Comparitive gene expression in skin between mice maintained in microgravity (0g) and normogravity (1g) environment. Six male C57Bl/J10 mice were housed for 91 days in the specially designed Mouse Drawer System in weightlessness aboard the International Space Station. Three wild-type mice (WT) and three transgenic mice overexpressing the osteogneic factor PTN/OSF1 under the control of the human bone specific ostecalcin promoter (Tg) were used in the experiment. During the 3-month stay on the ISS 3 mice unfortunately died leaving 2 Tg and 1 WT. MDS tissue sharing program allowed several teams to study various tissues from these mice. Our aim was to investigate the effect of such a long period of microgravity on skin physiology by morphological biochemical and genomewide analyses by comparison to similar mice on ground. Gene expression in the skin of 3 space mice and of 3 ground mice was analyzed by microarray. As this unique experiment performed on 3 mice limits the power of statistical analyis as the transgene PTN/OSF1 was not overexpressed in skin and as a pair wise Pearson s correlation rates between the individual levels of expressed transcripts in the WT and the Tg mice were not significantly different from each other in one experimental group (space or ground) data from the 3 mice were combined to compare results from the space an ground groups.

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).

The JAXA MHU-2 mission had two objectives: 1) To increase understanding of effects of spaceflight on the gut environment (microbiota and metabolites) and immune system using multi-omics based analysis; 2) To evaluate whether fructo-oligosaccharides added to the diet as prebiotics improve the gut environment and immune function during spaceflight. Twelve 16-18 week old male C57BL/6J mice were singly housed in the JAXA Habitat Cage Units (HCUs) on the ISS for 30 days. Six flight mice were housed in microgravity while six were exposed to simulated 1g by centrifugation. These two flight groups were further divided in half so that three mice in each group received standard JAXA chow while the other three were fed chow supplemented with fructooligosaccharides (FOS). Mice were returned live and euthanized and dissected <1 day after splashdown. Ground controls (n=6) were asynchronous and housed in HCUs. Vivarium controls (n=6) were asynchronous and housed in standard habitats. Three ground control and three vivarium animals received standard chow while the other three each ground control and vivarium animals received FOS-supplemented chow. Ground and vivarium samples were dissected by a separate dissection team than flight samples. Dorsal skin was dissected 30 minutes after euthanasia and snap frozen in liquid nitrogen. Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150). Study Factor Levels: 1)Spaceflight ug Std. Chow: 3; 2)Spaceflight ug FOS: 3; 3) Spaceflight Artificial 1g Std. Chow: 3; 4)Spaceflight Artificial 1g FOS: 3; 5)Ground 1g Std. Chow: 3; 6)Ground 1g FOS: 3; 7)Vivarium 1g Std. Chow: 3; 8)Vivarium 1g FOS: 3.

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).

Upon weightlessness and microgravity deleterious effects on the neurosensory and neurovestibular systems haematological changes and deconditioning of musculoskeletal cardiovascular and cardiopulmonary functions have been reported. In particular loss of muscle mass and strength are triggered by weightlessness in humans during space flights that is similarly observed as a result of physical inactivity conditions and aging on Earth. However skeletal muscle tissue is of paramount importance for health maintenance (e.g. being essential to locomotion heat production and metabolism). To better prevent or eventually treat microgravity-induced muscle atrophy its underlying mechanisms have first to be characterized in detail. Using cutting-edge quantitative proteomics the aim of the present study was therefore to get an in depth view of the molecular regulations triggered by space conditions in skeletal muscles of mice during the 30-day flight of the BION-M1 biosatellite. As muscles differing in their fiber type composition appear to respond differently to microgravity (see above) we characterized here the differential response of the soleus extensor digitorum longus and vastus lateralis muscles.

Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronaut xe2 x80 x99s health and safety. However consequences on B cell development and responses are still under-investigated. Up to now most studies addressing these questions were performed using an amphibian species. Consequently we used advanced proteomics analysis of femur bone and marrow of mice flown for one month on board the BION-M1 biosatellite to determine whether extreme conditions encountered during a real spaceflight affect B cell development in mice and to examine reversibility of the effects upon return to Earth. Our data revealed that adverse effects on B lymphopoiesis were more marked one week after landing and that this phenomenon was associated with a 41% reduction of B cells in the spleen. Thus the effects of spaceflight persisted during at least one week after landing. These reductions may contribute to explain increased susceptibility to infection even if we confirmed that animals were able to mount a humoral immune response.

Upon weightlessness and microgravity deleterious effects on the neurosensory and neurovestibular systems haematological changes and deconditioning of musculoskeletal cardiovascular and cardiopulmonary functions have been reported. In particular loss of muscle mass and strength are triggered by weightlessness in humans during space flights that is similarly observed as a result of physical inactivity conditions and aging on Earth. However skeletal muscle tissue is of paramount importance for health maintenance (e.g. being essential to locomotion heat production and metabolism). To better prevent or eventually treat microgravity-induced muscle atrophy its underlying mechanisms have first to be characterized in detail. Using cutting-edge quantitative proteomics the aim of the present study was therefore to get an in depth view of the molecular regulations triggered by space conditions in skeletal muscles of mice during the 30-day flight of the BION-M1 biosatellite. As muscles differing in their fiber type composition appear to respond differently to microgravity (see above) we characterized here the differential response of the soleus extensor digitorum longus and vastus lateralis muscles.

The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.

Articular and sterno-costal cartilages were isolated from skeletally-mature mice flown for 30 days on the BION-M1 mission. Samples were characterized histologically for proteoglycan loss and at the gene expression levels using Affymetrix gene arrays.

Microgravity exposure as well as chronic muscle disuse are two of the main causes of physiological adaptive skeletal muscle atrophy in humans and murine animals in physiological condition. The aim of this study was to investigate at both morphological and global gene expression level skeletal muscle adaptation to microgravity in mouse soleus and extensor digitorum longus (EDL). Adult male mice C57BL/N6 were flown aboard the BION-M1 biosatellite for 30 days on orbit (BF) or housed in a replicate flight habitat on Earth (BG) as reference flight control. In this study we investigated for the first time gene expression adaptation to 30 days of microgravity exposure in mouse soleus and EDL highlighting potential new targets for improvement of countermeasures able to ameliorate or even prevent microgravity-induced atrophy in future spaceflights. Overall Design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF) mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG) mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC) mice housed in a standard animal facility.