Metagenomic analysis of feces from mice flown on the RR-6 mission

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 β2 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 Spaceflight [LAR FLT]), or >50 days (N=20, ISS Terminal Spaceflight [ISS-T FLT]). After 29 days, the 20 LAR FLT 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 Basal (BSL) groups of animals sacrificed (LAR BSL & ISS-T BSL; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). LAR BSL animals were dissected, and samples were collected upon euthanasia. A Ground Control (GC) group, LAR GC, mimicked the LAR FLT group, which was housed at KSC, then shipped alive, to Novartis’s Facilities, where both the LAR FLT and LAR GC 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-T FLT 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 groups. Following blood draw and hind limb dissection, the ISS-T FLT animal carcasses were wrapped in aluminum foil, placed in a ziploc bag and placed in storage at -80˚C or colder until return. The ISS-T Ground Control (ISS-T GC) (at KSC) followed the same euthanasia timeline, methods, and preservation. The final processing of frozen ISS-T FLT, frozen ISS-T GC and frozen 0-day ISS-T BSL animals were completed at Houston Methodist Research Institute, in Houston, TX (5/21/18 until 5/24/18). GeneLab received feces from only sham treated animals (no drug treated animals) from the following groups. FLT: LAR (n=9), ISS-T (n=7); GC: LAR (N=7), ISS-T (N=9); BSL: LAR (n=7), ISS-T (n=9). DNA was extracted and analyzed by sequencing using a variety of different targeted and un-targeted metagenome profiling assays.

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