Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 97: 29-38, 2004. First published February 20, 2004; doi:10.1152/japplphysiol.01304.2003
8750-7587/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
97/1/29    most recent
01304.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pecaut, M. J.
Right arrow Articles by Gridley, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pecaut, M. J.
Right arrow Articles by Gridley, D. S.

Hypergravity-induced immunomodulation in a rodent model: hematological and lymphocyte function analyses

Michael J. Pecaut,1 Glen M. Miller,2 Gregory A. Nelson,1 and Daila S. Gridley1,2

1Department of Radiation Medicine, Radiobiology Program, and 2Department of Microbiology and Molecular Genetics, Loma Linda University School of Medicine and Medical Center, Loma Linda, California 92354

Submitted 5 December 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The major purpose of this study was to quantify hypergravity-induced changes in erythrocyte and thrombocyte characteristics, spontaneous and mitogen-induced lymphoblastogenesis, and capacity of splenocytes to secrete immunoregulatory cytokines. C57BL/6 mice were subjected to chronic 1, 2, and 3 G; subsets were euthanized after 1, 4, 7, 10, and 21 days of centrifugation. Erythrocyte counts, hematocrit, and hemoglobin were significantly reduced by day 21 in both centrifuged groups. Hemoglobin concentration and volume per red blood cell were generally low, but an early, transient spike above normal was noted in thrombocyte counts in the 3-G group. Fluctuations above and below normal in blood and spleen cell spontaneous blastogenesis were dependent on the length of centrifugation time and not on the level of gravity. Depression in splenocyte responses to phytohemagglutinin and lipopolysaccharide due to gravity were noted when the data were expressed as stimulation indexes. Cytokine production by spleen cells was primarily affected during the first week of centrifugation: IL-2, IL-4, and tumor necrosis factor-{alpha} increased, whereas interferon-{gamma} decreased. These findings, although not identical to those reported for spaceflight, indicate that altered gravity can influence both hematological and functional variables that may translate into serious health consequences during extended missions.

centrifugation; immune; cytokine; erythrocyte

INVESTIGATORS HAVE KNOWN FOR decades that the spaceflight environment can have dramatic effects on overall physiology. However, there are only limited reports on the mechanisms behind these changes. Given that recent events already include extended stays aboard the International Space Station by astronauts, cosmonauts, and the occasional space tourist, the gap in the literature is of critical importance as the human presence in space inevitably increases.

The spaceflight environment includes at least three factors that likely influence immunity. These include mission-related psychological stress, low-dose-to-low-dose rate radiation, and changes in inertial condition (i.e., microgravity and hypergravity) (43, 54). In the worst case, these environmental factors might act synergistically, compromising the ability of astronauts to mount an immune response to infection and possibly other diseases (18, 54). Clearly, the influence of the spaceflight environment, as well as any potential synergy between the various factors inherent to that environment, must be carefully considered when coordinating long-term missions (i.e., Mars or Lunar missions, International Space Station).

Despite the important pioneering work of earlier investigators, scientifically relevant and consistent immunological data are somewhat limited. However, there have been reports of spaceflight-induced changes noted in total body mass (1, 7, 9, 16, 43, 55, 59), thymus and spleen mass (16, 43), circulating corticosterone (7, 59), mitogen-induced lymphocyte proliferation (7, 28, 49), cytokine production and reactivity (16, 28, 31), and lymphocyte subpopulations (2, 22, 42, 43, 48, 49).

Due to the logistic difficulties involved and limited opportunity for these experiments, ground-based models have been developed to simulate many aspects of spaceflight. For example, low-dose-to-low-dose rate irradiation has been used to model the spaceflight radiation environment. The present investigators have demonstrated with C57BL/6 mice that low-dose radiation can also have dramatic short- and long-term effects on immunity. Low-dose {gamma}-ray (20, 40), proton (15, 17, 38, 39, 41), iron, and silicon ion (21) irradiation can have profound effects on immune parameters.

In the present study, the purpose was to model chronic changes in the inertial environment (as opposed to acute loads such as launch and landing) by using centrifugation. The early time points (days 1–7) were meant to explore the adaptation phase that astronauts undoubtedly experience soon after launch. Ideally, this study would be conducted in true spaceflight "microgravity." However, given the constraints of spaceflight research, we chose to use centrifugation to model a similar, although not identical, change in the gravitational environment.

To date, there have been very few reports on the effects of centrifugation on immunity in vivo. Sonnenfeld et al. (50) found no significant difference in response of bone marrow cells from control rats and rats continuously exposed to 2 G for 14 days when the cells were incubated with granulocyte-monocyte colony-stimulating factor. In the same study, little or no significant change was noted in leukocytes from bone marrow and spleen, based on expression of surface markers for T, B, and natural killer cells and IL-2 receptor. The investigators concluded that hypergravity did not greatly affect the same immunological parameters that were affected by spaceflight in the Cosmos 2044 mission. However, there were several differences between this study and the present investigation, including different animal species, times of assay, and inertial conditions.

Centrifugation, similar to antiorthostatic tail suspension, simulates many of the chronic stressors inherent to all current spaceflight experiments: exposure to a novel environment, changes in loads placed on the limbs, cephalic fluid distribution shifts, and orthostatic intolerance. The effects of these various stressors on physiological parameters have already been shown.

Circulating corticosterone generally increased (5–7, 59) or remained constant (10) in rats flown on the space shuttle. Similar results were found after tail suspension (37, 60) and centrifugation (37). That these are real stress-induced changes and are not simply due to handling or readaptation is supported by corresponding changes in stress-sensitive adrenal (5, 37, 59) and spleen (3, 4, 7, 9, 16, 19, 60) mass seen under all three gravitational conditions.

However, thymus mass, another indicator of stress, either did not change (7, 9) or increased (5, 6) in rats flown on the space shuttle. Adrenalectomy and basal corticosterone replacement increased this flight-induced change in thymus mass even further (5), suggesting that stress is not a factor in this increase. Because thymus mass decreased in rats after suspension (51) and remained unchanged after centrifugation (47), this measure may be more sensitive to fluid shifts and/or loading rather than psychological stress. This is further indicated by decreases in this measure in mice exposed to both spaceflight (16) and centrifugation (19). The fluid shifts and unloading in the much smaller mouse model are likely to be less severe than those of the larger rat model, thus making it more sensitive to psychological, rather than physical, stressors.

Therefore, it appears that the effects of spaceflight on immune parameters may fall into two separate categories: those induced by spaceflight-related psychological stressors (e.g., exposure to a novel environment), and those induced by spaceflight-related shifts in physical parameters (e.g., fluid shifts and unloading). Those parameters that are influenced primarily by physical stressors may have very different responses to the inertial environment (i.e., responses to hypergravity are opposed to those of microgravity). In contrast, those parameters that are influenced primarily by psychological stressors will likely have similar responses to changes in inertial condition (i.e., similar responses to both hyper- and microgravity).

In an effort to further characterize the effects of inertial condition on immunity, female C57BL/6 mice were exposed to up to 3 G on the 24-ft-diameter centrifuge at the Ames Research Center (Center for Gravitational Biology Research) for up to 21 days. Previously, reports from this study focused on population distributions in the spleen and blood (19). The parameters described herein will provide insight on the energy balance of the animal, red blood cell (RBC), and platelet characteristics, and the ability of the immune system to respond to a mitogenic challenge in terms of proliferation and cytokine expression. These findings provide a wide spectrum of immunological and hematological information regarding the suitability of hypergravity as a model for the chronic inertial component of the spaceflight environment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal identification and temperature.   Female C57BL/6 mice (n = 6 per inertial condition per time point, for a total of 90 mice; 8 wk of age) were purchased from Charles River Breeding Laboratories, Hollister, CA, and housed (3–4 per cage) at the Ames Research Center. Approximately 1 wk before centrifugation (within 1 wk of arrival), trained Loma Linda University (LLU) personnel tattooed the tails for identification purposes. Additionally, animals were briefly made recumbent with 100% CO2 and identification and temperature transponders (BioMedic Data Systems, Seaford, DE) were injected subcutaneously behind the necks of the mice. Mouse body and ambient temperature, as well as humidity, data were gathered at various time points throughout the centrifugation run during regularly scheduled maintenance and at times of euthanasia. The Institutional Animal Care and Use Committees of LLU and Ames Research Center reviewed and approved the protocols for this study.

Centrifugation.   Mice were subjected to hypergravity at Ames in the 24-ft-diameter centrifuge designed for small-animal research. The centrifuge has 10 radial arms, each holding two large gondolas (23.5 in. high x 39.5 in. wide x 22 in. deep) capable of holding four polycarbonate cages (n = 5 mice/cage). The gondolas swing freely with centrifugation, ensuring that the gravitational force vector was perpendicular to the cage floor.

Control animals were housed in stationary gondola-like enclosures that are located in the same room as the centrifuge. Three smaller enclosures were located in the center of the centrifuge for rotation control animals. These animals experienced slow rotational effects but minimal centrifugal force.

All enclosures were illuminated by fluorescent lighting on a 12:12-h light-dark cycle. Several centrifuge gondolas were fitted with video cameras to allow in-cage TV monitoring of animals during centrifugation.

In the present study, mice were subjected to a maximum of 3 G for up to 21 days. Food and water were available ad libitum. The centrifuge was stopped daily each morning for ~1 h on the request of the veterinarian who assessed animal health by visual inspection. Experimental gravitational forces were achieved within ~1–2 min after initiation of centrifugation. Provision of clean bedding two to three times each week and selection of animal subsets for euthanasia were synchronized as closely as possible with the times of health inspection to minimize downtime.

Euthanasia and tissue collection.   On days 1, 4, 7, 10, and 21, subsets of animals in each group were removed from the centrifuge and euthanized with 100% CO2 (44). Whole blood was collected immediately by cardiac puncture with K2-EDTA-coated syringes. To protect the tissues during transport from the Ames Research Center to LLU, spleens were removed, cut into four pieces, and placed in sterile screw-capped tubes containing complete RPMI-1640 medium (with 20% heat-inactivated bovine calf serum) on wet ice. The samples were shipped with ice packs by overnight express to LLU for evaluation. At LLU, single-celled suspensions of spleen leukocytes were obtained, as described previously (17, 40).

Hematological analysis.   Hematological parameters were quantified by using an ABC Vet Hematology Analyzer (Heska, Waukesha, WI). Parameters characterized include RBC, platelet, and white blood cell counts, Hb concentration, and mean corpuscular volume (MCV, mean volume per RBC). Based on these measurements, standard formulas were used to obtain Hct (percentage of whole blood consisting of RBC), mean corpuscular Hb (MCH; mean mass of Hb per RBC), MCH concentration (concentration of Hb per RBC), RBC distribution width (RDW; width of the RBC histogram produced by cell number x cell size), and the mean platelet volume (size of the average platelet).

Spontaneous and mitogen-induced blastogenesis.   The spontaneous and mitogen-induced blastogenesis assays were performed as previously described (14). To determine spontaneous blastogenesis of peripheral blood leukocytes, a 50-µl aliquot of whole blood from each mouse was dispensed in triplicate into wells of 96-well flat-bottom microtiter plates. RPMI medium (150 µl) supplemented with 10% FCS (Hyclone), antibiotics, mercaptoethanol, and [3H]thymidine (specific activity = 46 Ci/mmol; ICN Radiochemicals, Irvine, CA) was immediately added to each well (1 µCi/50 µl). The plates were incubated for a total of 4 h at 37°C in 5% CO2. Single-cell splenocyte suspensions (after RBC lysis) were treated similarly except that leukocyte counts were first adjusted to 2 x 106 cells/ml.

For mitogen-induced blastogenesis in the spleen, 100-µl aliquots of 2 x 106/ml leukocytes were dispensed into wells of microtiter plates and 100 µl of phytohemagglutinin (PHA), concanavalin A (ConA), or LPS (Sigma Chemical, St. Louis, MO) were added; control wells with no mitogen were included (no stimulation). Mitogens were pretitrated for optimal response. After 44 h, [3H]thymidine (1 µCi/50 µl/well) was added, and cells were allowed to incubate for an additional 4 h.

For both assays, cells were harvested into a 96-well format filter with a Harvester 96 Mach III-m (Tomtec, Hamden, CT), and the incorporated radioactivity was quantified on a 1450 Microbeta Trilux Liquid Scintillation and Luminescence Counter (EG&G-Wallac, Turku, Finland). Spontaneous blastogenesis results for blood leukocytes were adjusted to account for the cell counts, based on the white blood cell counts obtained with the hematology analyzer (see above). Stimulation index (SI) for each mitogen was calculated as follows: SI = [counts per minute (cpm) with mitogen – cpm without mitogen]/(cpm without mitogen).

Quantification of cytokines.   Spleen leukocytes were quantified and diluted with supplemented RPMI-1640 medium (Irvine Scientific) to 2 x 106 cells/ml. Aliquots (0.1 ml) were placed into wells of 96-well microculture plates. PHA (Sigma) was immediately added (0.1 ml/well), and the cells were incubated for 48 h. Supernatants were then aspirated, cells and debris were removed by centrifugation, and the levels of IFN-{gamma}, TNF-{alpha}, IL-2, IL-4, and IL-5 were quantified by using the Cytometric Bead Array Assay (CBA Assay, Becton Dickinson Biosciences, San Diego, CA), according to the manufacturer's instructions via flow cytometry. Supernatants from nonstimulated cells were collected from three mice/group and tested for background cytokine levels. Cytokine concentrations in each test sample were interpolated from the appropriate standard curve.

Statistical analysis.   Data were normalized to the controls on each day of euthanization (actual control means given in Table 1) and evaluated by using two-way ANOVA with Tukey's pairwise multiple-comparison test (Systat, Systat Software, Richmond, CA). P values of <0.05 and <0.1 were selected to indicate significance and a trend, respectively. Main effects of gravity and day, as well as interactions between these variables, are reported. A significant main effect of gravity suggests that there was an overall effect of the inertial condition independent of the day the assay was performed. Similarly, a main effect of day suggests that there was an overall effect of day independent of gravitational condition. Finally, an interaction between gravity and day suggests that the effect of gravity depended on the day the assay was performed.


View this table:
[in this window]
[in a new window]
  		Table 1. Controls before normalization

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal temperature.   As shown in Fig. 1A, ANOVA results indicate that there were significant main effects of gravity on subcutaneous body temperature (P < 0.001). Post hoc Tukey's test indicated that this was due to a general decrease in both centrifuge groups (P < 0.001 for 2 G and P < 0.005 for 3 G). There were no dependencies on day nor any day x gravity interactions. Figure 1, B and C, describes the environmental temperature and humidity for the animals on the centrifuge. In general, the environment of the centrifuge remained fairly stable throughout the experiment. Between days 10 and 14, there were decreases in bucket humidity and a spike of roughly 3°C in bucket temperatures. This was likely due to a combination of changing weather outside the facility and a malfunction in the building air conditioning. Although the malfunction was repaired, the environmental factors within the buckets fluctuated for the rest of the experiment. However, all parameters are well within typical housing conditions. According to the Guide for the Care and Use of Laboratory Animals, the recommended ranges for dry-bulb temperature and humidity are 18–26°C and 30–70%, respectively (23).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1. Effects of centrifugation on subcutaneous temperature and environmental conditions. Values are means ± SE (n = 6 per time point per inertial condition). A: mouse subcutaneous body temperature. B: bucket temperature. C: bucket humidity. A: P < 0.001 for main effect of gravity. Post hoc Tukey's test: aP < 0.05 for 3 G vs. 1 G controls. bP < 0.05 for 2 G vs. 1 G controls. B: the transient increase of ~3°C in bucket temperature on day 14 was due to a malfunction in the air conditioning system. However, the temperature was still well within animal care guidelines and did not translate into a significant change in subcutaneous temperature.

 
Hematology.   Overall, there were gravity-dependent decreases in most RBC, but not platelet, measures. There was a significant main effect of day on the RBC count (P < 0.001; Fig. 2A). Although there appeared to be a slow decrease throughout the centrifugation period, Tukey's analysis indicated that there were no significant differences between any group until day 21. At this time point, RBC counts were significantly depressed in both 2- and 3-G groups compared with 1-G controls (P < 0.005 and P < 0.001, respectively). This time-dependent effect of gravity led to a statistically significant day x gravity interaction (P < 0.005). As expected, the pattern of changes in RBCs was similar to those of both the Hct and the Hb (Fig. 2, B and C). There were main effects of day (P values <0.001), as well as day x gravity interactions (P values <0.005), for both of these measures. Furthermore, there were significant main effects of gravity on both Hb (P < 0.05) and Hct (P < 0.05).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Effects of centrifugation on red blood cell (RBC; A), Hb (B), and Hct (C). Values are means ± SE (n = 6 per time point per inertial condition), normalized to controls run on each assay day. P < 0.001 for main effect of day (A, B, and C). P < 0.05 for main effect of gravity (B and C). P < 0.005 for gravity x day interaction (A, B, and C). aP < 0.001 for 3 G vs. 1 G controls. bP < 0.005 for 2 G vs. 1 G controls.

 
There were main effects of both day (P < 0.05) and gravity (P < 0.001) on MCH (Fig. 3A). In general, MCH was significantly lower in the 3-G group compared with both 1-G controls and 2-G animals (P < 0.001 and P < 0.005, respectively). Post hoc Tukey analyses indicate that there were consistent decreases in the 3-G group compared with 1-G controls throughout the run, reaching significance on days 1, 7, and 21 (P values <0.05). In contrast, the 2-G group was significantly lower than controls on only day 7 (P < 0.05). However, when normalized to a per-cell basis, as indicated by MCH concentration, the effect of day was not present, and the gravity effect was reduced to a trend (Fig. 3B).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Effects of centrifugation on mean corpuscular Hb (A), mean corpuscular Hb concentration (B), mean corpuscular volume (C), and RBC distribution width (D). Values are means ± SE (n = 6 per time point per inertial condition), normalized to controls run on each assay day. P < 0.001 (D) or P < 0.05 (A) for main effect of day. P < 0.001 for main effect of gravity (A, C, and D). P < 0.05 for day x gravity interaction (D). aP < 0.001, bP < 0.01, or cP < 0.05 for 3 G vs. 1 G controls. dP < 0.01 for 2 G vs. 1 G controls. eP < 0.01 or fP < 0.05 for 3 G vs. 2 G controls.

 
Figure 3C shows a trend for a main effect of day and a significant main effect of gravity (P < 0.001) on MCV. The gravity effect is mainly due to decreases in the 3-G animals, compared with both 1-G controls and 2-G animals (P values <0.001), throughout the centrifugation period.

There were main effects of both day and gravity on RDW (P values <0.001; Fig. 3D). In general, the 3-G group was significantly lower than both the 1-G controls and 2-G animals (P < 0.005 and P < 0.01, respectively). However, compared with 1-G controls, there were significant decreases in the 3-G group on only days 4 and 7 (P values <0.05) and in the 2-G group on day 4 (P < 0.05). This led to a significant day x gravity interaction (P < 0.05).

Platelet parameters were only acutely influenced by centrifugation. There was a significant main effect of day (P < 0.001), and a trend for an effect of gravity, on platelet counts (Fig. 4A). After 1 day of centrifugation, the 3-G animals had elevated platelet counts. Counts in these animals returned to control levels by day 4, and there were no significant differences in any group throughout the rest of the run. This resulted in a significant day x gravity interaction (P < 0.05). However, there was only a trend for a main effect of day on mean platelet volume (Fig. 4B).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 4. Effect of centrifugation on platelet parameters. A: platelet. B: mean platelet volume. Values are means ± SE (n = 6 per time point per inertial condition), normalized to controls run on each assay day. P < 0.001 for main effect of day (A). P < 0.05 for gravity x day interaction (A). aP < 0.005 for 3 G vs. 1 G controls. bP < 0.005 for 2 G vs. 1 G controls.

 
Spontaneous blastogenesis.   Gravity appears to have a stronger influence on splenic, rather than peripheral, blastogenesis. Furthermore, this effect appears to be transient. As shown in Table 2, there were day-dependent changes in spontaneous blastogenesis of splenocytes (P < 0.001). This was likely due to an increase in the 2-G animals compared with both 1-G controls and, surprisingly, the 3-G animals (P < 0.001 and P < 0.05, respectively) on day 10. This led to a significant day x gravity interaction (P < 0.05). There were no main effects of gravity on this measure. Similarly, there was a main effect of day in the peripheral blood (P < 0.05; Table 2). This appears to be due to a slight, but statistically insignificant, decrease in the centrifuged groups on day 7 compared with controls.


View this table:
[in this window]
[in a new window]
  		Table 2. Normalized spontaneous blastogenesis (4-h incubation period)

 
Mitogen-induced blastogenesis.   In general, the effects of gravity on mitogen-induced blastogenesis depended on both the mitogen in question and the method used to describe the data. When measured in cpm, there were day-dependent changes in ConA- (P < 0.001) and PHA-induced (P < 0.001) T-cell-dependent proliferation (Table 3), due to a gradual and consistent increase in the centrifuged animals, peaking on day 10. Post hoc Tukey comparisons between gravity groups on day 10 indicate that, after stimulation with ConA, both 2- and 3-G groups had a significantly greater blastogenic response than 1-G controls (P < 0.05 and P < 0.01, respectively). Similar results were found with PHA (P < 0.1 and P < 0.01 for 2- and 3-G groups, respectively). Although there were no significant main effects of gravity on these parameters, there was a significant day x gravity interaction (P < 0.01) for PHA-induced blastogenesis. This was reduced to a trend with ConA.


View this table:
[in this window]
[in a new window]
  		Table 3. Normalized splenic mitogen-induced blastogenesis (48-h incubation period)

 
In contrast to the T-cell mitogens, there were significant main effects of both day (P < 0.001) and gravity (P < 0.05), as well as a day x gravity interaction (P < 0.005), on LPS-induced B-cell-dependent blastogenesis. With a profile similar to that of ConA and PHA, the peak in the response to centrifugation occurred on day 10. However, only the 2-G group reached significance compared with 1-G controls on this day (P < 0.001). There also appeared to be a long-term effect of centrifugation on this parameter. On day 21, both 2-G (P < 0.05) and 3-G (P < 0.001) groups were significantly suppressed in this measure compared with controls.

At first glance, it appears that the response in the unstimulated cells was actually greater than that of the stimulated cells. However, this is simply due to normalization, with the means for the 1-G controls set at 1. Before normalization, the cpm for the mitogen-induced responses were much higher for the stimulated than for the unstimulated cells. Ultimately, this meant that even a slight inertia-dependent change in the response for the unstimulated cells could result in a large proportional change. For a similar proportional increase in the stimulated cells, there would have to be a considerably larger gravity response.

Surprisingly, there were unexpected effects of both day (P < 0.001) and gravity (P < 0.05) in unstimulated blastogenesis. In general, cpm values for the 2-G (P < 0.001) and 3-G (P < 0.1) groups were increased compared with those for 1-G controls. The pattern of these effects was similar to both spontaneous and mitogen-induced blastogenesis, peaking on day 10 (P < 0.001 for 2 G and P < 0.01 for 3 G).

Because of this unexpected result, we are also presenting blastogenesis data in the form of stimulation indexes (Table 4). In this form, there were no significant effects of day on the ability of any of these mitogens to induce blastogenesis. However, there was a significant effect of gravity on PHA (P < 0.05), with trends in ConA and LPS. For both of the T-cell mitogens, this appears to be due to overall increases in the 2-G group compared with 1-G controls (P > 0.05 for PHA and P < 0.1 for ConA). For LPS, this was due to overall decreases in the 3-G group compared with 1-G controls. However, post hoc Tukey's test did not indicate that the centrifuged groups were significantly different from 1-G controls on any of the test days.


View this table:
[in this window]
[in a new window]
  		Table 4. Normalized splenic mitogen-induced blastogenesis (48-h incubation period)

 
Cytokine expression.   Figure 5 shows the concentrations of the six measured cytokines, normalized to controls. Whereas there were no consistent changes, there were transient changes noted in four of the six cytokines characterized. There was a significant main effect of day on splenocyte IL-2 production (P < 0.05). Although there was no significant main effect of gravity, there was a day x gravity interaction (P < 0.05). Tukey analysis indicated that this was due to an increase in IL-2 expression in the 3-G animals compared with both 2-G and 1-G controls (P < 0.005 and P < 0.005, respectively) on day 4. There was a significant main effect only of day on IL-4 (P < 0.05), due to an increase in expression for the 2-G animals compared with 1-G controls on day 1 (P < 0.05). The effect of gravity was reduced to a trend. No significant effects were noted for IL-5 expression.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5. Effects of centrifugation on mitogen-induced cytokine expression. A: IL-2; B: IL-4; C: IL-5; D: IFN-{gamma}; E: TNF-{alpha}; F: TGF-{beta} – plasma. Values are means ± SE (n = 6 per time point per inertial condition), normalized to controls run on each assay day. P < 0.01 (D) or P < 0.05 (A, B, and E) for main effect of day. P < 0.01 (D) or P < 0.05 (F) for main effect of gravity. P < 0.005 (E) or P < 0.05 (A and D) for gravity x day interaction. aP < 0.005, bP < 0.01, or cP < 0.05 for 3 G vs. 1 G controls. dP < 0.05 for 2 G vs. 1 G controls. eP < 0.001, fP < 0.005, or gP < 0.05 for 3 G vs. 2 G controls. Brackets indicate concentration.

 
There were significant main effects of both day (P < 0.01) and gravity (P < 0.01), as well as a significant day x gravity interaction (P < 0.05) on IFN-{gamma} expression. In general, the 3-G group was significantly lower than both 1-G controls (P < 0.01). Tukey analysis indicated that there were significant differences between 3-G and 1-G controls on day 1 (P < 0.05), and between 3-G and 2-G on day 21 (P < 0.05). There was a significant effect of day (P < 0.05), as well as a significant day x gravity interaction (P < 0.005), on TNF-{alpha} expression. This was due to an increase in the 3-G animals on day 7 compared with both 2-G and 1-G controls (P < 0.001 and P < 0.01, respectively).

In the plasma, there were main effects of gravity (P < 0.05) on the level of transforming growth factor (TGF)-{beta}. The effect of day was reduced to a trend. In general, TGF-{beta} concentration was low in the 3-G group compared with 1-G controls and 2-G animals (P < 0.05 and P < 0.1, respectively). However, in post hoc Tukey's test, the 3-G group only trended toward a decrease compared with 1-G controls on days 1 and 4.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The data show that hypergravity-induced decreases occurred in subcutaneous body temperature that did not correspond to changes in ambient room temperature or humidity. Whereas decreases in body temperature have been reported in rat studies, this was transitory and returned to control levels after 4 days of centrifugation (24). In mice, recovery to a new baseline temperature occurred within 8 days (33), which was significantly lower than that in 1-G controls (12). This temperature decrease appears counterintuitive, as one would expect an increase in metabolic requirements in hypergravity (46). However, the hypothermia may reflect an acute decrease in overall activity. In mice, hypergravity-induced decreases in both locomotor activity and body temperature have been shown to occur even after 44–48 days of exposure to 2 G (12). Hypergravity is also reported to cause a transient reduction in food intake and oxygen consumption in rats (46, 58). Surprisingly, oxygen consumption, but not resting energy expenditure, appears to actually increase in rats exposed to 2 wk of 2.3 or 4.1 G (57). In contrast, according to a meta-analysis of the results of 15 shuttle flight experiments involving rats, the spaceflight environment appears to have no effect on energy expenditure or caloric intake (56), suggesting that centrifugation may be unique in its effect on body temperature and metabolism. Although we did not do a quantitative analysis on behavior, a video of the animals on the centrifuge suggests that any changes in activity were minimal. Furthermore, because centrifugation and all cage changes began at 9 AM every morning, we believe that the initial adaptation to the hypergravity environment occurred while the animals were less active.

The overall decreases in RBC, Hct, and Hb across the 21 days may reflect adaptation. The lack of early centrifugation effects on Hct is consistent with past mouse studies under similar conditions (47). Furthermore, the pattern of effects is similar, although not identical, to that of the spaceflight environment. After 7 days aboard an orbiting space shuttle, rats had higher RBC, Hct, and Hb values compared with ground controls (27). After 9 days, these differences were no longer present (1, 55). We found a slight increase in RBC count but no change in Hct or Hb in mice after 12 days on the shuttle (16). The initial increases in these parameters likely reflect an acute decrease in water intake as the animals adapt to new inertial conditions. The opposing long-term results between hyper- and hypogravity may reflect the long-term adaptation to opposing physical stressors.

Centrifugation consistently decreased the MCV, whereas the MCH response was bimodal. These results do not match what has been seen after spaceflight, suggesting a response to physical rather than psychological stressors. There was no change in either MCV or MCH in rats flown on the space shuttle for 7–9 days compared with ground controls (27, 55). Similarly, although we found a slight decrease in MCV, there were no changes in MCH in mice flown for 12 days on the shuttle compared with ground controls (16). It is possible that the apparent disparity between spaceflight and centrifugation is a result of the analysis technique. This is particularly true for MCV. The values produced by the hematology analyzer for MCV are strictly integers, forcing the data to be artificially "quantized." With relatively small sample sizes, this may influence statistical analysis by artificially minimizing variation between samples. However, because the ~2% decreases seen in MCV were consistent across the entire 21-day run (Fig. 3) and involve ~30 animals per centrifuge condition, the possibility exists that this difference is biologically relevant. If this is indeed the case, such a result would be consistent with a response to physical, rather than psychological, stressors.

Decreases in RBC characteristics would be consistent with anemia, a condition frequently reported in flight personnel (45). However, these measurements depend, at least in part, on a constant blood volume. Fluid shifts due to altered gravitational conditions typically result in blood volume changes. A decrease in MCV suggests a possible iron deficiency and/or passage of cells from the portal to the systemic core due to hepatic insufficiency. In addition, both MCV and MCH have been shown to be negatively correlated with transport ATPase activity (25). As gravity-dependent changes in muscle ATPase activity have been reported (11, 29), the disparity between models may be related to similarly dependent changes in muscle mass and protein. Finally, there may be a hypergravity-induced decrease in Hb production during erythropoiesis or an increase in its degradation. Both of these mechanisms would be consistent with decreased oxygen and/or energy consumption. However, because immature RBCs are larger than their mature counterparts, a significant increase in erythropoiesis should result in increased MCV. Because MCV decreased after centrifugation, it is unlikely that enough new RBCs were produced to cause a shift in cellular Hb. The low RDW is similar to what we found in mice flown on the space shuttle for 12 days (16). This suggests a burst of cell death or apoptosis in reticulocytes, initiated shortly after a change in inertial condition (i.e., launch or placement on the centrifuge). Platelet counts and volume were relatively unaffected by hypergravity. This is similar to what has been shown in rats exposed to 8 days of spaceflight (7). However, we found that, after 12 days on the shuttle, platelet counts were increased in mice (16).

After an early, slight decrease, spontaneous blastogenesis in the spleen was increased in the centrifuged animals on day 10. The temporal changes may reflect an initial insult, followed by overcompensation. The reason for lack of similar shifts in blood is not clear. However, because of these compartmental differences, it seems unlikely that this was simply an effect of dehydration. The early decrease is in agreement with the hypergravity-dependent decreases that we previously reported in splenic lymphocyte numbers (19). Hence, the subsequent increase in DNA synthesis may well reflect reconstitution following splenocyte apoptosis (19). There are reports that spontaneous blastogenesis of lymph node lymphocytes is not significantly different between controls and rats exposed to the spaceflight environment or suspension for 14 days (35). However, as we found differences between the blood and spleen, the lack of spaceflight effects on lymph node lymphocyte spontaneous blastogenesis may not be surprising.

As with spontaneous blastogenesis in spleen, T-cell mitogen-induced proliferation initially exhibited a downward trend followed by a significantly increased responsiveness on day 10. It is noteworthy that a similar pattern, with the peak shifted to day 7, was seen in the splenic helper, but not cytotoxic, T cells (19). These two phenomena suggest a temporal relationship between helper T cells, which provide activating cytokines, and T-cell blastogenesis.

In contrast, experiments using blood from astronauts and cosmonauts have shown that spaceflight leads to decreased mitogen-induced proliferation (52, 53). Similarly, results from spaceflight rodents are different from those of centrifugation. ConA-, but not PHA-, induced splenocyte blastogenesis trended toward an increase in rats flown on the shuttle for 4 days (36). Other studies show normal levels after 8 days (7) and depression after 10 days (6) and 14 days (28) on the shuttle. A similar pattern has been reported for lymph node lymphocytes (6, 7). Unlike centrifugation, results from rat antiorthostatic tail suspension models are remarkably similar to those of spaceflight. ConA- and PHA-stimulated proliferation either decreased or remained constant after 7 days of suspension in lymphocytes from rat blood, lymph nodes, or spleen (34). This suggests that blastogenic responses to changes in gravitational loads in rats may be due more to the loading of the limbs and fluid distribution shifts, rather than a general stress response. In mice, tail suspension results are much closer to those of centrifugation than those of spaceflight. Splenic T-cell blastogenesis decreased after 7 days (60) and increased after 11 days (26) of tail suspension. Although there are no comparable spaceflight blastogenesis results for mice, a species-dependent response to gravity is possible.

With the B-cell mitogen LPS, we found a peak in activity on day 10, followed by a decrease on day 21, when expressed as cpm. However, these variations were no longer evident when the data were presented in the form of an SI, which takes into account the background cpm without mitogen. While the lack of a B-cell response to gravity is similar to results found with rat (34, 35) and mouse (60) tail suspension models, the increased activity on day 10 once again opposed that of spaceflight. LPS-induced blastogenesis in splenocytes from rats flown on the shuttle remained unchanged after 8 days (7) and decreased after 10 days (6), compared with controls. A similar pattern was reported in the lymph nodes (6, 36). Combined with the T-cell mitogen data, this further suggests that the influence of gravity on overall blastogenic responses is more likely due to loading and fluid redistribution than a generalized stress response.

To our knowledge, this is the first report of cytokine expression after centrifugation. Overall, the greatest effect on splenic PHA-induced cytokines was noted for IFN-{gamma}, which tended to be low for the entire 3-wk period. The early increases in IL-2 (needed for T-cell proliferation) and IL-4 (a promoter of B-cell differentiation) may be in response to the significant drop in T and B cells noted in these animals (19). The depression in IFN-{gamma} that was noted in conjunction with an increase in IL-4 suggests a shift in the balance between the T-helper type 1 (Th1) and type 2 (Th2) lymphocyte subsets. Under normal conditions, activated Th1 cells secrete IFN-{gamma} and other cytokines that promote cell-mediated immune responses important in the control of viral infections. Similarly, the Th2 cells secrete IL-4 and other cytokines that facilitate humoral immunity (e.g., antibody production). IFN-{gamma} is also well known to be a potent activator of cells of the monocyte-macrophage lineage that function in removal of bacteria and present antigen to lymphocytes. Thus compromised ability to secrete a sufficient amount of IFN-{gamma} may result in increased risk for infections of various types. By days 10 and 21, the total Th cell population, as well as the B cells, had either rebounded above controls, or returned to within the normal control range (19), as did IL-2 and IL-4 expression. The slight decrease in plasma TGF-{beta}1 during the first week of centrifugation is consistent with these observations. A reduction in this highly immunosuppressive cytokine could enhance the probability of lymphocyte proliferation. Except for an acute increase in mitogen-induced IL-2 secretion after exposure to flight for 4 days (36), splenic cytokine expression has generally either remained constant or decreased after spaceflight (7, 16, 28) or suspension (34, 60). This again suggests that fluid shifts and limb unloading may influence some immune parameters more than psychological stressors.

These shifts in cytokines, and hence changes in the ability to respond to disease, are consistent with the antiorthostatic tail suspension literature. When mice were inoculated with Listeria monocytogenes 2 days before, or on the day of, initiation of antiorthostatic tail suspension, bacterial clearance was no different from that of controls. However, when mice were inoculated 2 or 4 days into a 7- to 9-day suspension, clearance was actually enhanced. When mice were suspended for 7 days before inoculation, clearance was still enhanced but not to the extent seen for days 2 and 4 (30, 32). Furthermore, EL-4-stimulated splenocytes from animals exposed to 4 days of suspension had significantly higher levels of IL-1 activity compared with vivarium controls in both naive and Listeria inoculated mice (30). In contrast, Gould et al. (13) found that mice inoculated with a normally resisted dose of encephalomyocarditis virus on day 0 began to die within 4 days of suspension. This was reported to be coordinated by changes in IFN production (13).

Clearly, changes in inertial condition can have effects on multiple hematological and immune parameters. However, a factor that must be taken into consideration when interpreting the results presented here is the daily "stop and start" of the centrifuge. This was done on the request of the veterinarian at the Ames Research Center so that a general health assessment could be performed by visual inspection of all mice, rather than by visualization of animals in selected cages by video camera. It is possible that the frequency of inertial changes exacerbated the differences observed between the centrifuged and noncentrifuged control groups.

By comparing results across spaceflight, centrifugation, and antiorthostatic tail suspension experiments, potential mechanisms may be further elucidated. However, this effort must be coordinated to be of value. Furthermore, results from in vitro studies must be verified in in vivo models. For example, Cogoli (8) has summarized potential mechanisms by which gravitational changes could affect cells of the immune system. These mechanisms include those that function in signal transduction pathways involving cell activation and/or cytokine secretion, synthetic pathways leading to expression of receptors for growth factors and other immunoregulatory molecules, and interactions between lymphocytes and accessory cells that present antigen (e.g., dendritic cells and monocyte-macrophages). However, these conclusions are based primarily on cells in culture, and it is not yet clear whether many of the in vitro observations hold true in vivo.

Finally, the immunological significance of these gravity-dependent phenomena must be characterized by utilizing pathogenic-challenge and transgenic models. Whereas there has been some progress in these areas with antiorthostatic tail suspension, there is very little data available for spaceflight or centrifugation. Similarly, to more closely simulate the interplanetary spaceflight environment, these gravitational models must be combined with low-dose-to-low-dose-rate irradiation. Until these goals are met, astronaut risk assessment paradigms will be less than complete.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by NASA Cooperative Research Agreements NCC9-79 and NCC9-149, the NASA/Ames Research Center, and the Chan Shun International Foundation.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Melba L. Andres, Radha Dutta-Roy, Dong Won Kim, Anna L. Smith, Lora Benzatyan, and Tamako A. Jones for expert technical assistance. The valuable assistance of Charles E. Wade, Tianna Shaw, Tom Luzod, and other personnel at the National Aeronautics and Space Administration (NASA) Ames Research Center is also greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Pecaut, Chan Shun Pavilion, Rm. A-1010, 11175 Campus St., Loma Linda Univ. School of Medicine, Loma Linda, CA 92354 (E-mail: mpecaut{at}dominion.llumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Allebban Z, Gibson LA, Lange RD, Jago TL, Strickland KM, Johnson DL, and Ichiki AT. Effects of spaceflight on rat erythroid parameters. J Appl Physiol 81: 117–122, 1996.[Abstract/Free Full Text]
  2. Allebban Z, Ichiki AT, Gibson LA, Jones JB, Congdon CC, and Lange RD. Effects of spaceflight on the number of rat peripheral blood leukocytes and lymphocyte subsets. J Leukoc Biol 55: 209–213, 1994.[Abstract]
  3. Armstrong JW, Balch S, and Chapes SK. Interleukin-2 therapy reverses some immunosuppressive effects of skeletal unloading. J Appl Physiol 77: 584–589, 1994.[Abstract/Free Full Text]
  4. Armstrong JW, Nelson KA, Simske SJ, Luttges MW, Iandolo JJ, and Chapes SK. Skeletal unloading causes organ-specific changes in immune cell responses. J Appl Physiol 75: 2734–3739, 1993.[Abstract/Free Full Text]
  5. Blanc S, Somody L, Gharib A, Gauquelin G, Gharib C, and Sarda N. Counteraction of spaceflight-induced changes in the rat central serotonergic system by adrenalectomy and corticosteroid replacement. Neurochem Int 33: 375–382, 1998.[CrossRef][ISI][Medline]
  6. Chapes SK, Simske SJ, Forsman AD, Bateman TA, and Zimmerman RJ. Effects of space flight and IGF-1 on immune function. Adv Space Res 23: 1955–1964, 1999.[CrossRef][ISI][Medline]
  7. Chapes SK, Simske SJ, Sonnenfeld G, Miller ES, and Zimmerman RJ. Effects of space flight and PEG-IL-2 on rat physiological and immunological responses. J Appl Physiol 86: 2065–2076, 1999.[Abstract/Free Full Text]
  8. Cogoli A. The effect of hypogravity and hypergravity on cells of the immune system. J Leukoc Biol 54: 259–268, 1993.[Abstract]
  9. Congdon CC, Allebban Z, Gibson LA, Kaplansky A, Strickland KM, Jago TL, Johnson DL, Lange RD, and Ichiki AT. Lymphatic tissue changes in rats flown on Spacelab Life Sciences-2. J Appl Physiol 81: 172–177, 1996.[Abstract/Free Full Text]
  10. Davidson JM, Aquino AM, Woodward SC, and Wilfinger WW. Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat. FASEB J 13: 325–329, 1999.[Abstract/Free Full Text]
  11. De Luca A, Liantonio A, Pierno S, Desaphy JF, Leoty C, and Conte Camerino D. Potential targets for skeletal muscle impairment by hypogravity: basic characterization of resting ionic conductances and mechanical threshold of rat fast- and slow-twitch muscle fibers. J Gravit Physiol 5: P75–P76, 1998.[Medline]
  12. Fuller PM, Warden CH, Barry SJ, and Fuller CA. Effects of 2-G exposure on temperature regulation, circadian rhythms, and adiposity in UCP2/3 transgenic mice. J Appl Physiol 89: 1491–1498, 2000.[Abstract/Free Full Text]
  13. Gould CL, Lyte M, Williams J, Mandel AD, and Sonnenfeld G. Inhibited interferon-gamma but normal interleukin-3 production from rats flown on the space shuttle. Aviat Space Environ Med 58: 983–986, 1987.[Medline]
  14. Gridley DS, Andres ML, and Slater JM. Enhancement of prostate cancer xenograft growth with whole-body radiation and vascular endothelial growth factor. Anticancer Res 17: 923–928, 1997.[ISI][Medline]
  15. Gridley DS, Mackensen DG, Slater JB, Moyers MF, and Slater JM. Effects of proton irradiation on radiolabeled monoclonal antibody uptake in human colon tumor xenografts. J Immunother Emphasis Tumor Immunol 17: 229–237, 1995.[Medline]
  16. Gridley DS, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA, Morony S, Stodieck LS, Lacey DL, Simske SJ, and Pecaut MJ. Genetic Models in Applied Physiology: Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets. J Appl Physiol 94: 2095–2103, 2003.[Abstract/Free Full Text]
  17. Gridley DS, Pecaut MJ, Dutta-Roy R, and Nelson GA. Dose and dose rate effects of whole-body proton irradiation on leukocyte populations and lymphoid organs. Part I. Immunol Lett 80: 55–66, 2002.[CrossRef][ISI][Medline]
  18. Gridley DS, Pecaut MJ, Green LM, Miller GM, Andres ML, Smith AL, Dutta-Roy R, Kim DW, Jones TA, Murray DK, Mao XW, and Nelson GA. Immune system modulation by altered gravity: similarities with radiation effects? In: Joint DOE/NASA Radiation Investigators' Workshop (Low Dose Radiation Res Program, Workshop II and the 12th Annual Space Radiation Health Investigators' Workshop). Washington, DC: NASA, 2001, p. 229–231.
  19. Gridley DS, Pecaut MJ, Green LM, Miller GM, and Nelson GA. Hypergravity-induced immunomodulation in a rodent model: lymphocytes and lymphoid organs. J Gravit Physiol 9: 15–27, 2002.[Medline]
  20. Gridley DS, Pecaut MJ, Miller GM, Moyers MF, and Nelson GA. Dose and dose-rate effects of whole-body {gamma}-irradiation. II. Hematological variables and cytokines. In Vivo 15: 209–216, 2001.[ISI][Medline]
  21. Gridley DS, Pecaut MJ, and Nelson GA. Total-body irradiation with high-LET particles: acute and chronic effects on the immune system. Am J Physiol Regul Integr Comp Physiol 282: R677–R688, 2002.[Abstract/Free Full Text]
  22. Ichiki AT, Gibson LA, Jago TL, Strickland KM, Johnson DL, Lange RD, and Allebban Z. Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells. J Leukoc Biol 60: 37–43, 1996.[Abstract]
  23. Institute of Laboratory Animal Resources. Commission on Life Sciences, National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy, 1996.
  24. Ishihama LM, Murakami DM, and Fuller CA. Temperature regulation in rats exposed to a 2 G field. Physiologist 32: S61–S62, 1989.[Medline]
  25. Katyukhin LN, Kazennov AM, Maslova MN, and Matskevich Yu A. Rheologic properties of mammalian erythrocytes: relationship to transport ATPases. Comp Biochem Physiol B Biochem Mol Biol 120: 493–498, 1998.[CrossRef][Medline]
  26. Kopydlowski KM, McVey DS, Woods KM, Iandolo JJ, and Chapes SK. Effects of antiorthostatic suspension and corticosteroine on macrophage and spleen cell function. J Leukoc Biol 52: 202–208, 1992.[Abstract]
  27. Lange RD, Andrews RB, Gibson LA, Congdon CC, Wright P, Dunn CD, and Jones JB. Hematological measurements in rats flown on Spacelab shuttle, SL-3. Am J Physiol Regul Integr Comp Physiol 252: R216–R221, 1987.[Abstract/Free Full Text]
  28. Lesnyak A, Sonnenfeld G, Avery L, Konstantinova I, Rykova M, Meshkov D, and Orlova T. Effect of SLS-2 spaceflight on immunologic parameters of rats. J Appl Physiol 81: 178–182, 1996.[Abstract/Free Full Text]
  29. McDonald KS and Fitts RH. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity. J Appl Physiol 74: 2949–2957, 1993.[Abstract/Free Full Text]
  30. Miller ES, Bates RA, Koebel DA, and Sonnenfeld G. Antiorthostatic suspension stimulates profiles of macrophage activation in mice. Neuroimmunomodulation 6: 160–167, 1999.[CrossRef][ISI][Medline]
  31. Miller ES, Koebel DA, and Sonnenfeld G. Influence of spaceflight on the production of interleukin-3 and interleukin-6 by rat spleen and thymus cells. J Appl Physiol 78: 810–813, 1995.[Abstract/Free Full Text]
  32. Miller ES and Sonnenfeld G. Influence of antiorthostatic suspension on resistance to murine Listeria Monocytogenes infection. J Leukoc Biol 55: 371–378, 1994.[Abstract]
  33. Murakami DM and Fuller CA. The effect of 2G on mouse circadian rhythms. J Gravit Physiol 7: 79–85, 2000.[Medline]
  34. Nash PV, Bour BA, and Mastro AM. Effect of hindlimb suspension simulation of microgravity on in vitro immunological responses. Exp Cell Res 195: 353–360, 1991.[CrossRef][ISI][Medline]
  35. Nash PV, Konstantinova IV, Fuchs BB, Rakhmilevich AL, Lesnyak AT, and Mastro AM. Effect of spaceflight on lymphocyte proliferation and interleukin-2 production. J Appl Physiol 73: 186S–190S, 1992.[Abstract]
  36. Nash PV and Mastro AM. Variable lymphocyte responses in rats after space flight. Exp Cell Res 202: 125–131, 1992.[CrossRef][ISI][Medline]
  37. Ortiz RM, Wang TJ, and Wade CE. Influence of centrifugation and hindlimb suspension on testosterone and corticosterone excretion in rats. Aviat Space Environ Med 70: 499–504, 1999.[Medline]
  38. Pecaut M, Gridley D, and Nelson G. Long-term effects of low dose whole-body proton irradiation on immunity: shielded vs. unshielded. Aviat Space Environ Med 74: 115–124, 2003.[Medline]
  39. Pecaut MJ, Gridley DS, Smith AL, and Nelson GA. Dose and dose rate effects of whole-body proton-irradiation on lymphocyte blastogenesis and hematological variables. Part II. Immunol Lett 80: 67–73, 2002.[CrossRef][ISI][Medline]
  40. Pecaut MJ, Nelson GA, and Gridley DS. Dose and dose-rate effects of whole-body {gamma}-irradiation. I. Lymphocytes and lymphoid organs. In Vivo 15: 195–208, 2001.[ISI][Medline]
  41. Pecaut MJ, Nelson GA, Moyers MF, Rabin B, and Gridley DS. "Out-of-field" effects of head localized proton irradiation on peripheral immune parameters. In Vivo 17: 513–521, 2003.[ISI][Medline]
  42. Pecaut MJ, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA, Morony S, Stodieck LS, Lacey DL, Simske SJ, and Gridley DS. Genetic Models in Applied Physiology: Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions. J Appl Physiol 94: 2085–2094, 2003.[Abstract/Free Full Text]
  43. Pecaut MJ, Simske SJ, and Fleshner M. Spaceflight induces changes in splenocyte subpopulations: effectiveness of ground-based models. Am J Physiol Regul Integr Comp Physiol 279: R2072–R2078, 2000.[Abstract/Free Full Text]
  44. Pecaut MJ, Smith AL, Jones TA, and Gridley DS. Modification of immunological variables by method of CO2 euthanasia. Comput Med Imaging Graph 50: 595–602, 2000.
  45. Poliakov VV, Ivanova SM, Noskov VB, Labetskaia OI, Iarlykova IV, Karashtin VV, Legen'kov VI, Sarycheva TG, Shishkanova ZG, and Kozinets GI. Hematological investigations in conditions of long-term space flights. Aviakosm Ekolog Med 32: 9–18, 1998.[Medline]
  46. Robinson EL and Fuller CA. Gravity and thermoregulation: metabolic changes and circadian rhythms. Pflügers Arch 441: R32–R38, 2000.[CrossRef][ISI][Medline]
  47. Scibetta SM, Caren LD, and Oyama J. The unresponsiveness of the immune system of the rat to hypergravity. Aviat Space Environ Med 55: 1004–1009, 1984.[Medline]
  48. Sonnenfeld G. Immune responses in space flight. Int J Sports Med 19: S195–S202, 1998.
  49. Sonnenfeld G, Foster M, Morton D, Bailliard F, Fowler NA, Hakenewerth AM, Bates R, and Miller ES, Jr. Spaceflight and development of immune responses. J Appl Physiol 85: 1429–1433, 1998.[Abstract/Free Full Text]
  50. Sonnenfeld G, Koebel DA, and Davis S. Effects of hypergravity on immunologic function. Microgravity Sci Technol 7: 323–326, 1995.[ISI][Medline]
  51. Steffen JM and Musacchia XJ. Thymic involution in the suspended rat: adrenal hypertrophy and glucocorticoid receptor content. Aviat Space Environ Med 57: 162–167, 1986.[Medline]
  52. Taylor GR and Dardano JR. Human cellular immune responsiveness following space flight. Aviat Space Environ Med 54: S55–S59, 1983.[Medline]
  53. Taylor GR, Neale LS, and Dardano JR. Immunological analyses of US Space Shuttle crewmembers. Aviat Space Environ Med 57: 213–217, 1986.[Medline]
  54. Todd P, Pecaut MJ, and Fleshner M. Combined effects of space flight factors and radiation on humans. Mutat Res 430: 211–219, 1999.[ISI][Medline]
  55. Udden MM, Driscoll TB, Gibson LA, Patton CS, Pickett MH, Jones JB, Nachtman R, Allebban Z, Ichiki AT, Lange RD, and Alfrey CP. Blood volume and erythropoiesis in the rat during spaceflight. Aviat Space Environ Med 66: 557–561, 1995.[Medline]
  56. Wade CE, Miller MM, Baer LA, Moran MM, Steele MK, and Stein TP. Body mass, energy intake, and water consumption of rats and humans during space flight. Nutrition 18: 829–836, 2002.[CrossRef][ISI][Medline]
  57. Wade CE, Moran MM, and Oyama J. Resting energy expenditure of rats acclimated to hypergravity. Aviat Space Environ Med 73: 859–864, 2002.[Medline]
  58. Warren LE, Hoban-Higgins TM, Hamilton JS, Horwitz BA, and Fuller CA. Effects of 2G exposure on lean and genetically obese Zucker rats. J Gravit Physiol 7: 61–69, 2000.[Medline]
  59. Wronski TJ, Li M, Shen Y, Miller SC, Bowman BM, Kostenuik P, and Halloran BP. Lack of effect of spaceflight on bone mass and bone formation in group-housed rats. J Appl Physiol 85: 279–285, 1998.[Abstract/Free Full Text]
  60. Yamauchi K, Hales NW, Robinson SM, Niehoff ML, Ramesh V, Pellis NR, and Kulkarni AD. Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog. J Appl Physiol 93: 161–166, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
97/1/29    most recent
01304.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow