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 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.

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.

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 Rodent Reasearch-1 National Lab (RR-1 CASIS) experiment was performed to study the effect of microgravity on muscle wasting. RNA, DNA, and protein were purified from RR-1 CASIS (the Center for the Advancement of Science in Space) liver samples. Groups included: Flight (FLT) dissected on-orbit (21 or 22 days after launch); age-matched Ground Controls (GC); and Basal Controls (BC, euthanized at time of launch). RNA-Seq, whole genome BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed.

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated. or implanted with vehicle or treatment-filled nDS and launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of dorsal skin from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=9) ISS Terminal (n=9); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=6). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018,. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis Facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of liver from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=10) ISS Terminal (n= 10); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=10). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

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).