Rodent Research-1 (RR-1) was the first mission in which animals were delivered and maintained on the ISS for a long duration mission in modified Animal Enclosure Module (AEM) hardware. Both RR validation and commercial science objectives were pursued during the RR-1 mission. Twenty adult female mice were launched Sept 21, 2014 in RR hardware within a Dragon Capsule (SpaceX-4) as the flight group (FLT), then after 4 days in transit, were transferred for habitation on the ISS for a total time in space of 21-22 days (10 commercial mice) or 37-38 days (10 validation mice), when animals were euthanized and select tissues recovered on orbit. Three control groups at Kennedy Space Center consisted of: 1) Basal mice from the same cohorts as FLT mice euthanized at the time of launch, 2) Vivarium (VIV) controls housed in standard vivarium cages, and 3) Ground Controls (GC) housed in RR flight hardware within an environment chamber that controlled temperature, CO2 and humidity to ISS levels. The health and behavior of all mice on the ISS were monitored by video feed on a daily basis. Mice were euthanized by injection of Euthasol, then either fast frozen intact or dissected to preserve livers (fast frozen) and spleens (RNAlater™). Samples were stored at = -80°C until their return to Earth for later analyses.
Hardware performed nominally throughout the mission and the planned in-flight science operations were completed successfully. FLT mice appeared more physically active on orbit than respective GC groups. After 37-38 days in space, mean body weights of FLT mice did not differ from GC, with both groups showing a 6% rise compared to time of launch, while VIV mice showed an 8% rise over the same period. Importantly, there were no significant differences in body weights between groups at the end of 37-38 days in microgravity, providing an indication that the RR hardware supported the health of the mice both on Earth and in space. Based on the data obtained from the livers and spleens of mice after 21-22 days in microgravity, purified RNA was of high quality (RIN values of spleen: FLT = 9.48 + 0.40, GC = 9.28 + 0.44, n=5/group). Therefore, RNA quality from samples retrieved on orbit was acceptable for even the most demanding transcriptomic analyses. In addition, liver enzyme activity levels (units/mg protein) of FLT mice and all control mice were similar in magnitude to samples that were optimally prepared by freezing in liquid nitrogen in the laboratory (enzymes analyzed included catalase, glutathione reductase and glyceraldehyde-3-phosphate dehydrogenase).
Validation analyses included behavior and tissue biochemistry as well as optimization of science return by post-flight recovery of tissues for biospecimen sharing and global expression analyses. Quantitative behavioral results and quality of image collection was based on analysis of the daily health check video record from two groups of five C57BL/6 female adult mice (Validation mice) via fixed camera views compared with identically housed Ground Controls utilizing the health surveillance video. Flight and Ground Control mice exhibited the same range of behaviors, including eating, drinking, exploratory behavior, self- and allo-grooming, and social interactions indicative of healthy animals. Mice propelled themselves freely and actively throughout the Habitat using their forelimbs to push off or by floating from one cage area to another, and they quickly learned to anchor themselves using tails and/or paws. We also observed spontaneous ambulatory behavior, including the development of organized circling or ‘Race-Tracking’ behavior that emerged within the first few days of flight. The qualitative and quantitative analysis reported herein provides unique new insights into behavioral phenotypes of space-flown mice important for guiding future research on rodents in space.
Together, these findings demonstrate new science capability for supporting long duration rodent research on the ISS to achieve both basic science and biomedical objectives.