In this postdoctoral fellowship, we propose to evaluate the impact of sex hormones on neuronal and musculoskeletal health in micro and Martian gravity environments by assessing naive and castrated/ovariectomized male and female rats. We hypothesize micro- and fractional gravity will result in differential aberrations to sex hormone status in female and male rats; these sex hormone aberrations will moderate neurological and musculoskeletal declines in micro- and fractional gravity environments. We will study these rats over a 4-week period and investigations will include a series of neurophysiological and functional measures, blood analyses, and a detailed series of post-mortem histological studies. With completion of this work, we will have a far deeper understanding of the relationship between hormonal status and neuronal and musculoskeletal function. This proposed work will be specifically responsive to the following subtopics in Appendix D:

• (AB1-A): Behavior and underlying neural function, including circadian effects, controlling ability of animals to sense and respond to their environment.

• (AB1-B): Studies to characterize interactions between multiple physiological systems.

• (AH1-E): Effects of fractional gravity provided by spaceflight centrifugation or ground microgravity/partial gravity analogs to gain insights into mechanisms of how animals sense, respond, and adapt to gravity shifts that are less than 1G.

In this proposal, we have male and female Fischer rats divided into different gravitational and hormonal conditions. Specifically, animals are divided into either ovariectomized/castrated or sham operated. Animals are then further sub-divided into three different gravitational loads 1g (control), 0.4g (simulating Martian gravity), or 0g (simulating micro-gravity). This design results in a total of 12 groups, including: Male-Sham-1g, Male-Sham-0.4g, Male-Sham-0g, Male-Castrated-1g, Male- Castrated-0.4g, Male-Castrated-0g, Female-Sham-1g, Female-Sham-0.4g, Female-Sham-0g, Female-Ovarectomized-1g, Female-Ovarectomized-0.4g, and Female-Ovarectomized-0g. Animals have respective surgeries and then have a 2 week recovery period. After the recovery period, animals had baseline testing of various musculoskeletal and neurological parameters. Animals were then assigned to their appropriate gravitational loads (1g, 0.4g, or 0g) and remain in those interventions for 28 days. After 28 days, animals again undergo testing for musculoskeletal and neurological parameters. Animals are then sacrificed and tissues collected for additional analysis.

Additionally, in the female animals, after surgeries we also monitor estrous cycle in female rats through daily vaginal lavages. Vaginal cells are collected using sterile water and a pipette. Then cells are placed on a microscope slide and allowed to dry. Slides are then stained in crystal violet staining solution and visualized with a microscope. We are happy to note that we have completed all animal work for the proposed study.

Upon receiving notification of selection in September 2020, we immediately started planning so the study could start as soon as funds became available. We originally planned to complete the surgeries in our own laboratory; however, we learned that Charles River could complete the surgeries before shipping animals. We opted for this option because completing the surgeries in our home laboratory would require borrowing shared surgical spaces with other researchers and this option would allow us to complete more animals in a shorter time period. In a minor change from the original proposal, animals had surgeries completed at 11 weeks of age and arrived at Beth Israel Deaconess Medical Center at 12 weeks of age (as opposed to surgeries at 12 weeks of age). After the appropriate acclimation period (48 hours), we then began estrous cycle monitoring of female rats. This two week measurement period allows for determination of baseline estrous cycle for each animal as well as confirms the ovariectomy surgery. Males still had this 2 week waiting period but were just gently handled without any additional monitoring/measurements.

Since starting data collection in January of 2021, we have thus far completed 120 animals over the 42 day protocol. We completed animal data collection in May of 2022. Since completion of animal data collection, we have been analyzing data collected for 120 animals across > 20 outcome variables. We have also completed hormone analysis in the serum of the animals. However, due to the quality of the data we have opted not to present those results.

Thus far, our data appears to suggest females tend to have greater relative muscle strength loss compared to males after exposure to simulated micro- or partial-gravity. These changes are also accompanied by greater overall muscle area loss in females compared to males. Interestingly, we note that sex differences between males and females tended to be greater during exposure to partial-gravity compared to micro-gravity. This may suggest both males and females reach a plateau in musculoskeletal losses with micro-gravity, possibly “hiding” sex difference in musculoskeletal alterations. However, with a slightly less aggressive atrophic stimuli (e.g., partial-gravity), there are gradations to muscle loss that can be detected between males and females. In essence, our results suggest aggressive stimuli such as micro-gravity elicit such dramatic muscle loss that potential sex differences may be lost. Though when there is more “wiggle room” in the degree of muscle loss with partial-gravity, sex differences between males and females become more apparent, with females tending to have greater muscle loss compared to males. It should be noted that both males and females experienced dramatic muscle loss at both micro- and partial-gravity and these sex differences were overall relatively subtle compared to the overall muscle loss detected across both males and females.

With regards to hormonal status, there were no differences between castrated males and sham males on many components of muscle size and strength at either micro- or partial-gravity. In females, there were generally no differences in relative muscle strength losses between ovariectomized and sham animals after exposure to micro- or partial-gravity. However, we did find dramatic alterations in the female rat’s estrous cycle after exposure to either simulated micro- or partial-gravity. Specifically, females spent more time in low estradiol phases of the estrous cycle (diestrus and metestrus). Moreover, these changes were detectable within one week of exposure to either micro- or partial-gravity. It is possible the clear alterations to estrous cycle in our females may have limited our ability to find differences between ovariectomized and sham females, though one of the goals of this project was to evaluate changes to the estrous cycle during exposure to micro- and partial-gravity. Overall, our data suggests either micro- or partial-gravity is capable of disrupting female estrous cycle and (presumably) estradiol secretion.

To assess cognition, we used a novel object recognition assessment. To test this, animals were exposed to two small plastic toys of similar size and shape. After a 5 minute familiarization phase, one toy was removed and replaced by a new object that was a different color and shape. Animals were given 5 minutes to investigate the objects (one familiar, one novel). All videos were assessed by at least two investigators. Overall, the simulated gravity conditions (0.4g and 0g) appeared to negatively influence animals’ cognitive function, though there were no obvious differences between either sexes or hormonal status.

Finally, we assessed skeletal health in these animals using microCT. We are still actively working to complete data analysis and manuscript submission for these data. For global bone mineral density (BMD), as expected, castrated and ovariectomized animals, tended to have lower BMD compared to sham animals. However, when we normalized the data to percent difference from 1g control animals within hormonal status (sham or castrated/ovariectomized), it appears that castrated and ovariectomized rats had greater relative loss in BMD, despite (presumably) starting a lower BMD at the initiation of interventions. These data suggest that gonadal hormones are very important for skeletal health, in particular during exposure to simulated micro- or partial-gravity environments. Rats lacking gonadal hormones not only started at a lower BMD, but also had greater relative loss in BMD during micro- and partial-gravity interventions.

In this postdoctoral fellowship, we propose to evaluate the impact of sex hormones on neuronal and musculoskeletal health in micro and Martian gravity environments by assessing naive and castrated/ovariectomized male and female rats. We hypothesize micro- and fractional gravity will result in differential aberrations to sex hormone status in female and male rats; these sex hormone aberrations will moderate neurological and musculoskeletal declines in micro- and fractional gravity environments. We will study these rats over a 4-week period and investigations will include a series of neurophysiological and functional measures, blood analyses, and a detailed series of post-mortem histological studies. With completion of this work, we will have a far deeper understanding of the relationship between hormonal status and neuronal and musculoskeletal function. This proposed work will be specifically responsive to the following subtopics in Appendix D:

• (AB1-A): Behavior and underlying neural function, including circadian effects, controlling ability of animals to sense and respond to their environment.

• (AB1-B): Studies to characterize interactions between multiple physiological systems.

• (AH1-E): Effects of fractional gravity provided by spaceflight centrifugation or ground microgravity/partial gravity analogs to gain insights into mechanisms of how animals sense, respond, and adapt to gravity shifts that are less than 1G.

Upon receiving notification of selection in September 2020, we immediately started planning so the study could start as soon as funds became available. We originally planned to complete the surgeries in our own laboratory; however, we learned that Charles River could complete the surgeries before shipping animals. We opted for this option because completing the surgeries in our home laboratory would require borrowing shared surgical spaces with other researchers and this option would allow us to complete more animals in a shorter time period. In a minor change from the original proposal, animals had surgeries completed at 11 weeks of age and arrived at Beth Israel Deaconess Medical Center at 12 weeks of age (as opposed to surgeries at 12 weeks of age). After the appropriate acclimation period (48 hours), we then began estrous cycle monitoring of female rats. This two week measurement period allows for determination of baseline estrous cycle for each animal as well as confirms the ovariectomy surgery. Males have this 2 week waiting period but are just gently handled without any additional monitoring/measurements.

Since starting data collection in January of 2021, we have thus far completed 120 animals over the 42 day protocol. We completed animal data collection in May of 2022. Since completion of animal data collection, we have been analyzing data collected for 120 animals across > 20 outcome variables. We have also completed hormone analysis in the serum of the animals.

With the preliminary data generated from this grant, we have submitted 5 abstracts for virtual and in-person scientific meetings. Abstracts submitted to various conferences have resulted in multiple scientific awards. Additionally, the Post-Doc Principal Investigator on the grant (Dr. Rosa-Caldwell) was invited to give a talk at the 3rd International Conference on Musculoskeletal and Neural Interactions in Atlanta, GA in August. Moreover, Dr. Rosa-Caldwell was also invited to give a symposium talk at the Canadian Society for Exercise Physiology Annual Meeting in Fredericton, New Brunswick, Canada.

Additionally, a Harvard Medical School/Massachusetts Institute of Technology M.D./Ph.D. student, Anna Wadhwa, completed an 8-week rotation in the laboratory during this project. Ms. Wadhwa assisted the data analysis of muscle and fat changes during the partial/micro-gravity interventions. Afterwards, she wrote and submitted an abstract to the New England Chapter of the American College of Sports Medicine. Her abstract was selected as a finalist for the Ph.D. student research award and Ms. Wadhwa won this award.

Overall, we have made significant progress in the completion of this project and believe, given the current trajectory, will successfully complete this project within our designated timeframe. We have begun writing scientific manuscripts with the data generated from this project. We anticipate the first publications from this project to be submitted by the end of 2022.

In this postdoctoral fellowship, we propose to evaluate the impact of sex hormones on neuronal and musculoskeletal health in micro and Martian gravity environments by assessing naive and castrated/ovariectomized male and female rats. We hypothesize micro- and fractional gravity will result in differential aberrations to sex hormone status in female and male rats; these sex hormone aberrations will moderate neurological and musculoskeletal declines in micro- and fractional gravity environments. We will study these rats over a 4-week period and investigations will include a series of neurophysiological and functional measures, blood analyses, and a detailed series of post-mortem histological studies. With completion of this work, we will have a far deeper understanding of the relationship between hormonal status and neuronal and musculoskeletal function. This proposed work will be specifically responsive to the following subtopics in Appendix D:

• (AB1-A): Behavior and underlying neural function, including circadian effects, controlling ability of animals to sense and respond to their environment.

• (AB1-B): Studies to characterize interactions between multiple physiological systems.

• (AH1-E): Effects of fractional gravity provided by spaceflight centrifugation or ground microgravity/partial gravity analogs to gain insights into mechanisms of how animals sense, respond, and adapt to gravity shifts that are less than 1G.

A.) Establish the impact of sex hormones on neurological and musculoskeletal systems in these gravitational analogues; B.) Identify potential sex differences in response to Martian fractional gravity; C.) Determine the influence of micro- and partial- gravity environments on circulating hormonal status in males and females.

After ~ 1 year of working on this project, we are happy to report substantial progress towards successfully completing this project.

In this proposal, we have male and female Fischer rats divided into different gravitational and hormonal conditions. Specifically, animals are divided into either ovariectomized/castrated or sham operated. Animals are then further sub-divided into three different gravitational loads 1g (control), 0.4g (simulating Martian gravity), or 0g (simulating micro-gravity). This design results in a total of 12 groups, including:

Male-Sham-1g, Male-Sham-0.4g, Male-Sham-0g, Male-Castrated-1g, Male- Castrated-0.4g, Male-Castrated-0g, Female-Sham-1g, Female-Sham-0.4g, Female-Sham-0g, Female-Ovarectomized-1g, Female-Ovarectomized-0.4g, and Female-Ovarectomized-0g.

Animals would have respective surgeries and then have a 2-week recovery period. After the recovery period, animals have baseline testing of various musculoskeletal and neurological parameters. Animals are then assigned to their appropriate gravitational loads (1g, 0.4g, or 0g) and remain in those interventions for 28 days. After 28 days, animals again undergo testing for musculoskeletal and neurological parameters. Animals are then euthanized and tissues collected for additional analysis.

Additionally, in the female animals, after surgeries we also monitor estrous cycle in female rats through daily vaginal lavages. Vaginal cells are collected using sterile water and a pipette. Then cells are placed on a microscope slide and allowed to dry. Slides are then stained in crystal violet staining solution and visualized with a microscope. Phase of estrous cycle is determined based on the relative amount of different cell types.

Upon receiving notification of selection in September, we immediately started planning so the study could start as soon as funds became available. We originally planned to complete the surgeries in our own laboratory; however, we learned that Charles River Laboratories (Charles River) could complete the surgeries before shipping animals. We opted for this option because completing the surgeries in our home laboratory would require borrowing surgical spaces shared with other researchers, and this option would allow us to complete more animals in a shorter time period. In a minor change from the original proposal, animals had surgeries completed at 11 weeks of age and arrived at Beth Israel Deaconess Medical Center at 12 weeks of age (as opposed to surgeries at 12 weeks of age). After the appropriate acclimation period (48 hours), we then began estrous cycle monitoring of female rats. This 2-week measurement period allowed for determination of baseline estrous cycle for each animal, as well as confirmation of the ovariectomy surgery. Males still had this 2-week waiting period, but were just gently handled without any additional monitoring/measurements.

Since starting data collection in January of 2021, we have thus far completed 86 through the entire protocol and will have 92 animals completed by the end of 2021. Given this current trajectory, we anticipate all animal studies will be completed no later than July 2022.

Thus far, our preliminary data appears to suggest that micro- and partial gravity may influence cognitive function as measured by Novel Object Recognition, though at this point it is too early to deduce if there are any sex or hormone related differences in these outcomes. Additionally, our preliminary data seems to imply females may have exacerbated losses in electrically stimulated muscle strength compared to males; however, as we still have ~1/2 of female rats left to complete, this finding is very preliminary. We look forward to further disseminating our final conclusions when all animals have completed the protocol and we can holistically examine all the data.

With the preliminary data generated from this grant, we have submitted 3 abstracts for virtual and in-person scientific meetings. Additionally, a symposium proposal on space physiology was accepted for the Northeastern Chapter of the American College of Sports Medicine regional meeting in October 2021, which includes data from this project. We anticipate once all animal studies are collected, scientific manuscripts will be submitted in 2022.

Additionally, during the primary animal studies associated with this grant, we also added analysis of estrous cycle in our laboratory’s other currently funded NASA project (80NSSC19K9518). Through the monitoring of estrous cycle in those hindlimb unloaded and partially recovered female rats, we found estrous cycle strongly correlated with muscle size and strength in hindlimb unloaded female rats. Moreover, the degree of estrous cycle alterations corresponded to the degree of muscle recovery after cessation of hindlimb unloading. These findings have since been published in the scientific journal Experimental Physiology. Since being published in September, this article already has an Almetric score of 7, placing it in the top 25% of all research outputs scored by Altmetric.

Overall, we have made significant progress in the completion of this project and believe, given the current trajectory, will successfully complete this project within our designated timeframe.

In this postdoctoral fellowship, we propose to evaluate the impact of sex hormones on neuronal and musculoskeletal health in micro and Martian gravity environments by assessing naive and castrated/ovariectomized male and female rats. We hypothesize micro- and fractional gravity will result in differential aberrations to sex hormone status in female and male rats; these sex hormone aberrations will moderate neurological and musculoskeletal declines in micro- and fractional gravity environments. We will study these rats over a 4-week period and investigations will include a series of neurophysiological and functional measures, blood analyses, and a detailed series of post-mortem histological studies. With completion of this work, we will have a far deeper understanding of the relationship between hormonal status and neuronal and musculoskeletal function. This proposed work will be specifically responsive to the following subtopics in Appendix D:

• (AB1-A): Behavior and underlying neural function, including circadian effects, controlling ability of animals to sense and respond to their environment.

• (AB1-B): Studies to characterize interactions between multiple physiological systems.

• (AH1-E): Effects of fractional gravity provided by spaceflight centrifugation or ground microgravity/partial gravity analogs to gain insights into mechanisms of how animals sense, respond, and adapt to gravity shifts that are less than 1G.