NOTE: End date changed to 06/01/2025 per S. Mack-Phillips/JSC (Ed., 2/19/25)

NOTE: End date changed to 06/01/2024 per NSSC information (Ed., 11/25/22)

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 2/8/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 4/7/21)

NOTE: End date changed to 12/15/2020 per NSSC information (Ed., 7/24/20)20)

Gait analyses: Mice exposed to 0G failed to perform post-flight, were unable to locomote and frequently rolled onto their backs: µg was excluded from quantitative analysis. Moreover, 50% of mice from the 0.33G group had similar performance failures, limiting testable group size (n=3). All other mice (0.67G and 1G) performed post-flight. In general, changes in gait parameters were altered in a gravity-dependent manner, with post-flight deficits in the 0.33G group greatest vs VIV, GC, and 1g; 0.67g attenuated the deleterious changes vs 0.33g.

Neuromuscular analyses: Front paw grip strength increased in HGC mice (+22% vs pre-flight) and decreased in all AG groups (up to - 35%, p=0.0032). All groups experienced an increase in rear paw grip strength that was directly related to the level of AG, with 0G, 1G, and HGC mice experiencing a 14.9±18.3%, 37.7±14.3%, and 47.7+ 12.0% increase versus pre-flight, respectively. Gastrocnemius and soleus wet mass were related to the level of AG (p<0.0001 and p=0.0002, respectively) with HGC animals having the largest muscles and the 0G mice having the smallest.

Bone analyses: We found that femoral bone mineral density (BMD) was lower in 0G mice (-12.3%, p<0.0001) than HGC. AG partially mitigated the effects of microgravity as 0.33G, 0.66G, and 1G mice all had significantly greater femoral BMD than 0G mice. At the distal femur and proximal tibia, 0G mice had ~30% less trabecular bone, with thinner and fewer trabeculae than HGC (p<0.05 for all). Mice exposed to AG at the 1G level had significantly greater trabecular bone volume at the distal femur (p=0.03) and proximal tibia (p=0.007) than 0G. 1G mice also had a greater number of trabeculae than 0G mice. The 1G and HGC groups had similar trabecular bone outcomes. However, AG did not completely prevent the loss of bone in the trabecular compartment, as the mice exposed to 0.67G had less trabecular bone, with thinner and fewer trabeculae than HGC mice. Vertebral strength was lower in 0G than HGC mice (-15%, p=0.001) while AG had mixed effects on estimated vertebral strength with 1G mice similar to HGC and 0.67G lower than HGC (p=0.0025). These results confirm the deleterious effects of microgravity on the murine skeleton. Artificial gravity induced by centrifugal force mitigated these deleterious effects, though there was no clear dose-dependent response.

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 2/8/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 4/7/21)

NOTE: End date changed to 12/15/2020 per NSSC information (Ed., 7/24/20)20)

To address this knowledge gap, an interdisciplinary project team of investigators, from the US and from Japan, designed an integrated protocol to assess the effects of varied levels of artificial gravity on various physiologic systems. Our team focused on the musculoskeletal system. Adult (12 weeks) male C57Bl/6J mice were exposed to either microgravity alone (0G), or to 0.33G, 0.67G, or 1G via centrifugation using the Japan Aerospace Exploration Agency (JAXA) Multiple Artificial-gravity Research System (MARS) during the Joint Partial-Gravity Rodent Research (JPG-RR)/Mouse Habitat Unit 8 (MHU-8) 30-day mission on the International Space Station (n=5-6/group). Ground controls were housed in an identical environment (HGC, n=12). The launch occurred in March 2023, with a successful live animal return in April 2023. Prior to launch and shortly after live animal return, we assessed gait performance, grip strength, bone mass and body composition. Bone and muscle tissues were harvested at necropsy for morphologic and transcriptomic analyses (via bulk RNASeq). In addition, we collected blood cell-depleted femoral bone marrow (n=5-6/group) postflight in the 0G and 1G groups for single-cell RNA sequencing using the Highly Integrated Virtual Environment (HIVE) platform (Honeycomb). Preliminary results indicate that artificial gravity was able to fully- or partially-mitigate deleterious effects of microgravity on the musculoskeletal system, including gait performance, bone mass and microstructure, and muscle morphology. Further, for the first time, we successfully collected spaceflight-flown specimens for analysis by single-cell RNA sequencing (RNASeq).

NOTE: End date changed to 12/31/2022 per NSSC information (Ed., 2/8/22)

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 4/7/21)

NOTE: End date changed to 12/15/2020 per NSSC information (Ed., 7/24/20)20)

We have worked extensively with the project team to revise the dissection schedule to better accommodate our scientific needs. Our team attended Radiation Safety Training sponsored by Kennedy Space Center (KSC) and completed paperwork to gain approval to be able to use an X-ray generating device at KSC.

Ground-based Tests: DXA device testing (completed in 2022) We compared the accuracy and precision of a new peripheral dual-energy X-ray absorptiometry (DXA) device for rodents, the InAlyzer, to that of an established device, the PIXImus. We measured bone mass and body composition of 18 male C57Bl/6J mice (6 each of ages 8, 14, and 24 weeks) on the two DXA devices. We compared the body composition measurements to those from whole-body magnetic resonance imaging (MRI), taken to be the gold standard. We found that the accuracy and precision of InAlyzer measurements were comparable to those from the PIXImus. Short-term precision was excellent for both devices, ranging between 0.39 and 2.4%. Given the safety and efficiency advantages of the InAlyzer device, we recommend that it be used for pre- and post-flight measurements in the MHU-8 mission. Further details are provided in the Technical Report submitted to the Project Team.

Ground-based Tests: Fluorochrome labels (completed in 2021) We previously reported the results of the ground-based test designed to determine if the fluorophores would be stable and taken up in the bone if prepared and frozen for 5 weeks and then injected into the animals. We used adult C57/Bl6J mice to test different combinations of fluorophores (Calcein green, Calcein Blue, Alizarin red, and Xylenol orange). In addition, we collected fecal samples and soleus muscle tissue for MHU-8 co-principal investigators M. Vitaterna and S. Takahashi, respectively, to determine whether the fluorochrome injections impacted their proposed outcomes. We found that calcein green, followed by alizarin red, demonstrated strong and clear labeling with both the fresh and frozen fluorophores preparations. There were no differences between the mice injected with PBS, or the fluorochromes, with both showing weight gain over time. In addition, Dr. Satoro Takahashi reported that there were no differences in gene expression in the soleus muscle when comparing mice injected with fluorochromes vs. those injected with PBS and Dr. Hotz-Vitaterna reported no impact of fluorochrome injections on the microbiome. We conclude that preparing the fluorophores and syringes ~5 weeks ahead and freezing at -80°C until the injections on orbit will be acceptable for dynamic histomorphometry measurements and do not interfere with other scientific objectives proposed in this study. We plan to use calcein green and alizarin red as these give the best images.

NOTE: End date changed to 12/31/2021 per NSSC information (Ed., 4/7/21)

NOTE: End date changed to 12/15/2020 per NSSC information (Ed., 7/24/20)20)

Project Accomplishments

Study Protocol & Administrative Tasks. We have worked with the multi-PI team to create a harmonized protocol that is able to complete the scientific aims of each individual PI. The PI team has met monthly. We reviewed, edited, and approved the Science Requirements Document. We attended a kick-off meeting with the service provider, Leidos.

Ground-based Test of Fluorochrome Labels. The primary aim of this technical experiment was to determine if the fluorophores would be stable and taken up in the bone if prepared and frozen for 5 weeks and then injected into the animals. We also tested different combinations of fluorophores (Calcein green, Calcein Blue, Alizarin red, and Xylenol orange). In addition, we collected fecal samples and soleus muscle tissue for Mouse Habitat Unit (MHU) MHU-8 co-principal investigators M. Vitaterna and S. Takahashi, respectively, to determine whether the fluorochrome injections impacted their proposed outcomes.

Reference: Shiba D, Mizuno H, Yumoto A, Shimomura M, Kobayashi H, Morita H, Shimbo M, Hamada M, Kudo T, Shinohara M, Asahara H, Shirakawa M, Takahashi S. Development of new experimental platform 'MARS'-Multiple Artificial-gravity Research System-to elucidate the impacts of micro/partial gravity on mice. Sci Rep. 2017 Sep 7;7(1):10837.

NOTE: End date changed to 12/15/2020 per NSSC information (Ed., 7/24/20)20)

The current study design includes four experimental groups of adult male C57BL/6J mice (12 weeks at launch): flight (FL, n=24), habitat ground control (HGC, n=12), vivarium ground control (VGC, n=12), and baseline ground control (n=12). Flight mice will be exposed to one of four gravity conditions (n=6 / group) between 0 and 1G while on a 30-day mission to the International Space Station. Mice will be implanted with an intraperitoneal datalogger pre-flight to measure body temperature for circadian rhythm analyses (Principal Investigator: Fuller). Additional pre- and post-flight in vivo testing includes microbiome analyses, gait analysis, neuromotor testing (e.g., balance beam walk), bone mineral density, and lower extremity bone microstructure by microcomputed tomography. Post-flight ex vivo analyses will include histology and transcriptomic analyses of multiple tissues. Altogether this comprehensive evaluation will provide new insights on the gravity dose vs physiologic response relationship for multiple tissues and biologic systems.

Additional work over the past year includes reviewing and editing of the Science Requirements Document (SRD), which outlines the detailed requirements related to pre- and post-flight animal care and data collection.