INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SPACEFLIGHT DECREASES BONE formation, increases bone resorption, and induces a progressive loss of bone mineral in adult humans (4, 7, 16, 20, 22, 25). Total body calcium losses can reach 300 mg/day (18, 19). Bones that receive the greatest gravitational loading (calcaneous, tibia, femur, vertebra) demonstrate the greatest losses, whereas non-weight-bearing bones (radius, ulna) tend to lose little or no mineral (7, 17). Mineral losses in the spine, femoral neck, trochanter, and pelvis on long flights average 1.0-1.6%/mo (13, 17). Despite the health implications for crew members on long-duration missions, progress has been slow in defining the mechanisms responsible for the changes in bone metabolism induced by spaceflight.

To examine the effects of spaceflight on skeletal structure and metabolism, the rat has been used extensively as a model system (2, 5, 6, 8, 11, 12, 14, 15, 21, 23, 24, 26-40). The effects of spaceflight on the rat skeleton, however, have not been consistent. In the Soviet Union's Cosmos 782, 936, 1129, and 1887 missions, Spacelab Life Sciences (SLS) 3 (SLS3), and Physiological Systems Experiment (PSE) 03 (PSE 03), spaceflight reduced cortical bone formation by 27-44% (P < 0.05) (14, 15, 23, 27, 29, 35, 38, 39) and diminished bone strength (21, 40). In the Soviet Cosmos 1129 and 1667 missions, significant losses in cancellous bone were also documented (11, 12, 33, 34), and in PSE 01 and 02 (2) and SLS2 (8), gene expression for bone matrix proteins was reduced. However, in Physiological and Anatomical Rodent Experiment 3, PSE 01, and the National Institutes of Health Rodent Experiment (NIH-R), significant decrements in cortical bone formation were not detected (26, 36; unpublished observations). In PSE 01 the flight duration was short (4 days), reducing the chance of observing a significant reduction in formation, but in NIH-R the flight duration was 18 days. A decrease in cancellous bone formation was observed in PSE 02, but loss of cancellous bone volume could not be detected in PSE 01, PSE 02, Cosmos 2044, or NIH-R (28, 32, 36). Furthermore, mechanical properties of the humerus were found to be unaltered in Cosmos 2044 (31). Thus, on some missions, bone formation is impaired, whereas on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. Previous spaceflight studies differed in housing conditions (singly or in groups), age, and genetic strain of the animals used, skeletal sites studied, flight duration, and time after the flight before the animals were euthanized. Although comparison of studies is difficult, review of 15 previous spaceflight missions suggests that animals housed singly were more prone to impaired bone formation during flight than animals housed in groups.

To determine directly whether animal housing can affect the response of bone to spaceflight, rats were group housed in the animal enclosure module (AEM) or singly housed in the research animal holding facility (RAHF) on the same flight. The results indicate that group housing can protect bone from the detrimental effects of spaceflight in juvenile rats.

	MATERIALS AND METHODS

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Animal protocols. The studies were conducted aboard National Aeronautics and Space Administration (NASA) shuttle Columbia (STS 40) as part of the 9-day SLS1. Specific-pathogen-free, male Sprague-Dawley-derived rats (Taconic Farms, Germantown, NY) were used. Animals were 58 days old and weighed on average 285 g at launch. Beginning 13 days before launch (L  13) and throughout the experiment, animals were fed Teklad NASA Experimental Diet TD88179 (0.5% Ca, 0.414% P, 108 IU vitamin D/100 g) extruded into food bars. The food bars were dipped in 15% sorbate to retard mold growth, radiation sterilized, sealed in polyethylene bags, and stored at 4°C. Animals were provided food and water ad libitum and subjected to a 12:12-h light-dark cycle (9 AM to 9 PM EST).

View larger version (122K): [in this window] [in a new window]   Fig. 1.   Animal housing units. Left: animal enclosure module (group housing); right: research animal holding facility (single housing).

Bone histomorphometry. Calcein and demeclocycline (15 mg/kg) were given subcutaneously to all animals 13 and 2 days, respectively, before launch. The distance between these fluorochrome labels provided an assessment of cortical bone formation rate before flight. On the day of landing, one-half of the animals in each of groups 2-5 were given a second calcein injection. The distance between the demeclocycline and the second calcein label provided an assessment of cortical bone formation rate during the flight. The distance between the second calcein injection and the edge of the bone provided an assessment of cortical formation rate after the flight.

Postflight studies. A ground RAHF was not available during the flight period to permit inclusion of an RAHF-housed ground control. To determine whether housing in the RAHF unit on the ground (RAHF ground control) would affect bone metabolism, a delayed flight profile test (DFPT) was conducted. Animals were randomly divided into two groups: 1) vivarium control and 2) RAHF. The experimental protocol was identical to the flight study, except all animals remained on the ground during the "flight period." Bones and tissues were harvested and processed as described above.

Statistics. Values are means ± SD. Statistical analyses were performed using two-way ANOVA and the Newman-Keuls test or repeated-measures ANOVA where appropriate (Sigma-Stat, Jandel Scientific, San Rafael, CA).

	RESULTS

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Body weight during and after spaceflight in group- and single-housed animals is shown in Table 2. Weight gain was comparable during the flight period among the group-housed control and flight animals and among the single-housed control and flight animals. During the recovery period, however, weight gain in the flight animals was reduced (P < 0.05, 2-way ANOVA).

Bone formation rate on the periosteal surface before the flight in all animals taken collectively averaged 0.063 ± 0.003 mm²/day and did not vary significantly among the groups (Table 3). During and after the flight the average formation rate in all groups combined decreased to 0.049 ± 0.007 and 0.039 ± 0.003 mm²/day (P < 0.05 for both), respectively, as the growth rate of the animals gradually diminished. Taken as a combined group (i.e., group- and single-housed rats), bone formation in the flight animals during the mission was lower than in the ground control animals (P < 0.001). In the group-housed animals alone, however, the difference (6%) did not reach significance. In the single-housed animals alone, the difference (30%) was highly significant (P < 0.001; Fig. 2). During the postflight period, there was no difference in periosteal bone formation rates between the control and flight animals in the group- or single-housed rats.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Periosteal bone formation rate at tibiofibular junction before, during, and after flight. Values are means ± SD. 1 P < 0.001, single-housed flight vs. single-housed control (2-way ANOVA, Student-Newman-Keuls method).

Repeated-measures ANOVA was also used to analyze the changes in bone formation rate between the preflight and flight periods. During the flight period no significant differences in bone formation rate were detected in the group-housed rats, but a significant impairment of formation was observed in the single-housed rats. Expression of the change in bone formation rate from preflight to flight as a percentage of the preflight value, however, showed a significant blunting of formation in group-housed (28 ± 8%) and single-housed (37 ± 7%) animals, with the magnitude of the decrement being larger in the single-housed rats.

Bone formation rates on the endocortical surface of the tibial midshaft before flight in all animals taken collectively averaged 0.057 ± 0.002 mm²/day and did not differ significantly among the groups (Table 4). During and after flight the average formation rate in all groups combined decreased to 0.031 ± 0.002 and 0.027 ± 0.002 mm²/day, respectively. No differences in endosteal formation were found at any time between the control and flight animals in the group- or single-housed rats.

Femur dry weight was lower in flight than in ground control animals at landing (P < 0.05; Table 5). In the group- and single-housed animals after flight, deficits in bone mass of 3 and 6.8%, respectively, were found. Although not significant, there was a trend for the mass deficit to be greater (2.3-fold) in single-housed animals. After flight the differences in bone mass between the control and flight animals were minimal.

Adrenal weight was slightly higher (+14%) in the flight single-housed than in the control single-housed animals (Table 6). This was not the case for the group-housed animals. Thymus weight tended to be lower (17%) in the single-housed flight animals, but this difference did not reach significance. No difference in thymus weight was observed in the group-housed rats.

In the DFPT, no significant differences in body weight, bone formation rate (Table 3), bone fat-free weight, adrenal weight, or thymus weight (data not shown) were observed between the vivarium control and the RAHF housed animals.

	DISCUSSION

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The rat has been used extensively as an animal model to study human bone metabolism and disease (9). Despite its usefulness, however, the results of rat skeletal studies during spaceflight have been inconsistent. In an attempt to resolve these inconsistencies, we took advantage of a unique opportunity provided us by the NASA SLS1 mission. During SLS1, rats were housed in groups or individually. Group-housed rats were afforded social and physical interaction, whereas single-housed rats were isolated.

Body weight gain was not different among any of the groups during the flight. This suggests that neither spaceflight nor the modest confinement of both housing units sufficiently perturbed the animals to interrupt normal growth. It also suggests that any observed changes in bone metabolism cannot be attributed to growth impairment. During the postflight period, body weight gain was reduced by one-half or more in flight animals. The reason for this is not clear but may be related to changes in activity level or readaptation to normal gravitational loading.

Cortical bone formation on the periosteal surface in the single-housed animals was reduced by 30% during the flight. Animals housed in a group demonstrated only a small, insignificant reduction (6%) in periosteal bone formation rate during the flight. When the data were analyzed with each animal as its own control, the group-housed animals showed a small but significant impairment of bone formation (15%) during the flight. With this analytic approach, single-housed animals showed a 52% higher inhibition of bone formation than group-housed animals. These data clearly demonstrate that housing can influence the response of bone to spaceflight. They indicate that formation is likely to be impaired in group- and single-housed rats, but the degree of impairment is much greater if animals are housed alone. The data also help resolve many of the discrepancies among earlier missions. In all previous flights where animals were housed alone, bone formation was reduced (14, 15, 23, 27, 29, 38, 39). In some (26, 36) but not all (5, 35) previous flights in which animals were housed in groups, bone formation was normal or nearly normal.

Endocortical bone formation at the middiaphysis of the tibia was not significantly affected by spaceflight in group- or single-housed animals. These data emphasize that the response of bone to spaceflight is region specific. Bones or areas of bone that receive the greatest gravitational loading are likely to respond to spaceflight, whereas non-weight-bearing bones or regions of bone that experience little or no mechanical strain are likely to be unresponsive (7, 17).

The level of impaired periosteal bone formation in the group- and single-housed animals during flight should manifest itself as a difference in bone mass. Although 9 days is a relatively short period of time to influence bone mass, our data indicate a strong trend for femur mass to be lower in single-housed (7%) than in group-housed (3%) animals.

Although group-housed animals do not exhibit the same level of inhibition of bone formation as single-housed animals during flight, group housing may not completely prevent the inhibition of bone formation induced by spaceflight and is likely one of multiple factors that can influence the response of bone to weightlessness. We studied young growing juvenile rats. In these animals, group housing reduced the bone response to spaceflight. Existing data from previous missions, however, suggest that group housing may have little or no beneficial effect on protection of older rats from the detrimental effects of spaceflight on bone (5, 35). Genetic background or animal strain may also influence bone responsiveness to flight (5, 35).

It is not clear why group housing of young growing rats can blunt the response of bone to weightlessness. Group housing affords physical interaction between animals, which may be sufficient to physically load the skeleton and prevent the decrease in bone formation in young animals. Alternatively, group housing also provides social interaction between animals, and companionship may reduce stress. That adrenal gland weight was marginally greater in single-housed flight than in single-housed vivarium control animals suggests that stress may be increased in single-housed animals. This notion is supported by the trend for lower thymus weight in spaceflight rats. RAHF-housed flight animals, however, were euthanized several hours after the AEM flight animals. It is possible, therefore, that the stress of reentry may have had a greater effect on adrenal gland weight in RAHF than in AEM animals. Others factors that may have influenced the responsiveness of bone to spaceflight in the single- but not the group-housed animals during SLS1 include the physical makeup of the interior of the RAHF and AEM cages and the air temperature during the flight. A grid of wire mesh covers the floor and side walls of the AEM, thus permitting AEM animals a foothold during weightlessness. This was not available in the RAHF cages. Inflight, the AEM was kept between 27 and 32°C, while the RAHF was maintained at 25 ± 2°C. Although this temperature difference is small, it may have contributed to the difference in bone responsiveness between the group- and the single-housed animals (6).

Interestingly, when the effect of RAHF housing alone (DFPT study) was examined, no effect on adrenal, thymus, or bone could be detected. It appears that the isolation and modest confinement induced by the RAHF alone are not sufficient to perturb bone metabolism but, when combined with spaceflight, produce a consistent and substantial reduction in bone formation.

In summary, our data show that housing can have a dramatic effect on the response of bone to spaceflight. This fact explains many of the discrepancies in previous flight studies dealing with the rat skeleton and needs to be taken into account in planning future flight experiments. Additionally, the effects of age and genetic background are likely to be factors in determining the response of bone to spaceflight.