Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.     

Effects of spaceflight on the spermatogonial population of rat seminiferous epithelium

Testes from rats flown on Cosmos 1887 were compared with vivarium control and synchronous control samples. The mean weights of flight testes, normalized for weight per 100 g, were 6.4% less when compared with the vivarium controls. Counts of spermatogonia from tissue sections (seminiferous tubules in maturation stage 6) from five animals in each group revealed 4% fewer spermatogonia in flight testes compared with synchronous controls and 11% fewer spermatogonia in flight samples compared with vivarium controls.     

Effects of spaceflight on the proliferation of jejunal mucosal cells

The mitotic indices, villus heights, and crypt depths were determined in each of three jejunal regions (proximal, middle, and distal) for five animals each in the flight, vivarium, and synchronous groups. Because of the rapid turnover of intestinal mucosal cells and the delay in recovering the flight animals, it is not known whether the proliferation of jejunal mucosal cells is affected by microgravity conditions associated with spaceflight. However, since there were no consistent differences between animals in the flight group and those in the synchronous and vivarium control groups, it appears that any effects of microgravity on the turnover of jejunal mucosal cells are short-lived. Thus, this study represents an initial step in determining the effects of microgravity on the proliferation and turnover of intestinal mucosal cells.     

Lack of effect of spaceflight on bone mass and bone formation in group-housed rats

As part ofan experiment to study the role of corticosteroids in bone changesduring spaceflight, male Sprague-Dawley rats (6 wk old, 165 g bodyweight) were placed in orbit for 17 days, in groups of six, inanimal-enclosure modules (AEMs) aboard the space shuttle Columbia(STS-78). Control rats were group housed in a similar manner inground-based AEMs or standard vivarium cages. Adrenal hypertrophyoccurred in flight rats, but bone histomorphometric analyses revealed alack of significant changes in bone mass and bone formation in theseanimals. Cancellous bone volume and osteoblast surface in the proximaltibial metaphysis were nearly the same in flight and ground-based rats.Normal levels of cancellous bone mass and bone formation were alsodetected in the lumbar vertebrae and femoral necks of flight rats. Inthe tibial diaphysis, periosteal bone formation rate was found to beidentical in flight and ground-based rats. The results indicate that,under conditions of group housing in AEMs, spaceflight has minimaleffects on bone mass and bone formation in rapidly growingrats. These findings emphasize the need to investigate theimportance of rat age, strain, and especially housing conditions forstudies of the skeletal effects of spaceflight.

     

Effects of space flight on bone formation and resorption.

Samples of femurs and tibiae of male Wistar rats subjected to a 13 day space-flight on the biosatellite Cosmos 1887--were investigated and compared with vivarium and synchronous controls or immobilized rats, using histological and histomorphometric methods. 1. After flight in the metaphysis of bones the density and volume of the spongious trabeculae diminished significantly indicated by the Sv and Vv histomorphometric values and histological data comparing to the controls. In the diaphysis, the density of trabeculae decreased too. 2. In the flight group significant suppression of bone formation was determined by histological and histomorphometric (decrease of the OS, OB and OBI values) methods. 3. In the flight group according to the histological pictures the signs of bone resorption (increase of Hoswship's lacunae, osteoclastic activity, structural rarefication of spongious and cortical bones, osteon disintegration, osteocytic osteolysis) were revealed, which was substantiated by the histomorphometric results (increase of osteoclastic index: OCI). 4. Significant differences between flight and immobilized groups were not determined, except the osteoid value, which was increased in the case of immobilization. 5. Some histomorphometric values related to bone formation of synchronous control group showed close relationship rather to the flight group than to the vivarium control group.     

Effects of the spaceflight on organ-development in the neonatal rats: results in the Neurolab (STS-90)]

In the Neurolab mission, we found that spaceflight affects the development of the aortic baroreflex system and the body weight of the flight rats was significantly lighter [correction of lightess] than that of the control group. The aim of this study is to examine the structural and functional development in various tissues and organs. One hundred and eighteen nine-day old rats and seven fifteen-day old rats, which were launched at these ages and nursed by their dams in the space shuttle Columbia for 16 days, were served for this study. Two hundred and twenty one neonates were used as the ground controls (VIV: vivarium and AGC: asynchronous ground controls). On the landing day after they returned to the earth, the rats were perfused with a fixative under deep urethane anesthesia, and the organs were weighed and the ratio of the organ weight to the body weight was calculated. Six animals of the nine-day old group were reared on the ground for 30 more days after landing and also examined in the same protocol as the landing-day-examination. The organs obtained to examine were heart, lung, spleen, thymus, adrenal glands, kidney, liver, small intestine, large intestine, mesentery, pancreas, testis and ovary. Paraffin sections were made from some organ tissues and prepared for HE staining and immunohistochemistry. We compared these organs in the flight rat with those in the ground controls. All organs except the lung of nine-day old group were significantly smaller. In the ratio of organ weight to body weight, the lung and heart were significantly larger. The weight and ratio of the liver showed no significant difference. The thymus, spleen, mesentery and pancreas were smaller in the weight and the ratio. There were no differences in the body weight among 30-day reared groups, but the lung in the flight group is significantly heavier than the control groups and thymus also tends to be relatively heavy. In flight rats of the fifteen-day group, the kidney was heavy and the ovary was light as compared to the controls. The adipose tissue was macroscopically little found around the thoracic and abdominal organs in all rats of the flight group. These results suggest that the organs related to oxygen supply like as the lung and heart have priority in development over the mesentery and immune system organs even during spaceflight. Lightness of the mesentery in space rats is due to small contents of adipose tissues, and may reflect amounts of the food taken by the flight dams. Lightness of the organs like as the thymus, spleen and pancreas suggests that spaceflight may affect the immune system and also affect continuously the lung and thymus development even after landing.     

Blood pressure and mesenteric resistance arterial function after spaceflight.

Ground studies indicate that spaceflight may diminish vascular contraction. To examine that possibility, vascular function was measured in spontaneously hypertensive rats immediately after an 18-day shuttle flight. Isolated mesenteric resistance arterial responses to cumulative additions of norepinephrine, acetylcholine, and sodium nitroprusside were measured using wire myography within 17 h of landing. After flight, maximal contraction to norepinephrine was attenuated (P < 0.001) as was relaxation to acetylcholine (P < 0.001) and sodium nitroprusside (P < 0.05). At high concentrations, acetylcholine caused vascular contraction in vessels from flight animals but not in vessels from vivarium control animals (P < 0.05). The results are consistent with data from ground studies and indicate that spaceflight causes both endothelial-dependent and endothelial-independent alterations in vascular function. The resulting decrement in vascular function may contribute to orthostatic intolerance after spaceflight.     

Morphological and biochemical examination of Cosmos 1887 rat heart tissue: Part I--Ultrastructure

Morphological changes were observed in the left ventricle of rat heart tissue from animals flown on the Cosmos 1887 biosatellite for 12.5 days. These tissues were compared to the synchronous and vivarium control hearts. While many normal myofibrils were observed, others exhibited ultrastructural alterations, i.e., damaged and irregular-shaped mitochondria and generalized myofibrillar edema. Analysis of variance (ANOVA) of the volume density data revealed a statistically significant increase in glycogen and a significant decrease in mitochondria compared to the synchronous and vivarium controls. Point counting indicated an increase in lipid and myeloid bodies and a decrease in microtubules, but these changes were not statistically significant. In addition, the flight animals exhibited some patchy loss of protofibrils (actin and myosin filaments) and some abnormal supercontracted myofibrils that were not seen in the controls. This study was undertaken to gain insight into the mechanistic aspects of cardiac changes in both animals and human beings as a consequence of space travel (1). Cardiac hypotrophy and fluid shifts have been observed after actual or simulated weightlessness and raise concerns about the functioning of the heart and circulatory system during and after travel in space (2-4).     

Histomorphometric and electron microscopic analyses of tibial epiphyseal plates from Cosmos 1887 rats

Previous studies have shown that the changes seen in the bones of growing rats exposed to microgravity are due in part to changes that occur in the growth plate during spaceflight. In this study, growth plates of rats flown aboard Cosmos 1887 (12.5-day flight plus 53.5-h recovery at 1 g) were analyzed using light and electron microscopy and computerized planimetry. The proliferative zone of flight animals was found to be significantly (P less than or equal to 0.01) larger than that of controls, while the reserve and hypertrophic/calcification zones were significantly reduced. Flight animals also had more cells per column in the proliferative zone than did controls and less in the hypertrophic/calcification region. The total number of cells, however, was significantly greater in flight animals. No difference was found in perimeter or in shape factor, but area was significantly less in flight animals. Electron microscopy showed that collagen fibrils in flight animals were wider than in controls. Since the time required for a cell to cycle through the growth plate is 2-3 days at 1 g, the results reported here represent both the effects of exposure to microgravity and the initial stages of recovery from that exposure.     

Structural and metabolic properties of rat muscle exposed to weightlessness aboard Cosmos 1887.

Male Wistar rats were subjected to 12.5 days of weightlessness aboard Cosmos 1887. Histomorphometric and biochemical analyses were investigated in soleus (SOL), plantaris (PL) and extensor digitorum longus (EDL) muscles of flight rats (group F) and compared with data from two groups of terrestrial controls: one group living free in a vivarium (group V) and another subjected to a flight simulation except for the state of weightlessness (group S). Relative to groups V and S, no alteration in the percentage distribution of fibres had occurred in SOL, PL or EDL, after the flight. In SOL muscles from group F animals, cross-sectional areas of all fibre types were reduced to a greater extent (-40%) than capillary to fibre ratio (-24%) leading to a higher capillary density (+33%) than in V and S groups. In PL, type I, IIA and IIB fibre cross-sectional areas were less decreased (-25%). In EDL, only fast-twitch fibre cross-sectional areas showed an average decrease of 30%. Capillary per fibre ratio was reduced by 15% and 28% respectively in PT and EDL muscles from group F rats compared to control groups V and S. Citrate synthase and 3-hydroxyacyl-coenzyme A dehydrogenase activities remained unchanged in SOL, PL and EDL following spaceflight. These findings indicate greater atrophy and functional alterations (capillarity) compared to those observed after 7 days of microgravity on Cosmos 1667.     

Habitual throwing and swimming correspond with upper limb diaphyseal strength and shape in modern human athletes

Variation in upper limb long bone cross‐sectional properties may reflect a phenotypically plastic response to habitual loading patterns. Structural differences between limb bones have often been used to infer past behavior from hominin remains; however, few studies have examined direct relationships between behavioral differences and bone structure in humans. To help address this, cross‐sectional images (50% length) of the humeri and ulnae of university varsity‐level swimmers, cricketers, and controls were captured using peripheral quantitative computed tomography. High levels of humeral robusticity were found in the dominant arms of cricketers, and bilaterally among swimmers, whereas the most gracile humeri were found in both arms of controls, and the nondominant arms of cricketers. In addition, the dominant humeri of cricketers were more circular than controls. The highest levels of ulnar robusticity were also found in the dominant arm of cricketers, and bilaterally amongst swimmers. Bilateral asymmetry in humeral rigidity among cricketers was greater than swimmers and controls, while asymmetry for ulnar rigidity was greater in cricketers than controls. The results suggest that more mechanically loaded upper limb elements––unilaterally or bilaterally––are strengthened relative to less mechanically loaded elements, and that differences in mechanical loading may have a more significant effect on proximal compared to distal limb segments. The more circular humerus in the dominant arm in cricketers may be an adaptation to torsional strain associated with throwing activities. The reported correspondence between habitual activity patterns and upper limb diaphyseal properties may inform future behavioral interpretations involving hominin skeletal remains. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Effects of zero gravity on myofibril content and isomyosin distribution in rodent skeletal muscle

The purpose of this experiment was to investigate the effects of 12.5 days of zero gravity (0 g) exposure (Cosmos 1887 Biosputnik) on the enzymatic properties, protein content, and isomyosin distribution of the myofibril fraction of the slow-twitch vastus intermedius (VI) and the fast-twitch vastus lateralis (VL) muscles of adult male rats. Measurements were obtained on three experimental groups (n = 5 each group) designated as flight group (FG), vivarium control (VC), and synchronous control (SC). Body weight of the FG was significantly lower than that of the two control groups (P less than 0.05). Compared with the two control groups, VI weight was lower by 23% (P less than 0.10), whereas no such pattern was apparent for the VL muscle. Myofibril yields (mg protein/g muscle) in the VI were 35% lower in the FG than in controls (P less than 0.05), whereas no such pattern was apparent for the VL muscle. When myofibril yields were expressed on a muscle basis (mg/g x muscle weight), the loss of myofibril protein was more exaggerated and suggests that myofibril protein degradation is an early event in the muscle atrophy response to 0 g. Analysis of myosin isoforms indicated that slow myosin (Sm) was the primary isoform lost in the calculated degradation of total myosin. No evidence of loss of the fast isomyosins was apparent for either muscle following spaceflight. Myofibril ATPase activity of the VI was increased in the FG compared with controls, which is consistent with the observation of preferential Sm degradation. These data suggest that muscles containing a high percentage of slow-twitch fibers undergo greater degrees of myofibril protein degradation than muscles containing predominantly fast-twitch fibers in response to a relatively short period of 0 g exposure, and the primary target appears to be the Sm molecule.     

Effects of microgravity on organ development of the neonatal rat.

We analyzed various organs in the same rats to study effects of gravitational condition on organ development of the neonatal rat in this study. Eight-day old and 14-day old Sprague-Dawley rats were flown for 16 days on the Space Shuttle Columbia (April 17-May 3, 1998). The organs were weighed and the ratio of the organ weight to the body weight (organ weight ratio; OBR) was calculated. Tissues were analyzed using anatomical, immunohistochemical and molecular biological technique. Six animals of the 8-day old group were reared on the ground for 30 more days after landing. The differences between flight and control rats in 8-day group were drastic. The lung, heart, kidney and adrenal glands in flight rats were significantly larger than that of control rats in OBR comparison. However, only the lung and kidney were still larger after 30 more days on ground. The kidney in flight rats performed pelvis expansion with down-regulation of aquaporin-2 expression confirmed by immunohistochemistry. The thymus, spleen, mesentery and pancreas were smaller in OBR. But the thymus in flight rats was heavier after 30 more days. The organs in flight rats which had no differences in OBR showed normal characteristics in histological analysis. We also found that the number of unmyelinated fibers of the aortic nerve in flight rats of 8-day group was smaller than that in control rats. In flight rats of the 14-day group, only the kidney was heavier and the ovary was lighter as compared to the controls. These results implied the second week of life was important for development during spaceflight. And the sensitivity and the critical period on neonatal development under microgravity might differ in each organ.     

Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets.

This portion of the study quantified the effects of a 12-day space shuttle mission (Space Transport System-108/UF-1) on body and lymphoid organ masses, activation marker expression, cytokine secretion, and erythrocyte and thrombocyte characteristics in C57BL/6 mice. Animals in flight (Flt group) had 10-12% lower body mass compared with ground controls housed either in animal enclosure modules or under standard vivarium conditions (P < 0.001) and the smallest thymus and spleen masses. Percentages of CD25(+) lymphocytes, CD3(+)/CD25(+) T cells, and NK1.1(+)/CD25(+) natural killer cells from Flt mice were higher compared with both controls (P < 0.05). In contrast, CD71 expression was depressed in the Flt and animal enclosure module control mice compared with vivarium control animals (P < 0.001). Secretion of interferon-gamma, IL-2, and IL-4, but not tumor necrosis factor-alpha and IL-5, by splenocytes from Flt mice was decreased relative to either one or both ground controls (P < 0.05). Flt mice also had high red blood cell and thrombocyte counts compared with both sets of controls; low red blood cell volume and distribution width, percentage of reticulocytes, and platelet volume were also noted (P < 0.05) and were consistent with dehydration. These data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become significant during long-term space missions.     

Animal housing influences the response of bone to spaceflight in juvenile rats.

The rat has been used extensively as an animal model to study the effects of spaceflight on bone metabolism. The results of these studies have been inconsistent. On some missions, bone formation at the periosteal bone surface of weight-bearing bones is impaired and on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. To determine whether animal housing can affect the response of bone to spaceflight, we studied young growing (juvenile) rats group housed in the animal enclosure module and singly housed in the research animal holding facility under otherwise identical flight conditions (Spacelab Life Science 1). Spaceflight reduced periosteal bone formation by 30% (P < 0.001) and bone mass by 7% in single-housed animals but had little or no effect on formation (-6%) or mass (-3%) in group-housed animals. Group housing reduced the response of bone to spaceflight by as much as 80%. The data suggest that housing can dramatically affect the skeletal response of juvenile rats to spaceflight. These observations explain many of the discrepancies in previous flight studies and emphasize the need to study more closely the effects of housing (physical-social interaction) on the response of bone to the weightlessness of spaceflight.     

Reduction in myotendinous junction surface area of rats subjected to 4-day spaceflight.

The surface area of myotendinous junctions (MTJs), expressed relative to the cross-sectional area of myofibrils attached to them, was determined using established morphometric techniques in which the digitlike processes of the cell at MTJs are modeled as circular paraboloids. The relative area, called the folding factor, was measured for six rats after a 4-day spaceflight and six control rats maintained in a vivarium under otherwise identical conditions. Spaceflight resulted in a significant reduction in relative MTJ surface area, from 19.7 +/- 2.3 (SD) in control animals to 13.3 +/- 2.5 for animals after spaceflight. Furthermore, space animals displayed increased numbers of fibroblasts enriched in rough endoplasmic reticulum near the MTJ, a greater number of ribosomes and mitochondria within muscle at the MTJ, and increased occurrence of lesions in the connective tissue near the MTJ. The results indicate that spaceflight, possibly through the removal of gravity-associated loading from muscle, causes a modification in MTJ structure and may result in injuries at MTJs after return to normal loading.     

Size and myonuclear domains in Rhesus soleus muscle fibers: short-term spaceflight

The cross-sectional area (CSA), myonuclear number per mm of fiber length, and myonuclear domain (cytoplasmic volume/myonucleus) of mechanically isolated single fibers from biopsies of the soleus muscle of 5 vivarium control, 3 flight simulation and 2 flight (BION 11) Rhesus monkeys (Macaca [correction of Macacca] mulatta) were determined using confocal microscopy before and after a 14-day experimental period. Simulation monkeys were confined in chairs placed in capsules identical to those used during the flight. Fibers were classified as type I, type II or hybrid (containing both types I and II) based on myosin heavy chain (MHC) gel electrophoresis. A majority of the fibers sampled contained only type I MHC, i.e. 89, 62 and 68% for the control, simulation and flight groups, respectively. Most of the remaining fibers were hybrids, i.e. 8, 36 and 32% for the same groups. There were no significant pre-post differences in the fiber type composition for any of the experimental groups. There also were no significant pre-post differences in fiber CSA, myonuclear number or myonuclear domain. There was, however, a tendency for the fibers in the post-flight biopsies to have a smaller mean CSA and myonuclear domain (approximately 10%, p=0.07) than the fibers in the pre-flight biopsy. The combined mean cytoplasmic volume/myonucleus for all muscle fiber phenotypes in the Rhesus soleus muscle was approximately 25,000 micrometers3 and there were no differences in pre-post samples for the control and simulated groups. The cytoplasmic domains tended to be lower (p=0.08) after than before flight. No phenotype differences in cytoplasmic domains were observed. These data suggest that after a relatively short period of actual spaceflight, modest fiber atrophy occurs in the soleus muscle fibers without a concomitant change in myonuclear number.     

Effects of space flight on GLUT-4 content in rat plantaris muscle

 The effects of 14 days of space flight on the glucose transporter protein (GLUT-4) were studied in the plantaris muscle of growing 9-week-old, male Sprague Dawley rats. The rats were randomly separated into five groups: pre-flight vivarium ground controls (PF-VC) sacrificed approximately 2 h after launch; flight groups sacrificed either approximately 5 h (F-R0) or 9 days (F-R9) after the return from space; and synchronous ground controls (SC-R0 and SC-R9) sacrificed at the same time as the respective flight groups. The flight groups F-R0 and F-R9 were exposed to micro-gravity for 14 days in the Spacelab module located in the cargo bay of the shuttle transport system – 58 of the manned Space Shuttle for the NASA mission named ”Spacelab Life Sciences 2”. Body weight and plantaris weight of SC-R0 and F-R0 were significantly higher than those of PF-VC. Neither body weight nor plantaris muscle weight in either group had changed 9 days after the return from space. As a result, body weight and plantaris muscle weight did not differ between the flight and synchronous control groups at any of the time points investigated. The GLUT-4 content (cpm/μg membrane protein) in the plantaris muscle did not show any significant change in response to 14 days of space flight or 9 days after return. Similarly, citrate synthase activity did not change during the course of the space flight or the recovery period. These results suggest that 14 days of space flight does not affect muscle mass or GLUT-4 content of the fast-twitch plantaris muscle in the rat. Received: 25 March 1997 / Accepted: 18 August 1997     

Preosteoblast production 55 hours after a 12.5-day spaceflight on Cosmos 1887

The influence of 12.5 days of spaceflight and a 55 h stressful recovery period (at 1 g) on fibroblastlike osteoblast precursor cells was assessed in the periodontal ligament (PDL) of rats that were 91 days old at launch. Nuclear morphometry was used as a marker for precursor cell differentiation in 3 microns sections cut in the midsagittal plane from the maxillary first molar. According to nuclear volume, cells were classified as preosteoblasts (C + D cells, greater than or equal to 120 microns 3) and less differentiated progenitor cells (A + A' cells, 40-79 microns 3). Compared with synchronous controls (simulated flight conditions), the 55 h postflight recovery period at 1 g resulted in a 40% decrease in the A + A' cell population, a 42% increase in the C + D cells, and a 39% increase in the number of PDL fibroblastlike cells near the bone surface. These results are consistent with a postflight osteogenic response in PDL. This recovery response occurred despite physiological stress in the flight animals that resulted in a highly significant (P less than or equal to 0.001) increase in adrenal weight. The data suggest that after spaceflight there is a strong and rapid recovery mechanism for osteoblast differentiation that is not suppressed by physiological stress.     

Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats

Comparisons of soleus and extensor digitorum longus (EDL) muscles from male Sprague-Dawley rats (350-400 g) after 7 days of weightlessness, 7 and 14 days of whole body suspension (WBS), and 7 days of recovery from WBS and from vivarium controls were made. Muscle mass loss of approximately 30% was observed in soleus after 7 and 14 days of WBS. Measurement of slow- and fast-twitch fibers showed significant alterations. Reductions in cross-sectional areas and increases in fiber densities in soleus after spaceflight and WBS were related to previous findings of muscle atrophy during unloading. Capillary density also showed a marked increase with unloading. Seven days of weightlessness were sufficient to effect a 20 and 15% loss in absolute muscle mass in soleus and EDL, respectively. However, the antigravity soleus was more responsive in terms of cross-sectional area reductions. After 7 days of recovery from WBS, with normal ambulatory loading, the parameters studied showed a reversal to control levels. Muscle plasticity, in terms of fiber and capillary responses, indicated differences in responses in the two types of muscles and further amplified that antigravity posture muscles are highly susceptible to unloading. Studies of recovery from spaceflight for both muscle metabolism and microvascular modifications are further justified.