STEM Today,October 2018,No. 37 by Abhishek Sinha

STEM TODAY October 2018, No. 37

CONTENTS Degen 7: Are there synergistic effects from other spaceflight factors (e.g. altered gravity microgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiationinduced degenerative diseases in a clinically significant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after flight. It is currently unknown whether there are significant synergistic effects from other secondary spaceflight factors (altered gravity (μgravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late effects resulting from space radiation exposure. Activities todate have included retrospective data mining and flight and ground studies to identify the role of the risk factors outlined above on cardiovascular health.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

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Disclaimer ( Non-Commercial Research and Educational Use ) STEM Today is dedicated to STEM Education and Human Spaceflight. This newsletter is designed for Teachers and Students with interests in Human Spaceflight and learning about NASA’s Human Research Roadmap. The opinion expressed in this newsletter is the opinion based on fact or knowledge gathered from various research articles. The results or information included in this newsletter are from various research articles and appropriate credits are added. The citation of articles is included in Reference Section. The newsletter is not sold for a profit or included in another media or publication that is sold for a profit. Cover Page Hurricane Florence as it was making landfall iss056e162819 (Sept. 14, 2018) — Hurricane Florence is pictured from the International Space Station as a category 1 storm as it was making landfall near Wrightsville Beach, North Carolina. Image Credit: NASA

Back Cover The Space Station Transits Our Sun This composite image, made from nine frames, shows the International Space Station, with a crew of three onboard, in silhouette as it transits the Sun at roughly five miles per second, Sunday, Oct. 7, 2018. Onboard are Commander Alexander Gerst of the European Space Agency, Serena AuÃśÃşn-Chancellor of NASA, and Sergey Prokopyev of Roscosmos. The trio will soon be joined by Nick Hague of NASA and Alexey Ovchinin of Roscosmos, who are scheduled to launch on October 11 from the Baikonur Cosmodrome in Kazakhstan. Image Credit: NASA/Joel Kowsky

STEM Today , October 2018

Editorial Dear Reader

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All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Road map. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss

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Space Radiation (SR) Degen - 7: Are there synergistic e ects from other space ight factors (e.g. altered gravity (Âľgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiation-induced degenerative diseases in a clinically signi cant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after ight. It is currently unknown whether there are signi cant synergistic e ects from other secondary space ight factors (altered gravity (Âľ-gravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late e ects resulting from space radiation exposure.

Space Radiation Impact on Cells Genetic Apparatus on Board the ISS

The damage of genetics apparatus of cells in space flight conditions is primarily due to the impact of mixed space radiation. Indirect effects of radiations in water-equivalent medium account for 80-90 % of the total damage in eukaryotes and prokaryotes. These effects are responsible for cell inactivation. The Russian "PHOENIX" experiment is designed to study the effects of long-term space flight conditions on the genetic apparatus and viability of dried human lymphocytes and mouse bone marrow cells (BMC). The experiment is carried out onboard the Russian Segment of International Space Station (ISS). Sealed vials with human lymphocytes and mouse BMC were packed in 12 "Bio-ecology" boxes arranged at 3 containers. Each box was equipped with the temperature sensor and passive space radiation dosimeter. The containers were delivered to ISS in November 2014 and installed in Pirs, Poisk and Zvezda modules at panels No.436, No.103 and No.103 respectively. Control vials with cells and travel background dosimeters were stored at the laboratory. Three boxes (one from each module) were descended to the Earth in 199 days exposure.

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Results Cell viability and DNA damage Cell viability was evaluated by trypan blue exclusion test (Figure 1). The morphology of human lymphocytes and mouse BMC was analyzed after Romanovsky-Giemsa staining (Figure 2). MTT assay showed the presence of mitochondrial activity of the cells after rehydration.

DNAs were isolated from the cells and analyzed by electrophoresis followed by densitometry. Four zones in Figure 3 reveal the most pronounced differences between the biological samples from different boxes. In the material from Poisk and Pirs modules density peaks were found corresponding to 1500-2000 bp values of the DNA marker (GeneRuler 1kb DNA Ladder, Thermo Scientific). The samples from Zvezda and Poisk modules were characterized by 15000 bp peak. In all cases, the peaks of samples from the Pirs module are shifted to the right relatively to the samples from the Zvezda and Poisk modules. This indicates the formation of smaller DNA fragments. Similar results were obtained for the human lymphocytes.

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Double staining with fluorescent dyes ethidium bromide (necrosis) and acridine orange (visualizing live cells and specifically staining apoptotic cells) revealed cells in different physiological states. Standard methods for evaluation of the nucleus integrity revealed no apparent damage. Space Radiation Data LET distribution The LET spectra measured in different locations are presented in Figure 4. The contribution from primary and secondary high-LET space radiations are presented in Figure 5 separately. The relatively high flux of particles with LET < 100 keV/µm (H2 O) observed in the Pirs module (Figure 4) is supposedly due to the thinner shielding of compartment. The measured flux of secondaries exceeds the flux of primary component at LET > 100 keV/µm in all three modules of ISS. The measured flux of secondaries is 1-2 orders of magnitude higher than flux of long-range component at LET 100-600 keV/µm.

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Doses The dose rates measured by means of passive detectors are summarized in Table 1. Contribution of nuclear fragments (Dc and Hc ) into the total absorbed dose Dd and dose equivalent Hd varies in 2.0-5.6 % and 19.6-36.5 % respectively. This agrees well with the result.

The observed effect of DNA fragmentation depends on dose rate at the compartment. The smaller fragments of DNA were produced in the Pirs module due to the highest dose rates from all components of space radiation. Conversely, the fragments of bigger size were produced in Poisk and Zvezda modules due to relatively small dose rates. The preliminary results of the "PHOENIX" space experiment shows a good survival of human lymphocytes and mouse bone marrow cells nuclei after 199 days space flight, when the doses and dose equivalents varied in the range 57-145 mGy and 140-319 mSv respectively. The degree of DNA fragmentation depends on the dose rate directly. The smallest DNA fragments were detected in the samples exposed at the Pirs module with the maximum dose rates.

Functional Genomic and Signaling Responses to HZE Particle Radiation (NNJ04HF41G) Recent advances in gene expression profiling have broadened understanding of the molecular mechanisms of response to a wide range of environmental insults, including toxic chemicals and radiation. Such studies hold great promise for the development of exposure and diagnostic biomarkers, as well as pointing the way to intervention strategies through mechanistic understanding. The majority of such studies, however, have considered 6

only low linear energy transfer (LET) radiation, and it is unknown to what extent this knowledge may be applicable to the high LET heavy ions prevalent in the space radiation environment. This proposal seeks to apply functional genomic analysis in conjunction with the irradiation capabilities of the NASA Space Radiation Laboratory (NSRL) to address questions of the molecular responses to highly-charged high-energy heavy ion (HZE particle) irradiation. A more comprehensive understanding of the molecular signaling pathways activated in response to damage by HZE particles will identify biomarkers of exposure and damage, and molecular targets for modification of the radiation response, as well as enhancing our understanding of the mechanisms by which HZE particle exposure may lead to the development of cancer and other late effects.

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RESULTS Funding was provided for one year in order to obtain preliminary data with heavy ions and to demonstrate feasibility of the proposed studies. The experiment team was able to obtain beam-time and complete three independent irradiations with 1 GeV 56Fe ions at NSRL during the two runs (NSRL-5 and NSRL-6) that were available during the funding period. While microarray analysis is ongoing, the data so far demonstrate clearly that the experiments were successful. This data and will provide a solid basis for shaping future studies. Survival While culturing cells in the presence of activated medium did not significantly affect cloning efficiency (p>0.6), the lowest dose used (10 cGy) resulted in a nearly 20% decrease in cell survival (p<0.05). The overall TK6 survival curve showed a linear decrease in survival with increasing dose, with overall survival levels in agreement with what had been predicted from the literature. The NH32 cells were treated with only two doses of iron ions, so the shape of the survival curve could not be accurately determined. Survival of the p53 knock-out cells was similar to that of the parental TK6, with perhaps a slight enhancement at the higher dose. Apoptosis Nuclear morphology was scored in DAPI-stained cells as a measure of apoptosis at 3 hours after irradiation, and at 24-hour intervals for the first week after exposure. The activated medium did not produce any significant deviations from the level of apoptosis in the unirradiated controls. The lowest (10 cGy) dose produced a slight elevation of apoptosis, but this was only significant at 2 days post-irradiation (p<0.05). Induction of apoptosis increased with increasing dose, but in all cases had returned to near-baseline levels by one week after exposure. No further elevation in apoptosis was observed up to 3 weeks post-treatment. Interestingly, the p53 knock-out cells showed nearly the same peak levels of apoptosis as the p53 wild-type parent cell line, but with different kinetics of expression. Apoptotic morphology took longer to manifest in the absence of wild-type p53 protein, and apoptotic cells persisted in culture somewhat longer. Cell cycle distribution and mitotic index Culture in the presence of activated medium did not alter cell cycle distribution. No significant alterations in the mitotic index (p=0.8) were seen in cells cultured with activated medium. In contrast, the lowest dose, 10 cGy, did induce a transient cell cycle disturbance, quantified as a decrease in mitotic index. This decrease was significant at 3 and 8 hours after treatment, with the cultures recovering normal levels of mitosis by 24 hours. Increasing doses resulted in more profound and more prolonged loss of cells from M-phase. Doses of 167 cGy or above essentially eliminated the presence of mitotic cells. Although the initial effects were virtually identical with or without p53, the knock-out cell line appeared to resume normal cell cycle slightly more rapidly than the wild-type TK6. All cultures had recovered normal levels of mitosis by one week post-exposure, and further disturbances were not noted two or three weeks later. Expression of CDKN1A Expression of the CDKN1A gene was measured at multiple time-points. Incubation of cells in activated medium did not result in any significant change in CDKN1A expression at any time-point measured (p=0.85). Peak expression in TK6 cells exposed to 56Fe ions occurred between 3 and 4 hours post-irradiation, consistent with low LET experiments. However, in contrast to the rapid return to near normal gene expression levels expected with low LET, 56Fe ion exposure yielded a prolonged elevation of CDKN1A expression, with little decrease from maximum levels during the first 24 hours. By one week after treatment, levels had returned to normal, and did not increase further through week three. This protracted elevation was observed even at the lowest dose used, which significantly elevated CDKN1A expression throughout the first 24 hours (p<0.0001). The dose response remained fairly linear at all times between 2 and 24 hours. The dose-response is illustrated for TK6 at the peak induction time of 3 hours. There is a slight flattening of the dose-response curve at the highest dose, as is typically seen with highly toxic treatments. Interestingly, there is also a discontinuity evident 7

at the low end of the dose-response curve. This mirrors the response we have seen previously with low LET, where gene induction responses for several genes were found to be linear within a certain dose range, but did not extrapolate back to baseline levels at zero dose. The ionizing radiation response of CDKN1A is dependent on TP53, and as expected, no induction was observed at early times in NH32, the p53 knock-out cell line. At 24 hours, there was a slight elevation in CDKN1A levels in the 56Fe ion treated NH32 cultures, however. This was significant at the 167 cGy dose, although the magnitude of induction remained far below that observed in TK6, the TP53 wild-type cell line. Preliminary microarray results Because the NSRL-6 beam-time, which provided the majority of our samples and data, did not occur until the very end of the one-year funding period, the microarray analysis is still ongoing. This data represents the heart of this project, and will provide the most insight for the direction of future proposed studies.

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In an initial comparison of early gene expression changes by microarray in TK6 cells treated with 56Fe ions, neutrons, and g-rays, both similarities and differences in the early response to HZE and low LET irradiation can be seen. As additional data is added to this analysis, a clearer picture of different response pathways should emerge. Careful analysis of dose and time dependence will be needed to determine if the differences observed represent some unique responses, or alterations in the time or dose responsiveness of genes commonly regulated by low LET. Genes responding to 56Fe ions and g-rays at 3-4 hours after treatment were annotated using EASE and compared to uncover commonly or differentially involved response pathways. Among down-regulated genes, there was a significant involvement of mitosis regulating genes in both the 56Fe ion- and g-ray-treated cells. Categories of up-regulated genes were more diverse, however. Although a slight over-representation of cell cycle regulatory genes was evident in the response to both radiation qualities, the major functional categories involved in the response to 56Fe ions were mitochondrial and other extra-nuclear genes. These same categories of genes were not significantly responsive to g-rays, indicating that different response pathways may indeed be activated by different radiation qualities. It should also be noted that although the initial analysis is focusing on the first 24 hours of response, high quality RNA and supporting cell cycle and apoptosis measurements were obtained at one, two and three weeks after exposure to 56Fe ions. This will enable the extension of these studies to later time-points, beyond the range of most previous studies, even those using only low LET.

Spaceflight environment induces mitochondrial oxidative damage in ocular tissue

More than 30% of the astronauts returning from Space Shuttle missions or the International Space Station (ISS) were diagnosed with eye problems that can cause reduced visual acuity. Xiao W. Mao, Michael J. Pecaut, Louis S. Stodieck, Virginia L. Ferguson, Ted A. Bateman,Mary Bouxsein, Tamako A. Jones, Maria Moldovan, Christopher E. Cunningham, Jenny Chieua and Daila S. Gridleya investigated whether spaceflight environment-associated retinal damage might be related to oxidative stress-induced mitochondrial apoptosis and published their findings in research paper "Spaceflight Environment Induces Mitochondrial Oxidative Damage in Ocular Tissue". Alterations in retinal structure and function, especially retinal vasculature and their association with parenchymal cells, could potentially impact visual acuity and compromise both mission goals and/or long-term quality of life. Retinal damage and degeneration can be accelerated by many factors including aging, ischemia, fluctuation in oxygen tension, oxidative stress and increased intraocular pressure. There is some evidence that cosmic rays induces cell death in the outer nuclear layer of rats flown in space. In addition, data suggest that microgravity induces intraocular pressure and vascular changes in the eye and promotes apoptosis in astrocytes. The study from the Space Shuttle Mission STS-118 showed a significant increase of apoptosis in the photore8

ceptor in the spaceflight mice compared to ground-control mice. Authors have also documented that radiation causes the death of retinal cells by oxidative stress-associated apoptosis. Studies by Yang et al. report that spaceflight is associated with oxidative stress to lipids and DNA. However, the pathophysiological consequences and cellular mechanisms of stress stimuli, especially in the spaceflight environment, in terms of facilitating retinal damage are less studied and remain unclear. The development and progression of retinal degeneration involves, at least in part, both acute and chronic oxidative stress. Oxidative stress-induced ocular tissue damage resulting from reactive oxygen species (ROS) has been associated with a variety of pathological conditions, including age-related macular degeneration, cataract and diabetic retinopathy. The retina contains a high level of polyunsaturated fatty acids, making it particularly susceptible to lipid peroxidation. Photoreceptor death is a central pathology of most forms of retinal degeneration and vascular dysfunction.

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In many cases, cell death induced by oxidative damage has been identified as occurring by the process of apoptosis. Photoreceptor loss can cause irreversible blindness in many retinal diseases. A large body of evidence also supports that increase in oxidative stress in retinal endothelial cell and microvasculature is a key factor for the development of retinal vascular disease, including diabetic retinopathy. Despite the established link between ROS overproduction and pathological neurodegeneration, the source of ROS and the target of oxidative damage remain unclear. Accumulating evidence suggests that oxidative stress-induced mitochondrial dysfunction might have profound implications in the pathogenesis of retinal cell death. The purpose of the investigation was to study spaceflight-induced oxidative damage to ocular tissue by evaluating expression of genes and proteins associated with mitochondria and endothelial cell biology. Female C57BL/6 mice were flown in the space shuttle Atlantis (STS-135), and within 3-5 h of landing, the spaceflight and ground-control mice, similarly housed in animal enclosure modules (AEMs) were euthanized and their eyes were removed for analysis. Results To test the hypothesis that mitochondria are involved in spaceflight-induced retinal cell damage due to oxidative stress; mitochondria-associated gene expression was examined. Of the 84 genes analyzed, 15 were significantly modulated with at least 1.5-fold changes in spaceflight ocular tissue compared to AEM ground controls: 14 up and 1 down-regulated genes (Table 1).

Several genes playing central roles in regulating mitochondrial associated (intrinsic) apoptotic pathways were significantly (P < 0.05) upregulated in mouse ocular tissue after spaceflight compared to the mice in ground

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AEMs. These included: Bak1, Bcl2l1, Bid, Pmaip1, Sh3glb1 and Trp53. The expression of several genes involved in molecular mitochondrial transport and translocations were also significantly enhanced (P < 0.05) after spaceflight compared to that of AEM ground controls. These genes were: Fxc1, Tspo, Opa1, Ucp1 and Ucp2. Several members of the mitochondrial small molecule transport family 25 (Slc25) genes were also significantly regulated.

Endothelial cells are of major importance in normal central nervous system (CNS) function and play a critical role in the reaction and response of the CNS to injury. Expressions of 10 out of 84 analyzed genes were significantly up-regulated with at least 1.5-fold increases compared to AEM using Endothelial Cell Biology RT2 Profilere PCR Array (Table 2). mRNA expressions of several genes playing central roles in regulating vascular endothelial cell response to oxidative stress and apoptosis were also significantly (P < 0.05) enhanced in mouse ocular tissue after spaceflight compared to AEM ground controls. These included: Bcl2l1, Ccl5, Cradd, Plg, Pgf, Tnfaip3 and Vegfa.

Furthermore, the Edn1 gene that is involved in vascular permeability and vessel vasoconstriction regulation was significantly up-regulated (P < 0.05). Proteins encoded by Ccl5, Edn1, Thbd, Plg and Vegfa have been shown to also be responsible for extracellular matrix (ECM) remodeling. In terms of oxidative stress, of 84 analyzed genes, 9 genes were significantly up-regulated and 1 was downregulated at least 1.5-fold compared to AEM ground controls (P <0.05). Many of the up-regulated genes are responsible for regulating the production of ROS including: Nox1, Nos2, Nudt15, Prnp, Txnrd3 and Xpa (Table 3).

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In contrast, mRNA expression of Gpx4, a gene involved in antioxidative defense, was significantly lowered in spaceflight mice compared to ground-control mice. Light microscopy examination revealed a well-organized layered structure in control retinas (Fig. 1A). The cell outline was clear, the structure was compact, and the cells were large with abundant cytoplasm. However, spaceflight mice, retinas appeared slightly disorganized (Fig. 1B). Cells were smaller and fuzzy.

Hyperpigmented cells with pyknosis (condensation of chromatin in nucleus), were also noted in the inner nuclear layer and ganglion cell layer. ROS are involved in lipid peroxidation and membrane lipids are among the major targets of ROS. The occurrence of lipid peroxidation was evaluated with antibody against 4-HNE, which is an indicative marker of oxidative damage to the retina. Increased 4-HNE staining was seen in the retinal ONL and INL after spaceflight (Fig. 2B) compared to AEM ground controls (Fig. 2A). As shown in Fig. 3, the fluorescent intensity, which reflects endogenous level of HNE was also significantly increased (P < 0.01). Immunocytochemical analysis by TUNEL assay showed that spaceflight conditions induced significant apoptosis in the retinal INL and GCL (Fig. 4B) in the spaceflight mice compared to AEM ground controls (Fig. 4A). The staining pattern for apoptosis was similar to that of the 4-HNE compound except that a few TUNEL-positive cells were present in the ONL layer. Our quantitative assessment (Fig.5) revealed that density of apoptotic cells in the retinal INL and GCL of space-flown mice was significantly higher (161.4/mm2 ) compared to the AEM ground-control group that had an average of 65.5/mm2 (P < 0.05). To confirm whether spaceflight-induced apoptosis in retina occurred by caspase signaling pathway, immunohistochemical labeling of activated caspase-3 in retinal sections was performed. Immunoreactivity for activated caspase-3 was visible in the ganglion cell layer and inner nuclear layer of the eyes from the spaceflight group (Fig. 6B). Only a sparse positive caspase-3 signal was found in sections from ground-control eyes (Fig. 6A).

As shown in Fig.7, the density profiles in GCL and INL, which reflect immunoactivity of active caspase-3 were significantly increased in the spaceflight group at 85.3/mm2 compared to the AEM ground-control group at 53.1/mm2 (P < 0.05).

Astronauts on missions have had short and longterm (potentially permanent) changes in vision during and after spaceflight, and there is also experimental evidence linking anomalies in ocular function with spaceflights. After missions in low-earth orbita, astronauts show signs of physiological disturbances including optic disc edema, globe flattening, choroidal folds, hyperopic shifts and cotton wool spots.

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Mader et al. showed that even in transient microgravity conditions produced by parabolic flight, changes in retinal vasculature occur. In this study authors showed for the first time in mice that spaceflight induced oxidative damage that resulted in mitochondrial apoptosis in retina endothelial cells and neurons, which may lead to visual impairment and retinal degeneration.

Other events during and after spaceflight including radiation, hypothermia, hypoxia or gravitation variations also may play important roles in contributing to the spread and ultimate expression of tissue injury. One mechanism that appears to be involved in response to spaceflight, is oxidative stress. Several studies have suggested that microgravity resulted in increased oxidative stress in the nervous system, although it is currently unknown, what amount of gravitational force needs to be maintained to prevent cellular and tissue damage. Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions and modifications in cell membrane composition and increase of lipid peroxidation products may be involved. Also, studies have shown that exposure to microgravity during spaceflight is associated with increased oxidative stress mark12

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ers reflecting damage in lipid, which results in lipid peroxidation in the brain of both humans and rodents. Fluid shifts due to the elimination of gravitational gradients are a fundamental consequence of the entrance into and existence in microgravity, which result in altered cerebral circulation followed by oxidative stress. Increased oxidative stress in simulated microgravity conditions induce increases in microvascular endothelial apoptosis.

A unique feature of the space radiation environment is the presence of high-energy charged particles, which present a significant hazard to spaceflight crews. Long-term exposure to low doses of radiation may contribute

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significantly to health risks for spaceflight crews including those residing on the ISS. Even though the number of these high-charge, high-energy (HZE) particles are relatively small they are particularly effective in inducing complex poorly repairable DNA damage. Although spacecraft shielding can significantly reduce radiation exposure, this protection is not present during extravehicular activities.

Authorâ&#x20AC;&#x2122;s previous data revealed that exposure to total doses of 0.1-1 Gy with low-dose-rate (LDR) photon radiation induced oxidative stress and ECM associated alterations in gene expression profiles in the skin. Chang et al. showed that alteration in the profile of genes known to be involved in neurotrophic functions in the hippocampal tissue appears to persist for up to 8 weeks after LDR radiation exposure at a total dose of 0.5 Gy. The development and progression of radiation-induced effects involves, at least in part, an acute and chronic oxidative stress. Indications of persistent oxidative stress after irradiation have been demonstrated in the brain of mice. A variety of effects in these cells, ranging from a dose-dependent apoptosis to activation of cell cycle checkpoints, were observed. Oxidative injury has been implicated as playing a causative or contributory role in a number of neurodegenerative conditions, e.g., aging, as well as ischemic and traumatic damage. The retinal layer is sensitive to oxidative injury due to its high oxygen consumption, content of oxidizable unsaturated lipids and low levels of antioxidant defenses. Many studies have shown that oxidative stress constitutes a biochemical mechanism regulating cellular function and the ultimate damage of ocular tissue. Just as lipid peroxidation was relatively greater in the retinal INL and GCL of spaceflight mice compared to AEM ground-control mice, apoptosis was also greater in the retinal INL of the spaceflight mice compared to their counterparts on the ground. The oxidative damage may result in cell death, at least in part by apoptosis, causing morphologic changes in the inner nuclear and ganglion cell layers, and may eventually lead to decline in retinal function. The data provides a model of retinal damage and degeneration due to oxidative damage occurring after the spaceflight mission, which can be used to further test antioxidant treatments. Oxidative stress triggers multiple signaling pathways, including some that are cytoprotective and others that contribute to cell death. Among these are the bcl-2 family proteins. The expression of pro- and anti-apoptotic Bcl-2 family proteins is regulated by oxidative stress, and these genes encode proteins that are a major factor in the outcome of apoptotic signaling.

The study showed that expression of Bak, Bid and Bcl2l were significantly enhanced shortly after landing. The proteins encoded by these genes are located at the outer mitochondrial membrane, and have been shown to regulate mitochondrial membrane potential, and thus controls the production of ROS and release of cytochrome C by mitochondria, and induces of cell apoptosis. This result implies that oxidative stress-induced changes in the expression of Bcl-2 genes may represent events upstream of the mitochondrial apoptotic step. Mitochondria are prominent in photoreceptors, clustering in the inner segment and the axon terminals. Among the various intracellular targets for ROS-mediated injury, mitochondria are thought to be particularly prone to ROS induced damage. These organelles consume >95% of the cellâ&#x20AC;&#x2122;s oxygen. Oxidative insult to the mitochondria can alter their membrane permeability transition and induce cytochrome c leakage from the mitochondria. The leakage of cytochrome c from the mitochondrial inner membrane compartment triggers a series of proapoptotic signal transduction processes, resulting in apoptotic cell death.

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Investigators have shown changes in the mitochondrial membrane potential are associated with the induction of photoreceptor apoptosis. The gene expression data reported here supports the notion that mitochondria are likely to be the target for spaceflight-associated oxidative stress induced cell death. Of the 84 evaluated genes associated with oxidative stress, the greatest enhancements (over threefold) after spaceflight occurred in nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) and nitric oxide synthase 2 (Nos2). There are several potential sources of ROS in the eye, including xanthine oxidase, mitochondrial enzymes, NADPH oxidase and enzymes involved in nitric oxide (NO) synthesis or arachidonic acid metabolism. A growing body of evidence supports roles for abnormal NADPH oxidase activation in retinal oxidative levels. In vascular cells, NADPH oxidases appear to be the important physiological factor involved in ROS production. Several reports have shown that the free radical NO appears to plays a role in mitochondrial dysfunction in the CNS by trigging mitochondrial fission, synaptic loss and neuronal death. The inducible isoform of NO synthase, termed Nos2 is expressed in macrophages, microglia and reactive astrocytes when these cells are activated. The data support the pathophysiological relationship between NO-mediated ROS production and retinal damage. The vertebrate retina contains the cells that are organized into 3 nuclear layers: ONL, INL and GCL. The increase of TUNEL-positive cells in retinal INL and ganglion cell layer from flight mice but not in ONL layer was notable. The ONL in retina is a tightly packed layer, in which the somas of rods and cones are crowded together, with relatively little cytoplasm. Larger organelles, such as mitochondria, are sparse in these somas. Most of the mitochondria of retina are located in the outer part of the inner segment. Mitochondra are a major source of signals that regulate apoptosis. Fewer numbers of mitochrondria in ONL probably account for few signals that converge on these organelles to trigger downstream pathways that lead to acute apoptotic cell death. Other potential sources of signals that regulate oxidative stress-induced apoptosis in the retina, including NADPH oxidase, and enzymes involved in nitric oxide synthesis may be responsible for releasing factors that cause damage to ONL cells at later time. This study showed that expression of genes and proteins involved in oxidative stress, mitochondrial and endothelial cell biology are significantly modified shortly after return from the spaceflight environment and resulted in increased apoptosis in the retina. However, this experiment did not allow us to determine whether the effects authors observed were transient, reversible or would deteriorate over time. Exposure to space conditions may result in a low level of retinal neuronal cell loss that can be compensated for by physiological adaptation, but if cell loss is significant, the capacity for physiological compensation may be exceeded. Degeneration of retinal neurons will ultimately lead to irreversible vision loss. Further spaceflight investigations should be conducted to evaluate the late effects of retinal response and association of visual function with spaceflight-induced retinal cell loss. One of the complications associated with determining the ocular response of stress insults is the latency between exposure and the expression of injury (e.g., cell loss or dysfunction). To obtain accurate data for development and progression of the injury response, it may be necessary to quantify changes over a long period of time. Regardless, this data does indicate that astronauts may be at risk for retinal damage and late degeneration. It is possible that reported changes envisioned by astronauts are trigged by the combination of factors including gravitational force, solar radiation, impact of launching, reentry into the Earthâ&#x20AC;&#x2122;s atmosphere, or fluctuations in the oxygen level aboard the space shuttle.

Impact of Spaceflight and Artificial Gravity on the Mouse Retina: Biochemical and Proteomic Analysis

The data from Space Shuttle studies showed a significant increase of apoptosis in retinal cells of flight mice compared to ground controls, which could lead to morphologic changes and impairment of retinal function. However, the pathophysiological consequences and cellular mechanisms of stress stimuli, especially those associated with a spaceflight environment, in facilitating retinal damage are less studied and remain unclear. Data on the response of biochemical systems to altered gravity is very limited in animal studies. Some lines of evidence suggest that one of the mechanisms involved in response to spaceflight, including changes in the gravity vector, is likely due to oxidative stress. Studies have shown that exposure to microgravity during spaceflights is associated with elevations in oxidative stress and production of lipid peroxidation in both humans and rodents.

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However, the knowledge about the duration or dose of microgravity exposure needed to induce oxidative stress or alterations in retinal structure and function is extremely limited. For example, one study has shown that even transient exposure to microgravity produced by parabolic flight can cause alterations in retinal vasculature to occur. One means to address the impact of microgravity conditions is through the application of artificial gravity (AG) during spaceflight. Creating AG by centrifugal force makes it possible to have a partial gravity or Earth-like gravity (1 g) control in space, where all other environmental conditions associated with spaceflight are present. A newly developed mouse habitat cage unit (HCU) by Japan Aerospace Exploration Agency (JAXA) has been installed in the Centrifuge-equipped Biological Experiment Facility (CBEF) on board the ISS. This rodent centrifugation capability provides opportunities to compare the effectiveness of AG prescription under spaceflight conditions. Since the HCU can reproduce Earth-like gravity, it can be used to test the effectiveness of AG to mitigate the effects of microgravity on physiological systems. The goal of this study was to investigate the effects of spaceflight on oxidative stress and apoptosis in retinal endothelial cells and to identify spaceflight-induced changes in protein expression profiles in mouse ocular tissue. Additionally, authors sought to determine whether the application of 1 g AG during spaceflight could mitigate any detrimental effects of microgravity on the retina. Authors hypothesized that spaceflight would induce elevations in oxidative stress and apoptosis in retinal endothelial cells, as well as alter ocular proteins associated with apoptosis, cell repair, inflammation and metabolic function. They further hypothesized that the application of 1 g AG would mitigate these changes. Results Apoptosis in Retinal Endothelial Cells Following Spaceflight Immunocytochemical analysis by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay showed that spaceflight conditions induced significant apoptosis in the retinal endothelial cells (Figure 1A) relative to that in the habitat and vivarium control conditions and in the AG (Âľg + 1 g) condition. The quantitative assessment revealed that the density of apoptotic cells in the retina was the highest in the Âľg group, and was 64% greater than that in the habitat control group (Figure 1B). 4-Hydroxynonenal (4-HNE) Immunoreactivity Following Spaceflight Reactive oxygen species (ROS) are involved in lipid peroxidation and membrane lipids are among the major targets of ROS. The occurrence of lipid peroxidation was evaluated with immunohistochemistry with an antibody against 4-HNE, which is an indicative marker of oxidative damage to the retina (Figure 1C). There were no significant differences among groups in the level of 4-HNE immunoreactivities (Figure 1D). Proteomics on Mouse Ocular Tissue Protein expression profile analysis were focused on comparisons between flight groups vs habitat control (HC). Analyzing the proteomic changes induced by flight conditions vs the HC group is more relevant for determining the effects of weightlessness and AG since HC mice were placed in the same flight hardware (cages) used in flight and environmental parameters such as temperature, humidity and carbon dioxide (CO2 ) levels were matched to that during spaceflight. Five micrograms of protein from each eyecup sample was resolved by 4-20% sodium dodecyl sulfate (SDS) Tris-Gly gel electrophoresis, visualized by Coomassie stain, in-gel trypsin digested, and analyzed by LC/MS on an LTQ Orbitrap Velos mass spectrometer. Figure 2A shows a gel image depicting one representative sample for each of the three sample groups. 16

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A MaxQuant database search (restricted to Mus musculus) identified a total of 4179 proteins from all 18 samples. A Venn diagram indicates that we identified 3174 (76%) common proteins in all groups and, therefore, differences between the groups are reflective of the intensities of the proteins detected (Figure 2B). A small percentage of proteins were uniquely identified in each of the Âľg + 1 g, Âľg, and HC samples and may reflect specific changes within each group. The MS1 precursor intensities were converted to relative iBAQ intensities by dividing the iBAQ intensity by the sum of all iBAQ intensities in the sample. The samples were normalized based on the median relative iBAQ intensities for each sample, log2 transformed, and missing values were imputed based on the normal distribution. The log2 normalized iBAQ intensities were used for analyses.

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Authors identified 250 and 171 significantly differentiating proteins comparing the µg versus HC and µg + 1 g versus HC sample groups, respectively, based on a Mann-Whitney U test with a false discovery rate (FDR) corrected p-value < 0.05. A hierarchical cluster was generated for both comparisons (Figure 3) and visually representing the significant protein intensities for each sample group. They further filtered the list of significant proteins by using a fold change >2 threshold. By using a p-value and a protein expression threshold, they were able to identify the top significant proteins. These top significant proteins are seen in the heat maps as the clusters of proteins with the largest changes in intensity (yellow to blue) (Figure 3). Volcano plots were used to visualize all identified proteins and highlight the significant proteins for each comparison in the study using R studio (Figure 4). The y-axis consists of -log10 p-values based on the Mann-Whitney U FDR adjusted p-values, while the x-axis consists of the log2 fold change. The vertical lines indicate up and down expression using a fold change >2 threshold. The horizontal line indicates a p-value of 0.05. They identified 77 and 23 significant proteins based on these two criteria for the µg versus HC and µg + 1 g versus HC, respectively. These differentially expressed proteins were then analyzed using Ingenuity Pathway Analysis. There were significant changes in canonical pathways and upstream regulators in the pathway analysis for flight groups compared to the HC controls. The proteomic analysis identified only 33 upstream regulator proteins based on the Z-scores used to predict either activation (Z-score > 0) or inhibition (Z-score < 0) in the µg + 1 g animals compared to HC mice that involves disease development, molecular/cellular function and cell signaling, while 60 regulators were identified in the µg group compared to HC mice. IPA analysis revealed many key pathways are affected that are responsible for cell death, cell repair, inflammation, carbohydrate metabolism, mitochondrial function, 19

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fatty acid metabolism, and oxidative phosphorylation when comparing the µg group with HC group. Pathways were considered significant based on the Fisher exact test with a -log10 p-value > 1.3 (corresponds to a p-value < 0.05).

Significantly affected canonical pathways between µg + 1 g vs. HC or µg group vs HC or are shown in Figure 5A,B, respectively. There were also significant changes in regulated protein expression in the pathway analysis for µg or µg + 1 g groups compared to HC group or µg group vs. µg + 1 g group. There was very little overlap of significantly regulated protein between µg vs. HC and µg + 1 g vs. HC. In the µg group, there was significant alteration in functions related to overall organismal survival, cellular assembly and metabolism. Significant increased or decreased protein expression levels are summarized and listed in Table 1, Table 2 and Table 3. This study demonstrates that spaceflight induces apoptosis in retinal vascular endothelial cells. Authors 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 AG provided by centrifugation during spaceflight as a countermeasure for mitigating putative effects of microgravity on ocular structure and function. In this study, several oxidative stress-related signaling pathways are significantly altered in the µg group, which includes fatty acid β-oxidation I, fatty acid activation and α-adrenergic pathway. However, they did not find a significant change in an oxidative stress marker for lipid peroxidation, 4-HNE when measured two days after splash-down.

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In the previous space shuttle Atlantis (STS-135) study, the level of HNE protein was significantly elevated in the retina after spaceflight compared to controls. This is not surprising as these two flight conditions are different. Female C57BL/6 mice were flown in the STS-135 for 13-day mission, and within 3-5 h of landing, eyes were removed for analysis.

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In the current study, male mice were flown to the ISS for a 35-day mission, and the mice were euthanized at about 40 h after splashing down. Differences in gender, mission duration and recovery time might contribute to their dynamic stress response. The level of oxidative stress can be affected by their stress reaction to a prolonged flight in space and in the period of re-adaptation to the normal gravity after landing. Another possible explanation for the lack of significant change in 4-HNE in the current study is that spaceflight might elicit protective effects in the central nervous system (CNS) by inhibiting some gravity-initiated stress signaling pathway. Of all the proteins significantly altered by spaceflight, the Cap-Gly domain containing linker protein 2(CLIP2), also known as CLIP-115, was significantly down-regulated in both µg and µg + 1 g groups. This protein is found predominantly in the CNS, where it likely plays a role in the normal structure and function of nerve cells. Within cells, this protein is thought to regulate cell cytoskeleton function that helps to determine cell shape, size, and movement. Figure 6A illustrates CLIP2 in the network of proteins responsible for cellular assembly and organization, cell signaling and interaction as defined by IPA in response to spaceflight.

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Methyl-CpG-binding protein 2 (MECP2) had the highest fold changes in flight mice (µg group) compared to HC. This protein binds to methylated DNA and regulates chromatin structure and transcription. MECP2 is most abundantly produced in the brain where it is expressed primarily in post-mitotic neurons. Previous work has also shown MECP2 mediated epigenetic regulation of senescent endothelial progenitor cells and promoted apoptosis. Furthermore, a recent study provides evidence that elevated MECP2 in mice causes neurodegeneration. Figure 6B shows the regulative role of MECP2 in a network of proteins that involves cell death and survival. The data from this study indicates that adding 1 g on the ISS is effective in reducing the endothelial cell damage and increasing cellular organization and function compared to the µg group. Another study of the same animals used in our present study also showed that 1 g AG served to maintain femur bone density and soleus/gastrocnemius muscle mass similar to that in the ground control group, whereas significant decreases in bone density and muscle mass occurred in the flight mice without centrifuge. These studies provide evidence that in-flight AG can mitigate some of the effects of weightlessness during spaceflight.

Quantification Of The Dose Response Of The Microvessel Parameters In Retina, Cortex And White Matter Following Iron-56 Irradiation And Proton Irradiation (NNJ04HD79G)

A main concern of NASA managers is to define the risks of tissue damage of brain and eye for astronauts associated with exposure to galactic cosmic ray particle (GCR) irradiation during a proposed space mission to Mars. Research is needed to specify the tissue effects produced by the high Z, high energy (HZE) particles that are found in space and to define the dose response of these tissues. Current histopathologic methods used to evaluate tissue changes in late-responding, non-proliferative tissues such as the brain and vessel endothelium rely on observations, word descriptions and arbitrary scoring. The approach is considered to be qualitative and lacks rigor. The proposed research introduces a detailed, step-by-step stereology program into radiation biology that will quantify the dose response of tissue population changes in situ following irradiation. These methods are known to anatomists and materials scientists but have been ignored by radiation biologists until this laboratory used them to quantify the dose response of the non-proliferative retinal vasculature following single and split time-dose schedules of protons. The brain and retina microvessel endothelial cell population number, vessel length, and number of micro vessel branching will be measured following single and split dose schedules of Iron-56 particles. The results will be compared with the changes produced by protons to define the dose response of the cortex and white matter microvessel endothelial populations and to specify the RBE quality factor of Iron-56 referenced to protons. Three-dimensional reconstructions of the micro vessels of the cortex and white matter will be compared. The objective is to utilize three dimensional reconstruction and stereology methods to quantify the micro vessel population and vessel cell kinetic parameters in the cortex and white matter regions of the brain in an effort to explain the difference in dose response between the two regions. 24

The significance of this research for NASA and for radiation biology is that it introduces into radiation biology a valuable quantitative technique that can be applied to quantify population morphologic, kinetic and identifiable functions in-situ following irradiation. It also defines the contribution (role) of the irradiated vasculature in producing radiation changes and the dose response of the brain and retina. RESULTS • Unbiased stereology was used to determine the population density of the pericytes and endothelial cells of the cortex and corpus callosum as well as the microvessel length density. • Using computer assisted 3 dimensional configurations and confocal immunohistochemical microscopy The branching of cortical and corpus callosum microvessels and location of the microvessel population nuclei was demonstrated. • The micro vessels of the corpus callosum are demonstrated to be nearly straight and less branching than those of the cortex.

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• Using the computer technology of our Czech and Slovak collaborators it was possible to fuse confocal small images into a composite image consisting of 9 adjacent fields of the hippocampus and adjacent corpus. • The dose response of the endothelial and pericyte cell density of the cortex and corpus callosum were determined following iron-56 ion and proton irradiation dose schedules. The endothelial cell population of the cortex and corpus and the microvessel length density were proliferative and increased their population. The pericyte population was not proliferative and their population decreased over a period of two years. • Using retinal digest techniques we determined the RBE of the iron-56 ion to be 3.45 compared to the proton density change as the standard.

Conclusion • It was documented that the dose response or cell density changes were the same using split dose schedules compared with single dose schedules of protons. This suggeststhat the dose response will not be markedly changed using fractionated irradiation. • It was shown that the microvessels of the cortex were more branched, and longer than the vessels of the white matter. The cortical cell density and branchings of the vessels of the cortex were larger than the same measurements for the corpus callosum. • The endothelial cells of the cortex and the corpus were proliferative and increased their number over the two year observational period following proton and Iron-56 ion brain irradiation with doses of 5 to 20Gy. Whereas, pericytes showed cell loss, but (surprisingly) there was an increase in vessel length density. • The data confirm the concerns of NASA managers that whole brain irradiation will with 5 to 20 Gy of protons or Iron-56 irradiation will produce an increase in cell density of endothelial cells and length density of microvessel, and will produce a significant loss of pericytes.

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