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.

Extraterrestrial Gynecology: Could Spaceflight Increase the Risk of Developing Cancer in Female Astronauts? An Updated Review.

Outer space is an extremely hostile environment for human life, with ionizing radiation from galactic cosmic rays and microgravity posing the most significant hazards to the health of astronauts. Spaceflight has also been shown to have an impact on established cancer hallmarks, possibly increasing carcinogenic risk. Terrestrially, women have a higher incidence of radiation-induced cancers, largely driven by lung, thyroid, breast, and ovarian cancers, and therefore, historically, they have been permitted to spend significantly less time in space than men. In the present review, we focus on the effects of microgravity and radiation on the female reproductive system, particularly gynecological cancer. The aim is to provide a summary of the research that has been carried out related to the risk of gynecological cancer, highlighting what further studies are needed to pave the way for safer exploration class missions, as well as postflight screening and management of women astronauts following long-duration spaceflight.

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.

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.

Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression.

Ovarian steroids dramatically impact normal homeostatic and metabolic processes of most tissues within the body, including muscle, bone, neural, immune, cardiovascular, and reproductive systems. Determining the effects of spaceflight on the ovary and estrous cycle is, therefore, critical to our understanding of all spaceflight experiments using female mice. Adult female mice (n = 10) were exposed to and sacrificed on-orbit after 37 days of spaceflight in microgravity. Contemporary control (preflight baseline, vivarium, and habitat; n = 10/group) groups were maintained at the Kennedy Space Center, prior to sacrifice and similar tissue collection at the NASA Ames Research Center. Ovarian tissues were collected and processed for RNA and steroid analyses at initial carcass thaw. Vaginal wall tissue collected from twice frozen/thawed carcasses was fixed for estrous cycle stage determinations. The proportion of animals in each phase of the estrous cycle (i.e., proestrus, estrus, metestrus, and diestrus) did not appreciably differ between baseline, vivarium, and flight mice, while habitat control mice exhibited greater numbers in diestrus. Ovarian tissue steroid concentrations indicated no differences in estradiol across groups, while progesterone levels were lower (p < 0.05) in habitat and flight compared to baseline females. Genes involved in ovarian steroidogenic function were not differentially expressed across groups. As ovarian estrogen can dramatically impact multiple non-reproductive tissues, these data support vaginal wall estrous cycle classification of all female mice flown in space. Additionally, since females exposed to long-term spaceflight were observed at different estrous cycle stages, this indicates females are likely undergoing ovarian cyclicity and may yet be fertile.

Validation of a New Rodent Experimental System to Investigate Consequences of Long Duration Space Habitation.

Animal models are useful for exploring the health consequences of prolonged spaceflight. Capabilities were developed to perform experiments in low earth orbit with on-board sample recovery, thereby avoiding complications caused by return to Earth. For NASA's Rodent Research-1 mission, female mice (ten 32 wk C57BL/6NTac; ten 16 wk C57BL/6J) were launched on an unmanned vehicle, then resided on the International Space Station for 21/22d or 37d in microgravity. Mice were euthanized on-orbit, livers and spleens dissected, and remaining tissues frozen in situ for later analyses. Mice appeared healthy by daily video health checks and body, adrenal, and spleen weights of 37d-flight (FLT) mice did not differ from ground controls housed in flight hardware (GC), while thymus weights were 35% greater in FLT than GC. Mice exposed to 37d of spaceflight displayed elevated liver mass (33%) and select enzyme activities compared to GC, whereas 21/22d-FLT mice did not. FLT mice appeared more physically active than respective GC while soleus muscle showed expected atrophy. RNA and enzyme activity levels in tissues recovered on-orbit were of acceptable quality. Thus, this system establishes a new capability for conducting long-duration experiments in space, enables sample recovery on-orbit, and avoids triggering standard indices of chronic stress.

Influence of Social Isolation During Prolonged Simulated Weightlessness by Hindlimb Unloading.

The hindlimb unloading (HU) model has been used extensively to simulate the cephalad fluid shift and musculoskeletal disuse observed in spaceflight with its application expanding to study immune, cardiovascular and central nervous system responses, among others. Most HU studies are performed with singly housed animals, although social isolation also can substantially impact behavior and physiology, and therefore may confound HU experimental results. Other HU variants that allow for paired housing have been developed although no systematic assessment has been made to understand the effects of social isolation on HU outcomes. Hence, we aimed to determine the contribution of social isolation to tissue responses to HU. To accomplish this, we developed a refinement to the traditional NASA Ames single housing HU system to accommodate social housing in pairs, retaining desirable features of the original design. We conducted a 30-day HU experiment with adult, female mice that were either singly or socially housed. HU animals in both single and social housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and hypothalamic-pituitary-adrenal (HPA) axis responses were differentially impacted by the HU social environment relative to matched NL controls. HU led to a reduction in % CD4+ T cells in singly housed, but not in socially housed mice. Unexpectedly, HU increased adrenal gland mass in socially housed but not singly housed mice, while social isolation increased adrenal gland mass in NL controls. HU also led to elevated plasma corticosterone levels at day 30 in both singly and socially housed mice. Thus, musculoskeletal responses to simulated weightlessness are similar regardless of social environment with a few differences in adrenal and immune responses. Our findings show that combined stressors can mask, not only exacerbate, select responses to HU. These findings further expand the utility of the HU model for studying possible combined effects of spaceflight stressors.

Author Correction: Behavior of mice aboard the International Space Station.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Behavior of mice aboard the International Space Station.

Interest in space habitation has grown dramatically with planning underway for the first human transit to Mars. Despite a robust history of domestic and international spaceflight research, understanding behavioral adaptation to the space environment for extended durations is scant. Here we report the first detailed behavioral analysis of mice flown in the NASA Rodent Habitat on the International Space Station (ISS). Following 4-day transit from Earth to ISS, video images were acquired on orbit from 16- and 32-week-old female mice. Spaceflown mice engaged in a full range of species-typical behaviors. Physical activity was greater in younger flight mice as compared to identically-housed ground controls, and followed the circadian cycle. Within 7-10 days after launch, younger (but not older), mice began to exhibit distinctive circling or 'race-tracking' behavior that evolved into coordinated group activity. Organized group circling behavior unique to spaceflight may represent stereotyped motor behavior, rewarding effects of physical exercise, or vestibular sensation produced via self-motion. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach yields a useful analog for better understanding human responses to spaceflight, providing the opportunity to assess how physical movement influences responses to microgravity.

Oxidative Stress as Cause, Consequence, or Biomarker of Altered Female Reproduction and Development in the Space Environment.

Oxidative stress has been implicated in the pathophysiology of numerous terrestrial disease processes and associated with morbidity following spaceflight. Furthermore, oxidative stress has long been considered a causative agent in adverse reproductive outcomes. The purpose of this review is to summarize the pathogenesis of oxidative stress caused by cosmic radiation and microgravity, review the relationship between oxidative stress and reproductive outcomes in females, and explore what role spaceflight-induced oxidative damage may have on female reproductive and developmental outcomes.

From the bench to exploration medicine: NASA life sciences translational research for human exploration and habitation missions.

NASA's Space Biology and Human Research Program entities have recently spearheaded communications both internally and externally to coordinate the agency's translational research efforts. In this paper, we strongly advocate for translational research at NASA, provide recent examples of NASA sponsored early-stage translational research, and discuss options for a path forward. Our overall objective is to help in stimulating a collaborative research across multiple disciplines and entities that, working together, will more effectively and more rapidly achieve NASA's goals for human spaceflight.

Intrauterine exposure to maternal stress alters Bdnf IV DNA methylation and telomere length in the brain of adult rat offspring.

DNA methylation (addition of methyl groups to cytosines) and changes in telomere length (TTAGGG repeats on the ends of chromosomes) are two molecular modifications that result from stress and could contribute to the long-term effects of intrauterine exposure to maternal stress on offspring behavior. Here, we measured methylation of DNA associated with the Brain-derived neurotrophic factor (Bdnf) gene, a gene important in development and plasticity, and telomere length in the brains of adult rat male and female offspring whose mothers were exposed to unpredictable and variable stressors throughout gestation. Males exposed to prenatal stress had greater methylation (Bdnf IV) in the medial prefrontal cortex (mPFC) compared to non-stressed male controls and stressed females. Further, prenatally-stressed animals had shorter telomeres than controls in the mPFC. Together findings indicate a long-term impact of prenatal stress on brain DNA methylation and telomere biology with relevance for behavioral and health outcomes, and contribute to a growing literature linking stress to intergenerational molecular changes.

Effects of sex and gender on adaptations to space: reproductive health.

In this report, sex/gender research relevant to reproduction on Earth, in conjunction with the extant human and animal observations in space, was used to identify knowledge gaps and prioritize recommendations for future sex- and gender-specific surveillance and monitoring of male and female astronauts. With overall increased durations of contemporary space missions, a deeper understanding of sex/gender effects on reproduction-related responses and adaptations to the space environment is warranted to minimize risks and insure healthy aging of the men and women who travel into space.

L-dopa reverses behavioral deficits in the Pitx3 mouse fetus.

Studies of fetal rodents have provided evidence that early emerging behaviors, such as the suckling response, are dependent on the developing dopaminergic system. Although connections have been made between manipulations of dopamine and altered behavioral responses, the specific neural pathways involved have yet to be discovered. In this study, we examined the neurobehavioral output of the nigrostriatal pathway, using the Pitx3ak/2J mouse model (Pitx3). Used extensively in the study of Parkinson's disease, the Pitx3 mouse has very specific prenatal loss of dopaminergic neurons solely in the nigrostriatal pathway. Because of this specificity, we hypothesized that behavioral deficits specific to the nigrostriatal pathway would be reversed with administration of the dopamine precursor 3,4-dihydroxyphenylalanine (L-dopa). To test this hypothesis, homozygous mutant and heterozygous control fetal subjects were administered 1 of 4 doses (0, 25, 50, or 75 mg/kg) of L-dopa on the day before birth. Quantification of fetal behavior was scored from video recordings of behavioral observations. The behavioral measures used were (a) spontaneous movement activity; (b) state organization, from quantifications of high- and low-amplitude movements; (c) interlimb movement synchrony, a measure of limb coordination; and (d) oral grasp, similar to a newborn infant suckling response. Specific behavioral deficits observed in the Pitx3 mutants were reversed by L-dopa administration in a dose-dependent manner. However, different deficits required dissimilar doses for reversal, suggesting that some early emerging behaviors may be more sensitive to the administration of L-dopa. Taken together, this study provides valuable information about prenatal behaviors dependent on the nigrostriatal pathway.

Prenatal ontogeny of the dopamine-dependent neurobehavioral phenotype in Pitx3-deficient mice.

Mouse models with prenatal alterations in dopaminergic functioning can provide new opportunities to identify fetal behavioral abnormalities and the underlying neural substrates dependent on dopamine. In this study, we tested the hypothesis that prenatal loss of nigrostriatal function is associated with fetal akinesia, or difficulty initiating movement. Specific behaviors were analysed in fetal offspring derived from pregnant Pitx3(ak) /2J and C57BL/6J dams on the last 4 days before birth (E15-18 of a 19-day gestation). Using digital videography, we analysed: (i) behavioral state, by quantification of high- and low-amplitude movements, (ii) interlimb movement synchrony, a measure of the temporal relationship between spontaneous movements of limb pairs, (iii) facial wiping, a characteristic response to perioral tactile stimulation similar to the defensive response in human infants, and (iv) oral grasp of a non-nutritive nipple, a component of suckling in the human infant. Pitx3 mutants showed a selective decrease in interlimb movement synchrony rates at the shortest (0.1 s) temporal interval coupled with significantly increased latencies to exhibit facial wiping and oral grasp. Collectively, our findings provide evidence that the primary fetal neurobehavioral deficit of the Pitx3 mutation is akinesia related to nigrostriatal damage. Other findings of particular interest were the differences in neurobehavioral functioning between C57BL/6J and Pitx3 heterozygous subjects, suggesting the two groups are not equivalent controls. These results further suggest that fetal neurobehavioral assessments are sensitive indicators of emerging neural dysfunction, and may have utility for prenatal diagnosis.

Longitudinal 1H MRS of rat forebrain from infancy to adulthood reveals adolescence as a distinctive phase of neurometabolite development.

This study represents the first longitudinal, within-subject (1) H MRS investigation of the developing rat brain spanning infancy, adolescence and early adulthood. We obtained neurometabolite profiles from a voxel located in a central location of the forebrain, centered on the striatum, with smaller contributions for the cortex, thalamus and hypothalamus, on postnatal days 7, 35 and 60. Water-scaled metabolite signals were corrected for T1 effects and quantified using the automated processing software LCModel, yielding molal concentrations. Our findings indicate age-related concentration changes in N-acetylaspartate + N-acetylaspartylglutamate, myo-inositol, glutamate + glutamine, taurine, creatine + phosphocreatine and glycerophosphocholine + phosphocholine. Using a repeated measures design and analysis, we identified significant neurodevelopment changes across all three developmental ages and identified adolescence as a distinctive phase in normative neurometabolic brain development. Between postnatal days 35 and 60, changes were observed in the concentrations of N-acetylaspartate + N-acetylaspartylglutamate, glutamate + glutamine and glycerophosphocholine + phosphocholine. Our data replicate past studies of early neurometabolite development and, for the first time, link maturational profiles in the same subjects across infancy, adolescence and adulthood.

Hypergravity disruption of homeorhetic adaptations to lactation in rat dams include changes in circadian clocks.

Altered gravity load induced by spaceflight (microgravity) and centrifugation (hypergravity) is associated with changes in circadian, metabolic, and reproductive systems. Exposure to 2-g hypergravity (HG) during pregnancy and lactation decreased rate of mammary metabolic activity and increased pup mortality. We hypothesize HG disrupted maternal homeorhetic responses to pregnancy and lactation are due to changes in maternal metabolism, hormone concentrations, and maternal behavior related to gravity induced alterations in circadian clocks. Effect of HG exposure on mammary, liver and adipose tissue metabolism, plasma hormones and maternal behavior were analyzed in rat dams from mid-pregnancy (Gestational day [G]11) through early lactation (Postnatal day [P]3); comparisons were made across five time-points: G20, G21, P0 (labor and delivery), P1 and P3. Blood, mammary, liver, and adipose tissue were collected for analyzing plasma hormones, glucose oxidation to CO(2) and incorporation into lipids, or gene expression. Maternal behavioral phenotyping was conducted using time-lapse videographic analyses. Dam and fetal-pup body mass were significantly reduced in HG in all age groups. HG did not affect labor and delivery; however, HG pups experienced a greater rate of mortality. PRL, corticosterone, and insulin levels and receptor genes were altered by HG. Mammary, liver and adipose tissue metabolism and expression of genes that regulate lipid metabolism were altered by HG exposure. Exposure to HG significantly changed expression of core clock genes in mammary and liver and circadian rhythms of maternal behavior. Gravity load alterations in dam's circadian system may have impacted homeorhetic adaptations needed for a successful lactation.

The experience of being born: a natural context for learning to suckle.

Understanding the developmental origins of congenital capabilities such as sucking is fundamental knowledge that can contribute to improving the clinical management of early feeding and facilitate the onset of oral ingestion. We describe analyses in rats showing that sensory stimulation in utero and during birth establishes the newborn's sucking responses to maternal cues. We mimicked elements of labor and delivery (viz., compressions simulating labor contractions, stroking simulating postnatal maternal licking of the newborn, and postnatal thermal flux), and used them to induce postnatal respiration and nipple attachment in caesarian-delivered pups. We report herein new data showing that, by simulating a fetal rat's experience of being born, specific components of vaginal birth provide sufficient conditions for the odor learning that guides newborn's sucking responses. In contrast, the absence of in utero compressions was associated with poor sucking onset. Knowing how birth stimuli contribute to the first nipple attachment and constitute a context for learning to suckle is an important step toward better management of some early feeding problems. It can serve also as a foundation for understanding the challenges of facilitating sucking by babies born prematurely so that they do not experience the typical contingencies mediating onset of oral ingestion.