NOTE: Extended to 6/4/2021 per F. Hernandez/ARC and NSSC information (Ed., 6/12/19)

NOTE: Extended to 7/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 2/14/19)

NOTE: Extended to 1/31/2019 per NSSC information (Ed., 3/12/18)

NOTE: Extended to 1/31/2018 per F. Hernandez/ARC (Ed., 2/12/17)

Rodent Research-1 (RR-1) Study 1: From the RR-1 mice, the retinas were isolated from the frozen eyes under rapid thaw process. RNA/DNA were extracted from the retina. QC data showed that the samples are suitable for RNA sequencing. These studies are currently in progress.

JAXA (Japan Aerospace Exploration Agency) MHU-1 Study 2: Results from this study have been published and demonstrate that spaceflight induces apoptosis in retinal vascular endothelial cells. We also identified spaceflight-induced changes in proteomic profiles and pathways in the ocular tissue. The results indicate that spaceflight induces changes in neuronal structure, cellular organization, mitochondrial function and oxidative phosphorylation and inflammation, which in turn, may lead to tissue injury and late neurodegeneration. This study is the first to investigate the role of artificial gravity provided by centrifugation during spaceflight as a countermeasure for mitigating putative effects of microgravity on ocular structure and function.

Further studies will be continued on the JAXA MHU-1 eyes to estimate the effects of spaceflight with and without artificial gravity on blood-retinal barrier function. These studies will be performed in parallel with the same measures performed on eyes obtained from a JAXA MHU-2 mission. Once we have the control tissue specimen in our possession we will commence with processing and data collection.

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NOTE: Extended to 7/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 2/14/19)

NOTE: Extended to 1/31/2019 per NSSC information (Ed., 3/12/18)

NOTE: Extended to 1/31/2018 per F. Hernandez/ARC (Ed., 2/12/17)

Conclusions from research during the reporting period:

a) The data demonstrated that spaceflight alone induced apoptosis in retinal endothelial cells and photoreceptors.

b) The number of apoptotic cells in the retina was reduced 24% during spaceflight with continuous artificial 1g while the animals were housed on the ISS.

c) Proteomic analysis showed that many proteins were significantly altered after spaceflight compared to that in habitat control mice; these proteins are involved in cell death, cell repair, inflammation, carbohydrate metabolism, and apoptosis.

d) Continuous artificial 1g showed lower organismal death and greater cellular organization and function signaling compared to the spaceflight alone group.

NOTE: Extended to 1/31/2019 per NSSC information (Ed., 3/12/18)

NOTE: Extended to 1/31/2018 per F. Hernandez/ARC (Ed., 2/12/17)

Study 1. The first study was a NASA mission testing the Rodent Research Hardware System, called the Rodent Research-1 (RR-1), where mice (female, C57Bl6/J, 16wk old at time of launch) and hardware payload was transported to the ISS on a SpaceX-4 CRS-4 Dragon cargo spacecraft. During this mission, which was launched September 20, 2014 and returned October 25, 2014, astronauts transferred ten flight mice (Group 1) to a habitat to validate NASA hardware and operations. A transporter and identical habitat were tested at Kennedy Space Center in Florida as ground control units based on a 4-day delay. Ground control groups consisted of environmental ground control mice (Group 2) housed in RR-1 flight hardware within an environmental simulator under the same conditions as the ISS, vivarium control mice (Group 3) housed in standard vivarium cages, and basal control mice (Group 4) from the same cohorts as the flight mice. At the end of the mission, the flight and ground control mice were sacrificed and some tissue (not eyes) was dissected from some of the mice (flight mice dissected on-orbit 37 days after launch). On-orbit dissections were conducted by astronauts using the Microgravity Science Glovebox. After dissection, the remaining carcass or whole carcass of the animals were frozen. Subsequently, at NASA Ames Research Center in California, the frozen carcasses were thawed and the eyes and other tissues were removed. The eyes were re-frozen once they were removed from the carcass and shipped to Florida State University on September 22, 2016.

Study 2. The second study was a collaborative arrangement between the Japan Aerospace Exploration Agency (JAXA) and NASA. This study was part of the larger “Mouse Epigenetics” program under the direction of JAXA Principal Investigator Dr. Satoru Takahashi, a Professor at Tsukuba University in Japan. This investigation included two flight groups that were transported to the ISS on the SpaceX-9 mission. These animals spent approximately 30 days on the ISS. The first group of flight mice (n=6, male) were exposed to µG, while the second group of flight mice (n=6, male) were exposed to continuous artificial-G (1G) while they were in the Habitation Cage Units through the use of a short-arm centrifuge. After 30 days on-orbit the animals were returned to Earth alive on August 26, 2016 and dissected two days later in San Diego, California.

Ground control mouse studies were completed in Japan after the return of the flight mice. Control mice (Transportation/Habitation cage controls, n=6; Vivarium controls, n=6) were acquired on August 31, 2016 at the JAXA animal facility in Tsukuba, Japan. Transportation/Habitation cage control mice were acclimated to the water lixit system and flight food from August 31 through September 22. They were then habituated to the Transportation Cage Unit (to simulate launch and flight to ISS housing) from September 22 – 26, and then they were placed in the Habitation Cage Units from September 26 – October 31 to simulate time on the ISS. They were then placed in the Transportation Cage Unit October 31 – November 3 to simulate the return to Earth flight. The mouse dissections took place on November 3. Control mouse eye tissue was shipped to the US and delivered on November 9, 2016.

During the dissection, the right eye from each animal (flight and control) was fixed and kept by Principal Investigator (PI) Delp. The left eye from each animal was sectioned into two separate halves; the front half of the eye (including the cornea, ciliary body, and muscle) and the back half of the eye (including retina, macula, choroid, optic nerve, and associated vasculature). Both halves were frozen, and the front half of the eye was maintained by JAXA investigators, while the back half of the eye and the lens were maintained by PI Delp.

Results: From the RR-1 mice, the retinas were isolated from the frozen eyes under rapid thaw process. RNA/DNA were extracted from the retina. QC data showed that the samples are suitable for RNA sequencing. These studies are currently in progress.

Proteins lysates have been prepared for multiplex proteomics from the frozen eyes of the JAXA flight and ground control mice. Assay and data analyses are underway. Fixed ocular sections are being stained for markers of oxidative stress and apoptosis.