The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c, 12-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age, and matching sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight, ground controls and basal groups had blood collected, then were euthanized, had one hind limb removed, and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection. Brain samples from three flight and three ground control animal groups were cut in half between hemispheres. One hemisphere of each brain was used for generating spatially resolved transcriptional profiling data. Hemispheres were cryosectioned so that 2 consecutive sections from the hippocampus of each brain was placed on Visium Gene Expression arrays. Samples were fixed, stained with Hematoxylin and Eosin and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, following the 10XGenomics Visium Gene Expression protocol, Spatial Transcriptomics RNA-seq libraries were prepared and sequenced. The other hemisphere of each brain was used for single nuclei RNA-seq and ATAC-seq using the 10X Multiome protocol. In addition, bulk RNA-seq (ribodepleted, target depth of 60 M clusters, PE 150 bp) was performed from a pool of RNA extracted from 10-20 sections from each of 3 flight and 3 ground control samples.

NASA's Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS), it is imperative to use crew time efficiently while maximizing high-quality science return. NASA's GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics, systems biology-based approach, and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA, DNA, and protein extracted from archived RR-1 tissue samples for epigenomic, transcriptomic, and proteomic assays. During the first RR spaceflight experiment, a variety of tissue types were harvested from subjects, snap-frozen or RNAlater-preserved, and then stored at least a year at -80C after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands, quadriceps, gastrocnemius, tibialis anterior, extensor digitorum longus, soleus, eye, and kidney. We report here protocols for and results of these tissue extractions, and thus, the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues, our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities.

The objective of the Rodent Research-23 mission (RR-23) was to better understand the effects of spaceflight on the eyes, specifically on the structure and function of the arteries, veins, and lymphatic vessels that are needed to maintain vision. To this end, twenty male, C57BL/6J, 16-17 weeks-old mice were delivered to the International Space Station (ISS) on SpaceX-21 in a single transporter, transferred to two rodent habitats, and maintained in microgravity for 38 days. Flight mice were then returned to Earth alive (January 13th, 2021). After splashdown in the Atlantic Ocean, mice were transported to Kennedy Space Center via helicopter. The 20 Flight, 20 Habitat Ground Control (HGC), and 20 Vivarium Ground Control (VGC) mice were removed from Rodent Transporters (Flight and HGC) or vivarium cages (VGC), placed into shipping containers, and flown to Texas A and M University. There, mice underwent post-flight procedures, before euthanasia and tissue collection. Flight, HGC and VGC animals were euthanized and dissected on Jan 14th, 17th or 20th of 2021, respectively. Right extensor digitorum longus muscle samples were preserved by immersion in liquid nitrogen and stored at -80C until RNA was extracted, and libraries generated and sequenced (target 60 M clusters per sample, PE 150 bp). This dataset features 9 samples from the Flight group, 9 samples from the Habitat Ground Control group, and 9 samples from the Vivarium Ground Control group.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c 18-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the Rodent Habitat for 39-42 days. Twenty mice of similar age and matching sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight ground controls and basal groups had blood collected then were euthanized had one hind limb removed and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection. Brain samples from three flight and three ground control animal groups were cut in half between hemispheres. One hemisphere of each brain was used for generating spatially resolved transcriptional profiling data. Hemispheres were cryosectioned so that 2 consecutive sections from the hippocampus of each brain was placed on Visium Gene Expression arrays. Samples were fixed stained with Hematoxylin and Eosin and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently following the 10XGenomics Visium Gene Expression protocol Spatial Transcriptomics RNA-seq libraries were prepared and sequenced.

The objective of the Rodent Research-5 (RR-5) study was to evaluate bone loss in mice during spaceflight and to determine if treatment with a modified version of NEL-like molecule-1 (NELL-1) can reduce or prevent bone loss that would otherwise occur during spaceflight. To this end, a cohort of forty 30-weeks-old female BALB/cAnNTac mice were flown to the ISS and housed in the Rodent Habitat. Six days after launch half of the mice were treated with NELL-1 (10 mg/kg in 0.3 ml PBS), while the other half were treated with vehicle control (0.3 mls PBS). Fourteen days after launch animals were again treated with NELL-1 or vehicle control as before, except that all animals were also injected with the bone marker, calcein green (20 mg/kg in 0.1 ml). Injections of vehicle, NELL-1, and bone markers were intraperitoneal. After all forty mice on orbit received two treatments; ten control mice and ten experimental mice were randomly selected for live animal return (LAR). At approximately 30 days after launch the twenty LAR mice were transported live back to Earth. Animals were allowed to recover for 30 days in standard habitats before euthanasia via intraperitoneal injection with ketamine/xylazine. During the recovery, the animals received another two treatments. GeneLab received RNA later preserved dorsal skin from ten live animal return and ten matching ground control mice. These were from the vehicle control animals only. RNA was extracted, libraries generated (stranded, ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight, particularly visual impairment, and joint tissue degradation. To this end, a flight group (FLT) of 10-week-old male C57BL/6J mice were launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean, the animals were transported to Loma Linda University (LLU) for testing, euthanasia, and dissection on 9/18/2018. Ground Control (GC) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However, all the GC mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018. Because this resulted in a different cohort of mice being used for the GC controls as compared to the flight (FLT) groups, two cohort controls were included in the study. The first, Cohort Control 1 (CC_C1), was from the same cohort as the FLT animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second, Cohort Control 2 (CC_C2), was from the same cohort as the GC, and was sacrificed and dissected 2-8 days after the GC, (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. Upon dissection, spleen tissues were preserved in liquid nitrogen and stored at 80 C before RNA was extracted. Only the flight (FLT, n of 10) and Ground Control (GC, n of 10) samples were processed and analyzed in this study. Libraries were generated using a 3’ Tag-seq approach and sequenced at a targeted depth of 40 M clusters (SE 93 bp).

The objective of the RR-23 mission was to better understand the effects of spaceflight on the eyes, specifically on the structure and function of the arteries, veins, and lymphatic vessels that are needed to maintain vision. To this end, twenty male, C57BL/6J, 16-17 weeks-old mice were delivered to the ISS on SpaceX-21 in a single transporter, transferred to two rodent habitats, and maintained in microgravity for 38 days. Flight mice were then returned to Earth alive (Jan 13th, 2021). After splashdown in the Atlantic Ocean, mice were transported to Kennedy Space Center via helicopter. The 20 Flight, 20 Habitat Ground Control (HGC), and 20 Vivarium Ground Control (VGC) mice were removed from Rodent Transporters (Flight and HGC) or vivarium cages (VGC), placed into shipping containers, and flown to Texas A and M University. There, mice underwent post-flight procedures, before euthanasia and tissue collection. Flight, HGC and VGC animals were euthanized and dissected on Jan 14th, 17th or 20th of 2021, respectively. Livers were preserved by flash freezing in liquid nitrogen and stored at -80 C until RNA was extracted, and libraries generated and sequenced (target 60 M clusters per sample, PE 150 bp). This dataset features 9 samples from the Flight and VGC groups, and 8 samples from the HGC group.

The Rodent Research-3 (RR-3) mission was sponsored by the pharmaceutical company Eli Lilly and Co. and the Center for the Advancement of Science in Space to study the effectiveness of a potential countermeasure for the loss of muscle and bone mass that occurs during spaceflight. Twenty BALB/c 18-weeks old female mice (ten controls and ten treated) were flown to the ISS and housed in the AEM-X habitat for 39-42 days. Twenty mice of similar age sex and strain were used for ground controls housed in identical hardware and matching ISS environmental conditions. Basal controls were housed in standard vivarium cages. Spaceflight ground controls and basal groups had blood collected then were euthanized had one hind limb removed and finally whole carcasses were stored at -80 C until dissection. All mice in this data set received only the control/sham injection.

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes single cell transcriptional profiling data from the spleens from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.