NOTE: Extended to 6/4/2022 per F. Hernandez/ARC (Ed., 7/27/21)

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)

Twelve male 9-week old C57BL/6 male mice, obtained from a United States (US) breeding colony, were launched July 18, 2016, at NASA Kennedy Space Center (KSC) on a SpaceX-9 rocket for the 35-day Mouse Habitat Unit-1 (MHU-1) mission to the International Space Station (ISS). The animals were housed in the mouse Habitat Cage Unit (HCU) located in the Japan Aerospace Exploration Agency (JAXA) "Kibo" facility on the ISS. The 12 flight mice were subdivided into two groups. The first group of flight mice (n=6) were exposed to ambient microgravity conditions (µg group), while the second group of flight mice (n=6) were exposed to continuous artificial Earth gravity (µg + 1g group) while they were in the HCU. AG was achieved through the use of a short-arm centrifuge for the duration of their stay on the ISS. The flight mice were then returned live to Earth and splashed down in the Pacific Ocean on August 26, 2016. It took approximately 40 hours for the mice to be recovered in the Pacific Ocean, brought to shore and transported to the testing and processing laboratory located in San Diego, California on August 28, 2016. The spaceflight mice were then sacrificed and their eyes were removed and prepared for analysis. Ground control mouse studies were completed in Japan after the return of the flight mice. Control mice (Habitat Controls, n=6; Vivarium controls, n=6) were acquired on August 31, 2016 from a breeding colony in Japan and shipped to the JAXA animal facility in Tsukuba, Japan. HC mice were acclimated to the water lixit system and the same special food bar diet as the spaceflown mice were fed. They were first housed in the Transportation Cage Unit (TCU) to simulate launch and flight to the ISS, and then placed in the HCUs to simulate the housing conditions experienced by µg mice on the ISS. They were again placed in the TCU to simulate the return to Earth flight. The control mouse dissections took place on November 3, 2016. Control mouse eye tissue was then shipped to the US for analysis. All mice received the same ad libitum access to food and water.

Study 1 Conclusions: 1) The data demonstrated that spaceflight alone induced apoptosis in retinal vascular endothelial cells, which suggests disruption in the integrity of the blood-retinal barrier. 2) 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. 3) 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. 4) Continuous artificial 1g showed lower organismal death and greater cellular organization and function signaling compared to the spaceflight alone group.

The purpose of Study 2 was to more directly address Specific Aim #2, i.e., to characterize the effects of spaceflight on the retinal vasculature and possible alterations in blood-retinal barrier (BRB) integrity, and to identify spaceflight-induced proteomic significance and biomarkers in mouse ocular tissue. The data demonstrate that spaceflight induces apoptosis in the retinal vascular endothelial cells and photoreceptors, as well as evokes alterations in vascular levels of aquaporin-4 (AQP-4), platelet endothelial cell adhesion molecule (PECAM-1) and zonula occludens-1 (ZO-1) proteins related to BRB integrity.

Study 2 Conclusions: 1. The results of this study demonstrate that exposure to a spaceflight environment is associated with increased retinal endothelial cell and photoreceptor cell death. 2. The changes in retinal microvasculature BRB integrity (i.e., vascular levels of AQP-4, PECAM-1, and ZO-1), indicate decrements in barrier function in the eye. 3. Protein expression profiles and pathway analysis provide evidence that spaceflight induces changes in cellular organization, cell cycle, mitochondrial function, circadian clock, and oxidative stress in the retina. 4. Collectively, these observations are consistent with, and extend, previous findings in rodents exposed to a weightless environment and suggest that spaceflight involves a complex combination of stressors that leads to alterations and impairment of ocular structure and function.

The purpose of Study 3 was to address Specific Aim #1, i.e., to characterize the effects of spaceflight on the retinal oxidative stress and extracellular matrix remodeling in the eye. Specifically, this study characterized the physical response of the retina, the degradation of photoreceptors, and the presence of oxidative stress markers. The findings also indicate that spaceflight induces a distinct gene expression signature in the retina of mice. This signature is enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Several genes with significant differential expression in the spaceflight condition are also differentially expressed in the disease retinitis pigmentosa.

Study 3 Conclusions: The results suggest that the differential expression induced by spaceflight may be pathological. Additionally, we suspect the changes observed during spaceflight are influenced by alternative splicing and chromatin reorganization.

The purpose of Study 4 was to determine the effects of spaceflight on possible alterations in DNA methylome and transcriptome of the retina, and to determine whether the primary impacted genes belong to physiologically relevant cellular processes and pathways. These include processes and pathways associated with oxidative stress, inflammation, mitochondrial function, tissue remodeling, fibrosis, and angiogenesis. This study represents a more sophisticated experimental approach than that originally proposed in the specific aims and, consequently, provides more information regarding the broad effects of spaceflight on the retina.

Female C57 BL/6J mice that were 16 weeks old were used in this study. Both spaceflight and ground control animals were housed in NASA’s animal enclosure modules (AEM), with control mice being exposed to the same environment conditions (12-hour light cycle, temperature and humidity) as those flown on the ISS. Control animals were kept inside an environmental simulator (ISSES) at the Space Life Science Laboratory (SLSL) at Kennedy Space Center, and the spaceflight animals were transported to the ISS by SpaceX4 on September 21, 2014. All animals were fed with a special NASA food bar diet and their health was checked daily. The spaceflight mice were sacrificed and frozen in orbit after 37 days of flight, while ground control mice were simultaneously sacrificed and frozen under identical conditions. After the frozen carcasses were returned to KSC, the ocular tissues were removed from both groups.

Study 4 Conclusions: Approximately one in three astronauts flying on long-duration space missions experience visual impairment and morphologic changes to their eyes that include choroidal and retinal folds, optic disc edema, focal areas of retinal ischemia (i.e., cotton wool spots), globe flattening, and hyperopic shifts. This collection of ocular disorders has been termed Spaceflight Associated Neuro-ocular Syndrome (SANS). A variety of potential mechanisms have also been proposed to account for the unusual physiologic and pathologic neuro-ophthalmic findings in astronauts, including elevations in intracranial pressure (ICP) from cephalad fluid shifts, altered autoregulation of cerebral perfusion, impaired cerebrospinal fluid drainage from the brain and orbital optic nerve sheath through venous, glymphatic, and lymphatic drainage systems, and disruption of blood-brain, blood-retinal, and blood-optic nerve barrier function. This seemingly multifaceted pathological process, which varies from astronaut to astronaut, indicates a complex origin for these neuro-ophthalmic findings associated with SANS. The integrated DNA methylome and RNA transcriptome analysis demonstrates that spaceflight had profound effects on extracellular matrix (ECM) / cell junction and cell proliferation/apoptosis signaling in the retina. Although these data do not address all the possible mechanisms involved in the etiology of SANS, they provide crucial insight into the potential adverse consequences of spaceflight on the retina that could be functionally important for maintaining proper visual acuity among astronauts.

NOTE: Extended to 6/4/2022 per F. Hernandez/ARC (Ed., 7/27/21)

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)

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.

(Ed. note June 2019: compiled from PI's annual report received June 2019; covers reporting period Feb 2018-May 2019)

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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.