Gene expression changes induced by acute skeletal muscle unloading which leads to physiological changes including muscle atrophy fibre-type switching and loss of ability to transition between lipid and glucose as energy source (metabolic inflexibility) was investigated by hind-limb suspension (HLS) treatment of Male ICR mice (28 x9632 g body wt; Harlan Indianapolis IN). Agilent Whole Mouse Genome Oligo Microarrays were utilised to examine the effects of HLS on mRNA expression profiles of the soleus muscle and the gastrocnemius muscle in the hindlimbs of freely ambulating control and 24h HLS treated mice.

Data from the NASA Rodent Research-1 (RR-1) mission showed that gene-expression levels in mouse livers are different depending on what tissue preservation protocol is used and that slow freezing is not an effective method for preserving signals in gene-expression data. In response to these and other observations the Rapid Freeze hardware was built for use on the International Space Station. The Rapid Freeze hardware freezes mouse tissues (Glovebox freezer) and whole carcasses (Cryochiller) at rates closely mimicking those attained with immersion in liquid nitrogen. Because this hardware will be used extensively on future rodent research missions it is crucial to understand whether or not it preserves signals in gene expression data in order to maximize the value of these rare and expensive spaceflight experiments. Therefore this study was designed with three goals: 1) To evaluate the temperature profile of the Cryochiller and Glovebox freezer cartridges (Rapid Freeze hardware) over time during mock on-orbit procedures; 2) To determine the freezing profiles of tissues and carcasses using Rapid Freeze hardware at both optimal and sub-optimal temperatures (to mimic on-orbit operations) compared with those frozen in liquid nitrogen (the laboratory gold standard) or frozen at -80 C (the current standard method); 3) To identify gene expression changes in a) tissues that were frozen via the Glovebox freezer and b) tissues dissected from whole or partial carcasses that were frozen via the Cryochiller versus tissues that were frozen via control methods (liquid nitrogen or -80C slow freeze) to assess how the Rapid Freeze hardware compares with laboratory gold standard practices and our current standard methods.

Analysis of effect of hindlimb suspension and reloading on C57Bl/6 mouse soleus muscle. Experimental groups examined: -Control mice 14 days -Hindlimb suspension for 7 days -Hindlimb suspension for 7 days and subsequent reloading for 1 day -Hindlimb suspension for 7 days and subsequent reloading for 7 days

A recent physiological study established that hindlimb unloading of rats at 3 and 7 weeks inhibits healing of injured ligaments resulting in a badly aligned discontinuous collagen matrix. Using tissue from these rats we focused on the 3-week time point employing microarray analysis to identify what cellular processes or lack of processes could account for these observed deficiencies. We used the Affymetrix RG_U34A GeneChip and performed image analysis with Microarray Suite 5.0. For normalization we used the MAS global normalization protocol with a default target mean signal of 500. Gene expression in medial collateral ligament tissue under 4 different treatment conditions was measured: loaded control loaded wound healing unloaded control and unloaded wound healing. From our results it appears that unloaded tissue lags behind loaded tissue in its progression through the healing process and at 3 weeks is still engaged in the proliferative phase whereas loaded tissue is actively remodeling its collagen matrix.

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-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).

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 (RR-1) 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 present study concerns the effects of 21 days of sustained recumbence (bedrest) on skeletal muscle gene expression. 14 male subjects participated in normoxic bedrest (NBR) with muscle unloading for 21 days this is a substudy of tha PlanHab-study which apart from normoxic bedrest also involved hypoxic bedrest and hypoxic ambulation. Throughout both bedrest interventions each subject remained in a horizontal position at all times. He was allowed one pillow for the head and to occasionally lean on an elbow while eating or being transferred to a gurney. Muscular exercise was prohibited. During the HAMB confinement each subject was allowed to move freely within the hypoxic area. To mimic a normal level of physical activity subjects performed 30 minutes of low-to-moderate-intensity exercise twice daily. Muscle samples from Vastus lateralis.

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.