Simulated Microgravity Alters Gene Regulation Linked to Immunity and Cardiovascular Disease.

Microgravity exposure induces a cephalad fluid shift and an overall reduction in physical activity levels which can lead to cardiovascular deconditioning in the absence of countermeasures. Future spaceflight missions will expose crew to extended periods of microgravity among other stressors, the effects of which on cardiovascular health are not fully known. In this study, we determined cardiac responses to extended microgravity exposure using the rat hindlimb unloading (HU) model. We hypothesized that exposure to prolonged simulated microgravity and subsequent recovery would lead to increased oxidative damage and altered expression of genes involved in the oxidative response. To test this hypothesis, we examined hearts of male (three and nine months of age) and female (3 months of age) Long-Evans rats that underwent HU for various durations up to 90 days and reambulated up to 90 days post-HU. Results indicate sex-dependent changes in oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) and antioxidant gene expression in left ventricular tissue. Three-month-old females displayed elevated 8-OHdG levels after 14 days of HU while age-matched males did not. In nine-month-old males, there were no differences in 8-OHdG levels between HU and normally loaded control males at any of the timepoints tested following HU. RNAseq analysis of left ventricular tissue from nine-month-old males after 14 days of HU revealed upregulation of pathways involved in pro-inflammatory signaling, immune cell activation and differential expression of genes associated with cardiovascular disease progression. Taken together, these findings provide a rationale for targeting antioxidant and immune pathways and that sex differences should be taken into account in the development of countermeasures to maintain cardiovascular health in space.

Sexual dimorphism during integrative endocrine and immune responses to ionizing radiation in mice.

Exposure to cosmic ionizing radiation is an innate risk of the spaceflight environment that can cause DNA damage and altered cellular function. In astronauts, longitudinal monitoring of physiological systems and interactions between these systems are important to consider for mitigation strategies. In addition, assessments of sex-specific biological responses in the unique environment of spaceflight are vital to support future exploration missions that include both females and males. Here we assessed sex-specific, multi-system immune and endocrine responses to simulated cosmic radiation. For this, 24-week-old, male and female C57Bl/6J mice were exposed to simplified five-ion, space-relevant galactic cosmic ray (GCRsim) radiation at 15 and 50 cGy, to simulate predicted radiation exposures that would be experienced during lunar and Martian missions, respectively. Blood and adrenal tissues were collected at 3- and 14-days post-irradiation for analysis of immune and endocrine biosignatures and pathways. Sexually dimorphic adrenal gland weights and morphology, differential total RNA expression with corresponding gene ontology, and unique immune phenotypes were altered by GCRsim. In brief, this study offers new insights into sexually dimorphic immune and endocrine kinetics following simulated cosmic radiation exposure and highlights the necessity for personalized translational approaches for astronauts during exploration missions.

Galactic Cosmic Irradiation Alters Acute and Delayed Species-Typical Behavior in Male and Female Mice.

Exposure to space galactic cosmic radiation is a principal consideration for deep space missions. While the effects of space irradiation on the nervous system are not fully known, studies in animal models have shown that exposure to ionizing radiation can cause neuronal damage and lead to downstream cognitive and behavioral deficits. Cognitive health implications put humans and missions at risk, and with the upcoming Artemis missions in which female crew will play a major role, advance critical analysis of the neurological and performance responses of male and female rodents to space radiation is vital. Here, we tested the hypothesis that simulated Galactic Cosmic Radiation (GCRSim) exposure disrupts species-typical behavior in mice, including burrowing, rearing, grooming, and nest-building that depend upon hippocampal and medial prefrontal cortex circuitry. Behavior comprises a remarkably well-integrated representation of the biology of the whole animal that informs overall neural and physiological status, revealing functional impairment. We conducted a systematic dose-response analysis of mature (6-month-old) male and female mice exposed to either 5, 15, or 50 cGy 5-ion GCRSim (H, Si, He, O, Fe) at the NASA Space Radiation Laboratory (NSRL). Behavioral performance was evaluated at 72 h (acute) and 91-days (delayed) postradiation exposure. Specifically, species-typical behavior patterns comprising burrowing, rearing, and grooming as well as nest building were analyzed. A Neuroscore test battery (spontaneous activity, proprioception, vibrissae touch, limb symmetry, lateral turning, forelimb outstretching, and climbing) was performed at the acute timepoint to investigate early sensorimotor deficits postirradiation exposure. Nest construction, a measure of neurological and organizational function in rodents, was evaluated using a five-stage Likert scale 'Deacon' score that ranged from 1 (a low score where the Nestlet is untouched) to 5 (a high score where the Nestlet is completely shredded and shaped into a nest). Differential acute responses were observed in females relative to males with respect to species-typical behavior following 15 cGy exposure while delayed responses were observed in female grooming following 50 cGy exposure. Significant sex differences were observed at both timepoints in nest building. No deficits in sensorimotor behavior were observed via the Neuroscore. This study revealed subtle, sexually dimorphic GCRSim exposure effects on mouse behavior. Our analysis provides a clearer understanding of GCR dose effects on species typical, sensorimotor and organizational behaviors at acute and delayed timeframes postirradiation, thereby setting the stage for the identification of underlying cellular and molecular events.

Mitochondria-Targeted Human Catalase in the Mouse Longevity MCAT Model Mitigates Head-Tilt Bedrest-Induced Neuro-Inflammation in the Hippocampus.

Microgravity (modeled by head-tilt bedrest and hind-limb unloading), experienced during prolonged spaceflight, results in neurological consequences, central nervous system (CNS) dysfunction, and potentially impairment during the performance of critical tasks. Similar pathologies are observed in bedrest, sedentary lifestyle, and muscle disuse on Earth. In our previous study, we saw that head-tilt bedrest together with social isolation upregulated the milieu of pro-inflammatory cytokines in the hippocampus and plasma. These changes were mitigated in a MCAT mouse model overexpressing human catalase in the mitochondria, pointing out the importance of ROS signaling in this stress response. Here, we used a head-tilt model in socially housed mice to tease out the effects of head-tilt bedrest without isolation. In order to find the underlying molecular mechanisms that provoked the cytokine response, we measured CD68, an indicator of microglial activation in the hippocampus, as well as changes in normal in-cage behavior. We hypothesized that hindlimb unloading (HU) will elicit microglial hippocampal activations, which will be mitigated in the MCAT ROS-quenching mice model. Indeed, we saw an elevation of the activated microglia CD68 marker following HU in the hippocampus, and this pathology was mitigated in MCAT mice. Additionally, we identified cytokines in the hippocampus, which had significant positive correlations with CD68 and negative correlations with exploratory behaviors, indicating a link between neuroinflammation and behavioral consequences. Unveiling a correlation between molecular and behavioral changes could reveal a biomarker indicative of these responses and could also result in a potential target for the treatment and prevention of cognitive changes following long space missions and/or muscle disuse on Earth.

Neuro-consequences of the spaceflight environment.

As human space exploration advances to establish a permanent presence beyond the Low Earth Orbit (LEO) with NASA's Artemis mission, researchers are striving to understand and address the health challenges of living and working in the spaceflight environment. Exposure to ionizing radiation, microgravity, isolation and other spaceflight hazards pose significant risks to astronauts. Determining neurobiological and neurobehavioral responses, understanding physiological responses under Central Nervous System (CNS) control, and identifying putative mechanisms to inform countermeasure development are critically important to ensuring brain and behavioral health of crew on long duration missions. Here we provide a detailed and comprehensive review of the effects of spaceflight and of ground-based spaceflight analogs, including simulated weightlessness, social isolation, and ionizing radiation on humans and animals. Further, we discuss dietary and non-dietary countermeasures including artificial gravity and antioxidants, among others. Significant future work is needed to ensure that neural, sensorimotor, cognitive and other physiological functions are maintained during extended deep space missions to avoid potentially catastrophic health and safety outcomes.

Mammalian and Invertebrate Models as Complementary Tools for Gaining Mechanistic Insight on Muscle Responses to Spaceflight.

Bioinformatics approaches have proven useful in understanding biological responses to spaceflight. Spaceflight experiments remain resource intensive and rare. One outstanding issue is how to maximize scientific output from a limited number of omics datasets from traditional animal models including nematodes, fruitfly, and rodents. The utility of omics data from invertebrate models in anticipating mammalian responses to spaceflight has not been fully explored. Hence, we performed comparative analyses of transcriptomes of soleus and extensor digitorum longus (EDL) in mice that underwent 37 days of spaceflight. Results indicate shared stress responses and altered circadian rhythm. EDL showed more robust growth signals and Pde2a downregulation, possibly underlying its resistance to atrophy versus soleus. Spaceflight and hindlimb unloading mice shared differential regulation of proliferation, circadian, and neuronal signaling. Shared gene regulation in muscles of humans on bedrest and space flown rodents suggest targets for mitigating muscle atrophy in space and on Earth. Spaceflight responses of C. elegans were more similar to EDL. Discrete life stages of D. melanogaster have distinct utility in anticipating EDL and soleus responses. In summary, spaceflight leads to shared and discrete molecular responses between muscle types and invertebrate models may augment mechanistic knowledge gained from rodent spaceflight and ground-based studies.

Spaceflight Modulates the Expression of Key Oxidative Stress and Cell Cycle Related Genes in Heart.

Spaceflight causes cardiovascular changes due to microgravity-induced redistribution of body fluids and musculoskeletal unloading. Cardiac deconditioning and atrophy on Earth are associated with altered Trp53 and oxidative stress-related pathways, but the effects of spaceflight on cardiac changes at the molecular level are less understood. We tested the hypothesis that spaceflight alters the expression of key genes related to stress response pathways, which may contribute to cardiovascular deconditioning during extended spaceflight. Mice were exposed to spaceflight for 15 days or maintained on Earth (ground control). Ventricle tissue was harvested starting ~3 h post-landing. We measured expression of select genes implicated in oxidative stress pathways and Trp53 signaling by quantitative PCR. Cardiac expression levels of 37 of 168 genes tested were altered after spaceflight. Spaceflight downregulated transcription factor, Nfe2l2 (Nrf2), upregulated Nox1 and downregulated Ptgs2, suggesting a persistent increase in oxidative stress-related target genes. Spaceflight also substantially upregulated Cdkn1a (p21) and cell cycle/apoptosis-related gene Myc, and downregulated the inflammatory response gene Tnf. There were no changes in apoptosis-related genes such as Trp53. Spaceflight altered the expression of genes regulating redox balance, cell cycle and senescence in cardiac tissue of mice. Thus, spaceflight may contribute to cardiac dysfunction due to oxidative stress.

Overexpression of catalase in mitochondria mitigates changes in hippocampal cytokine expression following simulated microgravity and isolation.

Isolation on Earth can alter physiology and signaling of organs systems, including the central nervous system. Although not in complete solitude, astronauts operate in an isolated environment during spaceflight. In this study, we determined the effects of isolation and simulated microgravity solely or combined, on the inflammatory cytokine milieu of the hippocampus. Adult female wild-type mice underwent simulated microgravity by hindlimb unloading for 30 days in single or social (paired) housing. In hippocampus, simulated microgravity and isolation each regulate a discrete repertoire of cytokines associated with inflammation. Their combined effects are not additive. A model for mitochondrial reactive oxygen species (ROS) quenching via targeted overexpression of the human catalase gene to the mitochondria (MCAT mice), are protected from isolation- and/or simulated microgravity-induced changes in cytokine expression. These findings suggest a key role for mitochondrial ROS signaling in neuroinflammatory responses to spaceflight and prolonged bedrest, isolation, and confinement on Earth.

Placenta-Expanded Stromal Cell Therapy in a Rodent Model of Simulated Weightlessness.

Long duration spaceflight poses potential health risks to astronauts during flight and re-adaptation after return to Earth. There is an emerging need for NASA to provide successful and reliable therapeutics for long duration missions when capability for medical intervention will be limited. Clinically relevant, human placenta-derived therapeutic stromal cells (PLX-PAD) are a promising therapeutic alternative. We found that treatment of adult female mice with PLX-PAD near the onset of simulated weightlessness by hindlimb unloading (HU, 30 d) was well-tolerated and partially mitigated decrements caused by HU. Specifically, PLX-PAD treatment rescued HU-induced thymic atrophy, and mitigated HU-induced changes in percentages of circulating neutrophils, but did not rescue changes in the percentages of lymphocytes, monocytes, natural killer (NK) cells, T-cells and splenic atrophy. Further, PLX-PAD partially mitigated HU effects on the expression of select cytokines in the hippocampus. In contrast, PLX-PAD failed to protect bone and muscle from HU-induced effects, suggesting that the mechanisms which regulate the structure of these mechanosensitive tissues in response to disuse are discrete from those that regulate the immune- and central nervous system (CNS). These findings support the therapeutic potential of placenta-derived stromal cells for select physiological deficits during simulated spaceflight. Multiple countermeasures are likely needed for comprehensive protection from the deleterious effects of prolonged spaceflight.

The individual and combined effects of spaceflight radiation and microgravity on biologic systems and functional outcomes.

Both microgravity and radiation exposure in the spaceflight environment have been identified as hazards to astronaut health and performance. Substantial study has been focused on understanding the biology and risks associated with prolonged exposure to microgravity, and the hazards presented by radiation from galactic cosmic rays (GCR) and solar particle events (SPEs) outside of low earth orbit (LEO). To date, the majority of the ground-based analogues (e.g., rodent or cell culture studies) that investigate the biology of and risks associated with spaceflight hazards will focus on an individual hazard in isolation. However, astronauts will face these challenges simultaneously Combined hazard studies are necessary for understanding the risks astronauts face as they travel outside of LEO, and are also critical for countermeasure development. The focus of this review is to describe biologic and functional outcomes from ground-based analogue models for microgravity and radiation, specifically highlighting the combined effects of radiation and reduced weight-bearing from rodent ground-based tail suspension via hind limb unloading (HLU) and partial weight-bearing (PWB) models, although in vitro and spaceflight results are discussed as appropriate. The review focuses on the skeletal, ocular, central nervous system (CNS), cardiovascular, and stem cells responses.