The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing and spatially resolved transcriptional profiling data from hippocampi from 5 WT flight animals and 5 WT ground control animals. Hippocampi from the right hemisphere were embedded and cryosectioned. Cryosections were either processed for bulk RNA sequencing or placed on gene expression arrays, stained and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, spatial transcriptomics libraries were prepared and sequenced.

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 bulk RNA sequencing and spatially resolved transcriptional profiling data from cerebellums from 4 (bulk RNAseq) or 5 (spatial transcriptomics) old ISS-T flight animals, 3 old ISS-T ground control (GC) animals, 5 young ISS-T flight animals, 3 young ISS-T GC animals, 3 old LAR flight animals, 3 old LAR GC animals, 2 (bulk RNAseq) or 3 (spatial transcriptomics) young LAR flight animals, and 3 young LAR GC animals. Cerebellums from the right hemisphere were embedded and cryosectioned. Cryosections were either processed for bulk RNA sequencing or placed on gene expression arrays, stained and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, spatial transcriptomics libraries were prepared and sequenced.

The objective of the Rodent Research-10 Mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. At the end of RR-10 experiment, all frozen carcasses were partially thawed and kidney tissues were removed and preserved by flash freezing in LN2. Kidneys were kept at -80 C freezer until processing. To ensure that both transcriptional profiling and protein expression profiling datasets are representative of the entire kidney, whole kidneys were first pulverized into a fine powder on dry ice. This powder was then split into two equal fractions with half being used for RNA isolation and half being used for protein isolation. RNA was used to generate three different transcriptional profiling datasets: a 3' tag-Seq datasets (20 M clusters at SE 93 bp), a polyA enriched dataset (60 M clusters at PE 150 bp), and a ribodepleted dataset (60 M clusters at PE 150 bp). Protein was used to generate protein expression profiling, and phosphoprotein profiling using the iTRAQ method (Isobaric tags for relative and absolute quantitation). Transcriptional profiling dataset features WT samples from the Flight, Ground, Basal and Vivarium groups. Protein expression profiling and phosphoprotein profiling datasets exclude the Vivarium group.

The objective of the Rodent Research-10 Mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. At the end of RR-10 experiment, all frozen carcasses were partially thawed and fecal pellets were removed and preserved by flash freezing in LN2. DNA was extracted, shotgun metagenome libraries generated, and libraries sequenced (target 10 M clusters at PE 250 bp). This dataset features WT samples from the Flight (n=3), Ground (n=5), Basal (n=10) and Vivarium groups (n=12), as well as p21-null samples from Flight (n=5), Ground (n=5), Basal (n=10) and Vivarium groups (n=12).

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 humerus bone marrow from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

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-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-23 missions (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 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. Cerebellums were preserved by immersion in RNAlater 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 group, 9 samples from the HGC group, and 8 samples from the VGC group. A technical replicate is included for one sample in each group. These consist of an independent library preparation for a single RNA extraction.

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 bulk RNA sequencing and single nuclei transcriptomics and epigenomics from left cerebral hemispheres from 4 young ISS-T spaceflight animals, 5 old ISS-T spaceflight animals, 5 young ISS-T ground control animals, and 4 young ISS-T ground control animals.

The objective of the Rodent Research-7 mission (RR-7) was to study the impact of the space environment on the gut microbiota of two strains of mice and how any changes in-turn affect the immune system, metabolic system, and circadian or daily rhythms. To this end, ten 11-week-old female C57BL/6J and ten 11-week-old female C3H/HeJ mice were flown to the International Space Station on June 29, 2018 on SpaceX-15 and housed in two Rodent Habitats. Samples of food, swabs from living surfaces, and fecal pellets were collected from each animal before launch and regularly during the mission. The mission also involved extended video collection (48 hr video segments per Habitat) to monitor circadian rhythms, and on-orbit mass measurement. After 25 days on-orbit, half of the mice of each strain were euthanized on the ISS with Ketamine/Xylazine/Acepromazine and cardiac puncture, after which carcasses were segmented in three sections and preserved in RNA later. After 75-76 days the remaining 5 animals from each group were euthanized and processed in the same manner. The 25-day dissected carcasses returned on SpX-15, and the 75-day dissected carcasses returned on SpX-16. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (same habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups, which were euthanized and processed on the same schedule and in the same manner as the flight samples. Dissections for tissues from all experimental groups were completed by the PI groups along with NASA's Biospecimen Sharing Program in February 2019. GeneLab received dorsal skin samples from forty C57BL/6J mice: 10 Basal, 5 Ground (25 days), 5 Ground (75 days), 5 Flight (25 days), 5 Flight (75 days), 5 Vivarium (25 days), 5 Vivarium (75 days). GeneLab received dorsal skin samples from forty C3H/HeJ mice: 10 Basal, 5 Ground (25 days), 5 Ground (75 days), 5 Flight (25 days), 5 Flight (75 days), 5 Vivarium (25 days), 5 Vivarium (75 days). From these skin samples, RNA was extracted, libraries generated (stranded, ribodepleted) and sequenced (target 60 M clusters at PE 98 bp).