NOTE: End date changed to 1/30/2023 per F. Hernandez/ARC (Ed., 4/25/22)

NOTE: End date changed to 1/30/2022 per F. Hernandez/ARC (Ed., 1/18/21)

Humans are homeotherms and when exposed to a cold environment defend their core body temperature, whereas mice are obligatory daily heterotherms and experience cyclic changes in core temperature when subjected to cold stress (i.e., temperature below thermoneutral). Mice are typically housed at or near room temperature (~22°C), which is well below the thermoneutral zone for the species (~32°C). Therefore, mice must expend energy to maintain core body temperature. Cold stress induced by sub-thermoneutral housing increases sympathetic outflow to peripheral tissues, including brown adipose tissue, and has profound effects on metabolism. We have recently shown that cold stress induced by room temperature housing results in rapid cancellous bone loss in mice. Based on this finding, we hypothesize that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or postural adjustments) are less effective during spaceflight and simulated spaceflight. This results in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the thermogenic mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

This proposal will explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on the skeleton in C57BL6 (B6) mice, a strain commonly used in spaceflight and HLU studies. To accomplish our goal, we propose 2 Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

We will accomplish this aim by comparing HLU-induced bone loss in mice housed at room temperature (22°C) with mice housed at thermoneutral (32°C).

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We will accomplish this aim by performing temperature response studies (20-32°C) to evaluate induction of adaptive thermogenesis. Once we identify the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis, we will perform a long-term study to verify that minimal premature bone loss occurs and that magnitude of HLU-induced bone loss does not differ from mice housed at thermoneutral.

Successful completion of the proposed research will provide guidance to investigators for insuring housing conditions minimize the confounding effects of species-specific differences in thermoregulation on experimental outcomes with the ultimate goal of optimizing the mouse to model human responses to spaceflight.

We proposed to accomplish this aim, in part, by comparing HLU-induced bone loss in male and female mice housed at room temperature (22°C) with mice housed at thermoneutral (32°). We have completed the animal studies for female mice and are well into data collection and analysis.

The findings to date demonstrate remarkable differences in response to HLU between growing female mice housed at 22°C and those housed at 32°C.

Progress on Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We proposed to accomplish this aim by performing temperature response studies (20°C to 32°C) to measure adaptive thermogenesis as a function of housing temperature (Subaim 1). Once we identified the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis and premature bone loss, we proposed to perform a long-duration study to verify that the magnitude of HLU-induced bone loss in mice housed at this temperature does not differ from mice housed at thermoneutral (Subaim 2).

Summary of Results to Date

Specific Aim 1: We have evidence that adaptation to cold stress is responsible for ~50% of bone loss in the femur of growing HLU mice. When we consider the cancellous compartment, the impact of housing temperature is even more striking; HLU prevented bone accrual at 32°C and resulted in bone loss at 22°C. In contrast, HLU prevented bone accrual in mice housed at 32°C but did not induce cancellous bone loss. Even more striking was our finding that HLU induced bone loss in humerus, a bone subjected to near normal weight bearing in the HLU model. Taken together, these findings provide evidence that adaptation to cold stress induced by room temperature housing is an important modifier of the skeletal response to HLU in mice. In addition, analyses of splenocytes suggests that increased cytokine secretion may contribute to excess bone loss induced by HLU in room temperature-housed mice.

Specific Aim 2: Room temperature housing-induced premature cancellous bone loss in growing mice is not sex specific; although male mice have higher peak bone mass, the magnitude of bone loss in room temperature housed mice did not differ between male and female mice. Housing mice at 26°C reduced but did not prevent premature cancellous bone loss in either male or female growing mice. However, the observed differences between mice housed at 22°C and 26°C indicate that even small differences in housing temperature influence bone mass in growing mice.

NOTE: End date changed to 1/30/2022 per F. Hernandez/ARC (Ed., 1/18/21)

Humans are homeotherms and when exposed to a cold environment defend their core body temperature, whereas mice are obligatory daily heterotherms and experience cyclic changes in core temperature when subjected to cold stress (i.e., temperature below thermoneutral). Mice are typically housed at or near room temperature (~22°C), which is well below the thermoneutral zone for the species (~32°C). Therefore, mice must expend energy to maintain core body temperature. Cold stress induced by sub-thermoneutral housing increases sympathetic outflow to peripheral tissues, including brown adipose tissue, and has profound effects on metabolism. We have recently shown that cold stress induced by room temperature housing results in rapid cancellous bone loss in mice. Based on this finding, we hypothesize that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or postural adjustments) are less effective during spaceflight and simulated spaceflight. This results in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the thermogenic mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

This proposal will explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on the skeleton in C57BL6 (B6) mice, a strain commonly used in spaceflight and HLU studies. To accomplish our goal, we propose 2 Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

We will accomplish this aim by comparing HLU-induced bone loss in mice housed at room temperature (22°C) with mice housed at thermoneutral (32°C).

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We will accomplish this aim by performing temperature response studies (20-32°C) to evaluate induction of adaptive thermogenesis. Once we identify the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis, we will perform a long-term study to verify that minimal premature bone loss occurs and that magnitude of HLU-induced bone loss does not differ from mice housed at thermoneutral.

Successful completion of the proposed research will provide guidance to investigators for insuring housing conditions minimize the confounding effects of species-specific differences in thermoregulation on experimental outcomes with the ultimate goal of optimizing the mouse to model human responses to spaceflight.

Mice are currently the preferred animal model for evaluating adaptive responses to spaceflight. They have several important advantages over other animal models for microgravity studies, including small size and ease of genetic manipulation. Although mice and humans share many common characteristics, thermoregulation differs markedly between the two species. We hypothesized that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into spaceflight/simulated spaceflight studies. This hypothesis was based on evidence that adaptation to room temperature housing in mice (1) results in increases in sympathetic tone, thermogenesis, glucocorticoid production, energy expenditure, blood pressure, and heart rate, and that (2) the collateral effects of these adaptive responses include rapid bone loss, immune suppression, and altered tissue and tumor response to ionizing radiation. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling and postural adjustments) are hypothesized to be less effective during spaceflight and simulated spaceflight. This would result in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of microgravity.

The studies reported here were designed to explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on premature bone loss in C57BL6 (B6) mice, a strain commonly used in spaceflight/simulated spaceflight studies.

To accomplish our goal, we proposed two Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature. Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

Progress on Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature. We proposed to accomplish this aim, in part, by comparing HLU-induced bone loss in male and female mice housed at room temperature (22°C) with mice housed at thermoneutral (32°). We have completed the animal studies and are well into data collection and analysis. The findings to date demonstrate remarkable differences in response to HLU between growing female mice housed at 22°C and those housed at 32°C.

Specific Aim 2: Room temperature housing-induced premature cancellous bone loss in growing mice is not sex specific. Although male mice have higher peak bone mass, the magnitude of bone loss in room temperature housed mice did not differ between male and female mice. Housing mice at 26°C reduced but did not prevent premature cancellous bone loss in either male or female growing mice. However, the observed differences between mice housed at 22°C and 26°C indicate that even small differences in housing temperature influence bone mass in growing mice.

To date, we have published 6 peer-reviewed manuscripts describing our results. See Cumulative Bibliography hyperlink.

Humans are homeotherms and when exposed to a cold environment defend their core body temperature, whereas mice are obligatory daily heterotherms and experience cyclic changes in core temperature when subjected to cold stress (i.e., temperature below thermoneutral). Mice are typically housed at or near room temperature (~22°C), which is well below the thermoneutral zone for the species (~32°C). Therefore, mice must expend energy to maintain core body temperature. Cold stress induced by sub-thermoneutral housing increases sympathetic outflow to peripheral tissues, including brown adipose tissue, and has profound effects on metabolism. We have recently shown that cold stress induced by room temperature housing results in rapid cancellous bone loss in mice. Based on this finding, we hypothesize that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or postural adjustments) are less effective during spaceflight and simulated spaceflight. This results in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the thermogenic mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

This proposal will explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on the skeleton in C57BL6 (B6) mice, a strain commonly used in spaceflight and HLU studies. To accomplish our goal, we propose 2 Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

We will accomplish this aim by comparing HLU-induced bone loss in mice housed at room temperature (22°C) with mice housed at thermoneutral (32°C).

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We will accomplish this aim by performing temperature response studies (20-32°C) to evaluate induction of adaptive thermogenesis. Once we identify the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis, we will perform a long-term study to verify that minimal premature bone loss occurs and that magnitude of HLU-induced bone loss does not differ from mice housed at thermoneutral.

Successful completion of the proposed research will provide guidance to investigators for insuring housing conditions minimize the confounding effects of species-specific differences in thermoregulation on experimental outcomes with the ultimate goal of optimizing the mouse to model human responses to spaceflight.

Humans are homeotherms. When exposed to cold environment humans resist decreases in interior body temperature. In contrast, mice are facultative daily heterotherms. Mice experience cyclic changes in body temperature when subjected to temperatures below thermoneutral, the temperature range where heat produced by metabolism is equal to heat loss to the environment. Mice are nocturnal and exhibit pronounced diurnal differences in activity, feeding, and sleep. Mice are typically housed at room temperature (~22°C) which is comfortable for humans but cold for mice. Room temperature housing has profound effects on energy allocation. To maintain body temperature when awake, mice housed at room temperature eat more food, increase their heart rate, are physically more active, metabolize fat reserves, shiver, huddle together, and when inactive (e.g., sleep) lower their body temperature to reduce heat loss. Not suppressing, these adaptations are regulated by stress hormones and central nervous system.

It has been known for some time that mice exhibit age related bone loss but in contrast to aging humans, much of the bone loss occurs in mice while they are still growing. Furthermore, we noticed that the bone loss in female mice is prevented if the mice are housed at 32°C, which is thermoneutral for a mouse. Based on our observation we hypothesized that the premature aging related bone loss noted in mice was a response to cold temperature stress. Based on this concept, we hypothesized that cold stress in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies performed on the International Space Station or using Earth based models for spaceflight, such as hindlimb unloading (HLU). Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or other postural adjustments) may be less effective during spaceflight and not possible during simulated spaceflight. This would results in increased dependence on increasing metabolism, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the heat generating mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

The studies reported here were designed to explore the individual and combined effects of (1) mild cold stress induced by room temperature housing and (2) HLU on premature bone loss in C57BL6 (B6) mice, a strain commonly used in spaceflight/simulated spaceflight studies. To accomplish our goal, we proposed two Specific Aims: Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature. Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

Progress on Specific Aim 1 We proposed to accomplish this aim by comparing HLU-induced bone loss in male and female mice housed at room temperature (22°C) with mice housed at thermoneutral (32°). We have completed the animal studies for female mice and are well into data collection and analysis. The findings to date demonstrate remarkable differences in response to HLU between growing female mice housed at 22°C and those housed at 32°C. Adaptation to cold stress was found to be responsible for ~50% of bone loss in the femur of growing HLU mice. When we evaluated a cancellous compartment (spongy bone located at the ends of long bones), the impact of housing temperature is even more striking; HLU prevented bone gain in growing mice at 32°C and resulted in bone loss at 22°C. In contrast, HLU prevented bone accrual in mice housed at 32°C but did not induce cancellous bone loss. Even more striking was our finding that HLU-induced bone loss in fore arm (humerus), a bone subjected to near normal weight bearing in the HLU model. Taken together, these findings provide evidence that adaptation to cold stress induced by room temperature housing is an important modifier of the skeletal response to HLU in mice. Analyses of splenocytes (immune cells in spleen) suggest that increased cytokine secretion may contribute to excess bone loss induced by HLU in room temperature-housed mice.

Progress on Specific Aim 2 As mentioned, the premature (prior to cessation of growth) cancellous bone loss in female B6 mice housed conventionally (room temperature) was prevented when the mice were housed at thermoneutral (32°C). We performed additional studies to determine whether these findings apply to males. Specifically, we compared growing mice housed at room temperature (22°C) to mice housed at 32°C. We found that room temperature housing-induced premature cancellous bone loss in growing mice is not sex specific; although male mice have higher peak bone mass, the magnitude of bone loss in room temperature-housed mice did not differ between male and female mice. The work is complete and a manuscript published.

We are in the process of performing studies to establish the lowest housing temperature able to prevent premature bone loss. To date, we have completed the animal portion for one long duration study where male and female growing mice were housed at either 22°C or 26°C until adulthood. We chose 26°C because this is the highest temperature recommended in animal care and use guidelines. We have completed collection of data related to energy expenditure, oxygen consumption, carbon dioxide emission, respiratory exchange ratio, food consumption, and distance traveled but have not completed statistical analysis. We have also analyzed bone mass, density, and microarchitecture. The data clearly indicates that housing mice at 26°C reduced but did not prevent adaptive thermogenesis or premature bone loss. Importantly, the observed differences between mice housed at 22°C or 26°C indicate that even small differences in housing temperature influence bone mass in growing mice.

Humans are homeotherms and when exposed to a cold environment defend their core body temperature, whereas mice are obligatory daily heterotherms and experience cyclic changes in core temperature when subjected to cold stress (i.e., temperature below thermoneutral). Mice are typically housed at or near room temperature (~22°C), which is well below the thermoneutral zone for the species (~32°C). Therefore, mice must expend energy to maintain core body temperature. Cold stress induced by sub-thermoneutral housing increases sympathetic outflow to peripheral tissues, including brown adipose tissue, and has profound effects on metabolism. We have recently shown that cold stress induced by room temperature housing results in rapid cancellous bone loss in mice. Based on this finding, we hypothesize that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or postural adjustments) are less effective during spaceflight and simulated spaceflight. This results in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the thermogenic mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

This proposal will explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on the skeleton in C57BL6 (B6) mice, a strain commonly used in spaceflight and HLU studies. To accomplish our goal, we propose 2 Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

We will accomplish this aim by comparing HLU-induced bone loss in mice housed at room temperature (22°C) with mice housed at thermoneutral (32°C).

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We will accomplish this aim by performing temperature response studies (20-32°C) to evaluate induction of adaptive thermogenesis. Once we identify the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis, we will perform a long-term study to verify that minimal premature bone loss occurs and that magnitude of HLU-induced bone loss does not differ from mice housed at thermoneutral.

Successful completion of the proposed research will provide guidance to investigators for insuring housing conditions minimize the confounding effects of species-specific differences in thermoregulation on experimental outcomes with the ultimate goal of optimizing the mouse to model human responses to spaceflight.

Humans are homeotherms. When exposed to cold environment humans resist decreases in interior body temperature. In contrast, mice are facultative daily heterotherms. Mice experience cyclic changes in body temperature when subjected to temperatures below thermoneutral, the temperature range where heat produced by metabolism is equal to heat loss to the environment. Mice are nocturnal and exhibit pronounced diurnal differences in activity, feeding and sleep. Mice are typically housed at room temperature (~22°C), which is comfortable for humans but cold for mice. Room temperature housing has profound effects on energy allocation. To maintain body temperature when awake, mice housed at room temperature eat more food, increase their heart rate, are physically more active, metabolize fat reserves, shiver, huddle together, and when inactive (e.g., sleep) lower their body temperature to reduce heat loss. Not suppressing, these adaptations are regulated by stress hormones and central nervous system.

It has been known for some time that mice exhibit age related bone loss but in contrast to aging humans, much of the bone loss occurs in mice while they are still growing. Furthermore, we noticed that the bone loss in female mice is prevented if the mice are housed at 32°C, which is thermoneutral for a mouse. Based on our observation we hypothesized that the premature aging related bone loss noted in mice was a response to cold temperature stress.

Based on this concept, we hypothesized that cold stress in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies performed on the International Space Station or using Earth based models for spaceflight, such as hindlimb unloading (HLU). Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or other postural adjustments) may be less effective during spaceflight and not possible during simulated spaceflight. This would results in increased dependence on increasing metabolism, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the heat generating mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

The studies reported here were designed to explore the individual and combined effects of (1) mild cold stress induced by room temperature housing and (2) HLU on premature bone loss in C57BL6 (B6) mice, a strain commonly used in spaceflight/simulated spaceflight studies. To accomplish our goal, we proposed two Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

Progress on Specific Aim 1: We proposed to accomplish this aim by comparing HLU-induced bone loss in male and female mice housed at room temperature (22°C) with mice housed at thermoneutral (32°). We have completed the animal studies for female mice and are well into data collection and analysis. The findings to date demonstrate remarkable differences in response to HLU between growing female mice housed at 22°C and those housed at 32°C. Adaptation to cold stress was found to be responsible for ~50% of bone loss in the femur of growing HLU mice. When we evaluated a cancellous compartment (spongy bone located at the ends of long bones), the impact of housing temperature is even more striking; HLU prevented bone gain in growing mice at 32°C and resulted in bone loss at 22°C. In contrast, HLU prevented bone accrual in mice housed at 32°C but did not induce cancellous bone loss. Even more striking was our finding that HLU-induced bone loss in forearm (humerus), a bone subjected to near normal weight bearing in the HLU model. Taken together, these findings provide evidence that adaptation to cold stress induced by room temperature housing is an important modifier of the skeletal response to HLU in mice.

Analyses of splenocytes (immune cells in spleen) suggests that increased cytokine secretion may contribute to excess bone loss induced by HLU in room temperature-housed mice.

Progress on Specific Aim 2: As mentioned, the premature (prior to cessation of growth) cancellous bone loss in female B6 mice housed conventionally (room temperature) was prevented when the mice were housed at thermoneutral (32°C). We performed additional studies to determine whether these findings apply to males. Specifically, we compared growing mice housed at room temperature (22°C) to mice housed at 32°C. We found that room temperature housing-induced premature cancellous bone loss in growing mice is not sex specific; although male mice have higher peak bone mass, the magnitude of bone loss in room temperature-housed mice did not differ between male and female mice. The work is complete and a manuscript published.

We are in the process of performing studies to establish the lowest housing temperature able to prevent premature bone loss. To date, we have completed the animal portion for one long duration study where male and female growing mice were housed at either 22°C or 26°C until adulthood. We chose 26°C because this is the highest temperature recommended in animal care and use guidelines. We have completed collection of data related to energy expenditure, oxygen consumption, carbon dioxide emission, respiratory exchange ratio, food consumption, and distance traveled but have not completed statistical analysis. We have also analyzed bone mass, density, and microarchitecture. The data clearly indicates that housing mice at 26°C reduced but did not prevent adaptive thermogenesis or premature bone loss. Importantly, the observed differences between mice housed at 22°C or 26°C indicate that even small differences in housing temperature influence bone mass in growing mice.

Humans are homeotherms and when exposed to a cold environment defend their core body temperature, whereas mice are obligatory daily heterotherms and experience cyclic changes in core temperature when subjected to cold stress (i.e., temperature below thermoneutral). Mice are typically housed at or near room temperature (~22°C), which is well below the thermoneutral zone for the species (~32°C). Therefore, mice must expend energy to maintain core body temperature. Cold stress induced by sub-thermoneutral housing increases sympathetic outflow to peripheral tissues, including brown adipose tissue, and has profound effects on metabolism. We have recently shown that cold stress induced by room temperature housing results in rapid cancellous bone loss in mice. Based on this finding, we hypothesize that activation of adaptive thermogenesis in mice housed at room temperature introduces unrecognized and uncontrolled confounding variables into mouse studies. Strategies used by weight-bearing mice to minimize heat loss during room temperature housing (e.g., huddling or postural adjustments) are less effective during spaceflight and simulated spaceflight. This results in increased dependence on adaptive thermogenesis, likely exaggerating the negative physiological effects of skeletal unloading on bone and immune cells. Additionally, the thermogenic mechanisms mediating cold stress-induced changes in metabolism in mice are unlikely to be directly translatable to astronauts and could therefore confound interpretation of experimental results as applicable to humans.

This proposal will explore the individual and combined effects of mild cold stress induced by room temperature housing and hindlimb unloading (HLU) on the skeleton in C57BL6 (B6) mice, a strain commonly used in spaceflight and HLU studies. To accomplish our goal, we propose 2 Specific Aims:

Specific Aim 1: Determine the contribution of increased adaptive thermogenesis to bone loss during HLU in mice housed at room temperature.

We will accomplish this aim by comparing HLU-induced bone loss in mice housed at room temperature (22°C) with mice housed at thermoneutral (32°C).

Specific Aim 2: Determine the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss.

We will accomplish this aim by performing temperature response studies (20-32°C) to evaluate induction of adaptive thermogenesis. Once we identify the lowest sub-thermoneutral housing temperature that does not induce adaptive thermogenesis, we will perform a long-term study to verify that minimal premature bone loss occurs and that magnitude of HLU-induced bone loss does not differ from mice housed at thermoneutral.

Successful completion of the proposed research will provide guidance to investigators for insuring housing conditions minimize the confounding effects of species-specific differences in thermoregulation on experimental outcomes with the ultimate goal of optimizing the mouse to model human responses to spaceflight.