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Description, Space radiation affects memory in male and not in female mice, and it is dependent on the innate immune system response., In the coming decade, astronauts will travel back to the moon in preparation for future Mars missions. Exposure to galactic cosmic radiation (GCR) is a major obstacle for deep space travel. Using multivariate principal components analysis, we found sex-dimorphic responses in mice exposed to accelerated charged particles to simulate GCR (GCRsim); males displayed impaired spatial learning, whereas females did not. Mechanistically, these GCRsim-induced learning impairments corresponded with chronic microglia activation and synaptic alterations in the hippocampus. Temporary microglia depletion shortly after GCRsim exposure mitigated GCRsim-induced deficits measured months after the radiation exposure. Furthermore, blood monocyte levels measured early after GCRsim exposure were predictive of the late learning deficits and microglia activation measured in the male mice. Our findings (i) advance our understanding of charged particle–induced cognitive challenges, (ii) provide evidence for early peripheral biomarkers for identifying late cognitive deficits, and (iii) offer potential therapeutic strategies for mitigating GCR-induced cognitive loss.

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/− 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4–19.3 mmHg vs 16.3–20.3 mmHg) (p

There is concern that degradation of vision as a result of space flight may compromise both mission goals and long-term quality of life after space travel. The visual disturbances may be due to a combination of intracerebral pressure changes and exposure to ionizing radiation. The retina and the retinal vasculature play important roles in vision, yet have not been studied extensively in relation to space travel and space radiation. The goal of the present study was to characterize oxidative damage and apoptosis in retinal endothelial cells after whole-body gamma-ray, proton and oxygen ((16)O) ion radiation exposure at 0.1 to 1 Gy. Six month old male C57Bl/6J mice were exposed whole-body to 600 MeV/n (16)O ions (0, 0.1, 0.25, 1 Gy), solar particle events (SPE)-like protons (0, 0.1, 0.25, 0.5 Gy) or (60)Co gamma-rays (0, 0.1, 0.25, 0.5 Gy). Eyes were isolated for examining endothelial nitric oxide synthase (eNOS) expression and characterization of apoptosis in retina and retinal endothelial cells at 2 weeks post irradiation. The expression of eNOS was significantly increased in the retina following proton and (16)O ions exposure. (16)O ions induced over 2-fold increase in eNOS expression compared to protons at the 2-week post irradiation time point (p

There are serious concerns about possible late radiation damage to ocular tissue from prolonged space radiation exposure, and occupational and medical procedures. This study aimed to investigate the effects of whole-body high-energy proton exposure at a single dose on apoptosis, oxidative stress, and blood-retina barrier (BRB) integrity in the retina and optic nerve head (ONH) region and to compare these radiation-induced effects with those produced by fractionated dose. Six-month-old C57BL/6 male mice were either sham irradiated or received whole-body high energy proton irradiation at an acute single dose of 0.5 Gy or 12 equal dose fractions for a total dose of 0.5 Gy over twenty-five days. At four months following irradiation, mice were euthanized and ocular tissues were collected for histochemical analysis. Significant increases in the number of apoptotic cells were documented in the mouse retinas and ONHs that received proton radiation with a single or fractionated dose (p < 0.05). Immunochemical analysis revealed enhanced immunoreactivity for oxidative biomarker, 4-hydroxynonenal (4-HNE) in the retina and ONH following single or fractionated protons with more pronounced changes observed with a single dose of 0.5 Gy. BRB integrity was also evaluated with biomarkers of aquaporin-4 (AQP-4), a water channel protein, a tight junction (TJ) protein, Zonula occludens-1 (ZO-1), and an adhesion molecule, the platelet endothelial cell adhesion molecule-1 (PECAM-1). A significantly increased expression of AQP-4 was observed in the retina following a single dose exposure compared to controls. There was also a significant increase in the expression of PECAM-1 and a decrease in the expression of ZO-1 in the retina. These changes give a strong indication of disturbance to BRB integrity in the retina. Interestingly, there was very limited immunoreactivity of AQP-4 and ZO-1 seen in the ONH region, pointing to possible lack of BRB properties as previously reported. Our data demonstrated that exposure to proton radiation of 0.5 Gy induced oxidative stress-associated apoptosis in the retina and ONH, and changes in BRB integrity in the retina. Our study also revealed the differences in BRB biomarker distribution between these two regions. In response to radiation insults, the cellular response in the retina and ONH may be differentially regulated in acute or hyperfractionated dose schedules.

Interplanetary exploration will be humankind's most ambitious expedition and the journey required to do so, is as intimidating as it is intrepid. One major obstacle for successful deep space travel is the possible negative effects of galactic cosmic radiation (GCR) exposure. Here, we investigate for the first time how combined GCR impacts long-term behavioral and cellular responses in male and female mice. We find that a single exposure to simulated GCR induces long-term cognitive and behavioral deficits only in the male cohorts. GCR exposed male animals have diminished social interaction, increased anxiety-like phenotype and impaired recognition memory. Remarkably, we find that the female cohorts did not display any cognitive or behavioral deficits after GCR exposure. Mechanistically, the maladaptive behavioral responses observed only in the male cohorts correspond with microglia activation and synaptic loss in the hippocampus, a brain region involved in the cognitive domains reported here. Furthermore, we measured reductions in AMPA expressing synaptic terminals in the hippocampus. No changes in any of the molecular markers measured here are observed in the females. Taken together these findings suggest that GCR exposure can regulate microglia activity and alter synaptic architecture, which in turn leads to a range of cognitive alterations in a sex dependent manner. These results identify sex-dependent differences in behavioral and cognitive domains revealing promising cellular and molecular intervention targets to reduce GCR-induced chronic cognitive deficits thereby boosting chances of success for humans in deep space missions such as the upcoming Mars voyage.

Microglia are the main immune component in the brain that can regulate neuronal health and synapse function. Exposure to cosmic radiation can cause long-term cognitive impairments in rodent models thereby presenting potential obstacles for astronauts engaged in deep space travel. The mechanism/s for how cosmic radiation induces cognitive deficits are currently unknown. We find that temporary microglia depletion, one week after cosmic radiation, prevents the development of long-term memory deficits. Gene array profiling reveals that acute microglia depletion alters the late neuroinflammatory response to cosmic radiation. The repopulated microglia present a modified functional phenotype with reduced expression of scavenger receptors, lysosome membrane protein and complement receptor, all shown to be involved in microglia-synapses interaction. The lower phagocytic activity observed in the repopulated microglia is paralleled by improved synaptic protein expression. Our data provide mechanistic evidence for the role of microglia in the development of cognitive deficits after cosmic radiation exposure.

PURPOSE: Exposure to proton irradiation during missions in deep space can lead to bone marrow injury. The acute effects of proton irradiation on hematopoietic stem and progenitor cells remain undefined and thus were investigated. MATERIALS AND METHODS: We exposed male C57BL/6 mice to 0.5 and 1.0 Gy proton total body irradiation (proton-TBI, 150 MeV) and examined changes in peripheral blood cells and bone marrow (BM) progenitors and LSK cells 2 weeks after exposure. RESULTS: 1.0 Gy proton-TBI significantly reduced the numbers of peripheral blood cells compared to0.5 Gy proton-TBI and unirradiated animals, while the numbers of peripheral blood cell counts were comparable between 0.5 Gy proton-TBI and unirradiated mice. The frequencies and numbers of LSK cells and CMPs in BM of 0.5 and 1.0 Gy irradiated mice were decreased in comparison to those of normal controls. LSK cells and CMPs and their progeny exhibited a radiation-induced impairment in clonogenic function. Exposure to 1.0 Gy increased cellular apoptosis but not the production of reactive oxygen species (ROS) in CMPs two weeks after irradiation. LSK cells from irradiated mice exhibited an increase in ROS production and apoptosis. CONCLUSION: Exposure to proton-TBI can induce acute damage to BM progenitors and LSK cells.

One of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.

The purpose of the current study was to characterize the effects of simulated microgravity and radiation-induced changes in retina and retinal vasculature, and to assess the accompanying early changes in immune cells and hematological parameters. To better understand the effects of spaceflight, we used a combination of treatments designed to simulate both the radiation and low-gravity aspects of space conditions. To simulate the broad energy spectrum of a large solar particle event (SPE) and galactic cosmic ray (GCR) radiation, male C57BL/6J mice were exposed to whole-body irradiation using fully modulated beams of 150-MeV protons containing particles of energy from 0 to 150 MeV and a uniform dose-vs.-depth profile. The mice were also hindlimb-unloaded (HLU) by tail suspension. Mice were unloaded for 7 days, exposed to 50 cGy, unloaded for an additional 7 days and then sacrificed for tissue isolation at days 4 and 30 after the combined treatments. Increases in the number of apoptotic cells were observed in the endothelial cells of mice that received radiation alone or with HLU compared to controls at both days 4 and 30 (P < 0.05). Endothelial nitric oxide synthase (eNOS) levels were significantly elevated in the retina after irradiation only or combined with HLU compared to controls at the 30-day time point (P < 0.05). The most robust changes were observed in the combination group, suggesting a synergistic response to radiation and unloading. For hematopoietic parameters, our analysis indicated the main effects for time and radiation at day 4 after treatments (day 11 postirradiation) (P < 0.05), but a smaller influence of HLU for both white blood cell and lymphocyte counts. The group treated with both radiation and HLU showed greater than 50% reduction in lymphocyte counts compared to controls. Radiation-dependent differences were also noted in specific lymphocyte subpopulations (T, B, natural killer cells). This study shows indications of an early effect of low-dose radiation and spaceflight conditions on retina and immune populations.

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The hippocampus is critical for learning and memory, and injury to this structure is associated with cognitive deficits. The response of the hippocampal microvessels after a relatively low dose of high-LET radiation remains unclear. In this study, endothelial population changes in hippocampal microvessels exposed to (56)Fe ions at doses of 0, 0.5, 2 and 4 Gy were quantified using unbiased stereological techniques. Twelve months after exposure, mice that received 0.5 Gy or 2 Gy of iron ions showed a 34% or 29% loss of endothelial cells, respectively, in the hippocampal cornu ammonis region 1 (CA1) compared to age-matched controls or mice that received 4 Gy (P0.05). We suggest that this "U-shaped" dose response indicates a repopulation from a sensitive subset of endothelial cells that occurred after 4 Gy that was stimulated by an initial rapid loss of endothelial cells. In contrast to the CA1, in the dentate gyrus (DG), there was no significant difference in microvessel cell and length density between irradiated groups and age-matched controls. Vascular topology differences between CA1 and DG may account for the variation in dose response. The correlation between radiation-induced alterations in the hippocampal microvessels and their functional consequences must be investigated in further studies.

Health risks due to exposure to high-linear energy transfer (LET) charged particles remain unclear. The major goal of this study was to confirm and further characterize the acute effects of high-LET radiation ((56)Fe(26)) on erythrocyte, thrombocyte and leukocyte populations in three body compartments after total-body exposure. Adult female C57BL/6 mice were irradiated with total doses of 0, 0.5, 2 and 3 Gy and killed humanely 4 days later. Body and organ masses were determined and blood, spleen and bone marrow leukocytes were evaluated using a hematology analyzer and flow cytometry. Spleen and thymus (but not body, liver and lung) masses were significantly decreased in a dose-dependent manner. In general, red blood cell (RBC) counts and most other RBC parameters were depressed with increasing dose (P0.05); the major exception was an increase in cell size at 0.5 Gy. Platelet numbers and volume, total white blood cell counts, and all three major types of leukocytes also decreased (P0.05). Lymphocyte populations in blood and spleen exhibited variable degrees of susceptibility to (56)Fe-particle radiation (BTNK and T cytotoxicT helper cells). In the bone marrow, leukocytes with granulocytic, lymphocytic ("dim" and "bright"), and monocytic characteristics exhibited proportional variations at the higher radiation doses in the expression of CD34 and/or Ly-6A/E. The data are discussed in relation to our previous investigations with iron ions, other forms of radiation, and space flight in this same animal model.

We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by RT-PCR differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density.

Our previous results with flight (FLT) mice showed abnormalities in thymuses and spleens that have potential to compromise immune defense mechanisms. In this study, the organs were further evaluated in C57BL/6 mice after Space Shuttle Atlantis returned from a 13-day mission. Thymuses and spleens were harvested from FLT mice and ground controls housed in similar animal enclosure modules (AEM). Organ and body mass, DNA fragmentation and expression of genes related to T cells and cancer were determined. Although significance was not obtained for thymus mass, DNA fragmentation was greater in the FLT group (P

Biological model: In this project, we investigated the control of radiation-induced genotoxic damage expression in somatic cells of the nematode Caenorhabditis elegans. We measured genotoxic damage in the C. elegans intestine by irradiating young larvae with 20 intestinal cells. Fourteen of these cells undergo exactly one nuclear division without cytoplasmic division leading to 14 binucleate cells. This nuclear division is synchronized and occurs at the first larval molt. Irradiation induces chromosome aberrations including dicentrics which we can quantify as stable anaphase bridges in the binucleate cells of young adult intestines. The endpoint is dose- and LET-dependent and we have demonstrated that individual intestinal cells have unique radiosensitivities. Results: The project has two components, a genetic screen for genes that control cell sensitivity and a microbeam component to directly probe individual cells. The genetic screen has identified several genes in NHEJ repair and telomere metabolism that modulate overall bridge frequency. Knockout mutants of cku-70, cku-80 and lig-4 greatly sensitize animals for anaphase bridge induction. A statistical method was used to determine whether induction of bridges was strictly random and cell autonomous and we determined that expression of bridges in pairs of cells was, in fact, non-random which suggested that signaling between cells affected the pattern of bridge expression. This allowed us to conduct an RNAi and mutation screen for genes that control the signaling (block non-random distributions) and several candidates have been identified. To directly test the notion that signaling of genotoxic damage occurs, we conducted experiments with alpha particles collimated through slits in metal foils and showed that genotoxic damage could be expressed many cell diameters away from a partial body exposure site. Thus, an in vivo bystander effect was demonstrated. Dose targeting was then improved to small regional exposures and eventually to individual cell targeting using 2 MeV protons from the microbeam facility at Texas A&M University. We now employ a green fluorescent protein (GFP)-expressing transgenic worm (rrIs1[elt-2::GFP]) to target GFP-positive gut cells via the gut-specific transcription factor elt-2. This allows alignment of the cell of interest over the microbeam aperture under appropriate fluorescence illumination. Microbeam irradiation experiments for many pairwise combinations of cell signal transmission and reception (observed as expression of anaphase bridges) have been conducted and several interesting patterns emerge. (i) The signaling pattern is cell-specific and does not simply reflect cell–cell distance or require direct contact between cell pairs. (ii) The signal range can be as far as from cell pair 2 to cell pair 8 (>100 µm). (iii) There appears to be a functional compartment boundary at the pharynx/intestine valve as even high-dose exposures to the posterior pharyngeal bulb fail to induce bridges in nearby intestinal cells. (iv) The frequency of signal transmission and reception corresponds broadly to the overall frequency of bridges observed during whole-body irradiations which suggests that direct irradiation and ‘out-of-field’ effects may be additive. These patterns have been analyzed in terms of a cellular logic circuit map for signal transmission and reception. A dose–response for a subset of microbeam-targeted cells was measured over the range of 5–20 Gy. Controlled cell pair targeting was used to test the potential additivity of signals and we found that effects were supra-additive. Finally, preliminary measurements were conducted on GFP-expressing transgenic strains that bore cku-70(tm1524) III and smk-1(mn156) V mutations which confer enhanced radiosensitivity. Cku-70 is a Ku-70 ortholog while smk-1 is orthologous to the mammalian and Dictyostelium discoideum SMEK (suppressor of MEK null) protein. In the cku-70(0/0) strain, the severity of the bridges in bystander cells was enhanced, suggesting that signal recipient cells employ NHEJ repair pathways in the expression of anaphase bridges. Clinical trial registration number: Not applicable.