t-test (equal variables)

Student t-test (unequal variables)

Wilcoxan (Z approximate)

Median (Z approximate)

The 012 experiment was carried out to make measurements of the various
erythropoiesis factors in a group of rats and their appropriate
controls. It was hoped to determine whether the rat was an appropriate
model for the hematological changes which occur in astronauts during
space flight.

1\. There were no significant differences between flight and control
animals for many parameters.

2\. Differences were found for the following parameters

a\. The BFU-E in the bone marrow of flight animals were decreased in
number an R+O

b\. the CFU-E of flight animals bone marrow were found to be decreased
in response to EPO of the EPO dose levels of 0.25 and 0.50/u/ml.

These differences had returned to normal by R+9.

c\. The white blood cell counts of flight animals were decreased.

d\. The absolute number of the following lymphocyte subsets in flight
animals were decreased: B cells, T-helper and T-suppressor cells.

e\. Flight animals decreased their food intake post flight.

f\. The flight of control animals from Florida to California affected
their food intake.

3\. The red blood cell parameters (RBC, Hgb, Hct) of the rats increased
as they grew.

4\. The DFPT results were variable and indicated that the rat population
differed from the flight animals and were perhaps heterogeneous.

5\. Reticulocyte counts by the manual method were decreased in
comparison to counts enumerated by flow cytometry.

6\. It was pointed out that due to the number of tests performed that
some differences could have been due to chance.

7\. The literature was reviewed and the results indicate that further
studies are needed.

**[CONCLUSIONS]{.underline}**

The result of the present studies are at variance with previous studies.
In part this may be due to the different lengths of exposure to
microgravity, in part to the different strains of animals used and also
due to the different sex of the animals flown. The isotope experiments
of E141 indicate that when rats are exposed to microgravity that there
is a reduction in RBCM. Since actual counts depend on the plasma volume
the isotope studies are needed to confirm that the rat may serve as a
model for erythropoiesis changes due to exposure to microgravity. The
variable results from previous studies indicate that more studies need
to be performed.

**[RECOMMENDATIONS FOR FUTURE STUDIES]{.underline}**

1\. A strategy for space biology and medical science for the 1980\'s and
1990\'s, the following recommendations were listed for the study of the
anemia of space flight.

a\. There is a need for more detailed studies of the time course and
magnitude of the anemia.

b\. Erythropoietin levels in the blood

c\. Red blood cell survival studies

d\. Bone marrow studies

e\. Clinical implications

2\. An ad hoc committee of the Federation of American Societies of
Experimental Biology has recommended that the following

parameters be measured in ground based and in flight studies (pre, in
and post flight) red cell count\*, hemoglobin\*, hematocrit\*, red cell
mass\*, blood volume\*, plasma volume\*, reticulocyte count\*,
erythropoietin\*, plasma or serum haptoglobin, platelets\*, red cell
shape\*, red cell size, blood P50, blood PC02, red cell 2,3 -DPG, red
cell ATP, red cell sodium, bone marrow smear\*.

In flight animals the presence of skin petechiae\* and subcutaneous and
subserosal oozing of RBC\*.

3\. The items marked with an asterisk will be measured in SLS-2.

4\. Since the above recommendations were made, many technical advances
have been accomplished. These include growth factors, cytokines and
receptors. It should now be possible to measure many of these including
IL-3, IL-9, IL-11 an stem cell factor. In addition, all the organs
involved in the Selye adaptation syndrome could be examined. These
include: Thymus, other lymphatic tissues, adrenal weights and other
measures that relate to current concepts of acute and subacute stress.

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