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.

Immune response: For this, we evaluated immune cell subtypes in the spleen and carried out ex vivo stimulation of splenocytes with media control, Lipopolysaccharide (LPS), concanavalin A (ConA), and anti- CD3/CD28 to induce cell proliferation and cytokine production. Production of Th1/Th2/Th17 cytokines were measured (IL10, IL17, Tumor Necrosis Factor alpha (TNF alpha), Interferon-gamma (IFNG), IL 6, IL 4, and IL 2).

We detected IL 2, IFNG, and IL6. For each of these cytokines, cold stress induced by room temperature housing exaggerated the immune cell response to a T- cell mitogen following HLU. These data will be included in a future publication.

Bone mass and microarchitecture: The femur is an important weight bearing bone that is unloaded during HLU. We evaluated the effects of HLU on total femur and cancellous bone volume fraction in the distal femur metaphysis in growing female mice housed at 22°C and mice housed at 32°C. Compared to baseline, HLU resulted in lower total femur bone volume in mice housed at either temperature, but the reduction was ~2 fold greater (22 % versus 10 %) in mice housed at 22°C. The influence of housing temperature on cancellous bone response to HLU was even more profound. HLU resulted in relative osteopenia at the distal femur metaphysis in mice housed at 32°C by preventing bone accrual. This finding is in marked contrast to mice housed at 22°C, where the weight bearing controls failed to accrue additional bone and HLU mice lost cancellous bone.

The humerus is a weight bearing bone in the mouse. The femur is unloaded during HLU but the humerus should be subjected to near normal loading. Compared to baseline, HLU had minimal effect on total humerus bone volume in female mice housed at 32°C (-3% change), but resulted in bone loss in mice housed at 22°C (-11% change). These findings support the concept that factors in addition to reduced weight bearing contribute to bone loss in HLU mice housed at 22°C. Studies evaluating the impact of room temperature induced cold stress on skeletal response to HLU on growing mice are published. Similar studies in skeletally mature mice housed at room temperature and thermoneutral have been completed and we are now in the process of manuscript preparation.

We performed studies to further define the time course for bone loss in mice housed at room temperature. Importantly, transferring mice to thermoneutral prevents additional bone loss but does not restore bone that had been lost during room temperature housing. Consequently, to prevent cold temperature induced bone changes, it is important to begin housing mice at thermoneutral as early as possible.

We initiated studies to further define the mechanisms mediating premature bone loss induced by room temperature housing. Our initial focus was on the role of the sympathetic nervous system. This is because sympathetic outflow is increased at sub-thermoneutral temperatures. In the study summarized below, we evaluated the effect of the ß-blocker propranolol on the skeleton in female B6 mice housed at room temperature (22°C) and thermoneutral (32°C). The work is complete, and a manuscript published. 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 (Sub-Aim 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 (Sub-Aim 2). We previously reported that 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. The work is complete, and a manuscript published.

We performed a serious of studies to establish the lowest sub-thermoneutral housing temperature able to prevent adaptive thermogenesis-associated bone loss., including a 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, and are preparing a publication. Additionally, we analyzed bone mass, density, and microarchitecture. The data clearly indicate that housing mice at 26°C modestly reduced but did not prevent adaptive thermogenesis or premature bone loss. Compared to either baseline or mice housed at 32°C, housing male mice at 26°C marginally reduced premature cancellous bone loss. Similarly, bone loss was reduced but not prevented in female mice housed at 26°C. However, it is important to note that a 4°C difference in housing temperature was sufficient to significantly alter cancellous bone volume fraction in growing male and female B6 mice. In addition, we have performed analysis of brown and white adipose tissue of mice flown and sacrificed aboard the International Space Station (ISS). The results support our hypothesis that non-shivering thermogenesis is increased aboard the ISS. A manuscript describing this research is published.

COVID-19 delayed our progress on the funded proposal. However, it provided us an opportunity to evaluate archival tissue and unpublished data to explore generalizability of published results and test a novel hypothesis. In the first case, we evaluated the effects of spaceflight on the femoral head using tissue from two (2) space shuttle experiments. This manuscript is now published. In the second case, we used unpublished data from numerous spaceflight and ground-based studies to test the gravitostat hypothesis for weight control. This manuscript is now published. In the third case, Nrf2 signaling has been implicated in mediating some of the detrimental effects of microgravity on the musculoskeletal system. We therefore participated in a collaborative study evaluating bone from mice in which Nrf2 was deleted. This manuscript is now published.

Summary (Interpretation) of Results

Specific Aim 1: We have strong 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 suggest that increased cytokine secretion may contribute to excess bone loss induced by HLU in growing room temperature housed mice. We have extended these studies to skeletally mature mice, which show similarly dramatic housing temperature associated differences in response to HLU. We are in the process of preparing these data for publication.

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 1/30/2023 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.