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Bone loss caused by ionizing radiation is a potential health concern for radiotherapy patients, radiation workers and astronauts. In animal studies, exposure to ionizing radiation increases oxidative damage in skeletal tissues, and results in an imbalance in bone remodeling initiated by increased bone-resorbing osteoclasts. Therefore, we evaluated various candidate interventions with antioxidant or anti-inflammatory activities (antioxidant cocktail, dihydrolipoic acid, ibuprofen, dried plum) both for their ability to blunt the expression of resorption-related genes in marrow cells after irradiation with either gamma rays (photons, 2 Gy) or simulated space radiation (protons and heavy ions, 1 Gy) and to prevent bone loss. Dried plum was most effective in reducing the expression of genes related to bone resorption (Nfe2l2, Rankl, Mcp1, Opg, TNF-α) and also preventing later cancellous bone decrements caused by irradiation with either photons or heavy ions. Thus, dietary supplementation with DP may prevent the skeletal effects of radiation exposures either in space or on Earth.

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.

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., (Copyright © 2021. Published by Elsevier Ltd.)

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.

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.

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.

A comprehensive understanding of spaceflight factors involved in immune dysfunction and the evaluation of biomarkers to assess in-flight astronaut health are essential goals for NASA. An elevated neutrophil-to-lymphocyte ratio (NLR) is a potential biomarker candidate, as leukocyte differentials are altered during spaceflight. In the reduced gravity environment of space, rodents and astronauts displayed elevated NLR and granulocyte-to-lymphocyte ratios (GLR), respectively. To simulate microgravity using two well-established ground-based models, we cultured human whole blood-leukocytes in high-aspect rotating wall vessels (HARV-RWV) and used hindlimb unloaded (HU) mice. Both HARV-RWV simulation of leukocytes and HU-exposed mice showed elevated NLR profiles comparable to spaceflight exposed samples. To assess mechanisms involved, we found the simulated microgravity HARV-RWV model resulted in an imbalance of redox processes and activation of myeloperoxidase-producing inflammatory neutrophils, while antioxidant treatment reversed these effects. In the simulated microgravity HU model, mitochondrial catalase-transgenic mice that have reduced oxidative stress responses showed reduced neutrophil counts, NLR, and a dampened release of selective inflammatory cytokines compared to wildtype HU mice, suggesting simulated microgravity induced oxidative stress responses that triggered inflammation. In brief, both spaceflight and simulated microgravity models caused elevated NLR, indicating this as a potential biomarker for future in-flight immune health monitoring., (Copyright © 2020 Paul, Mhatre, Cekanaviciute, Schreurs, Tahimic, Globus, Anand, Crucian and Bhattacharya.)

Accumulation of oxidative damage from excess reactive oxygen species (ROS) may contribute to skeletal aging and mediate adverse responses to physiological challenges. Wild-type (WT) mice and transgenic mice (male, 16 wk of age) with human catalase targeted to the mitochondria (mCAT) were analyzed for skeletal responses to the remodeling stimuli of combined hind-limb unloading and exposure to ionizing radiation ( 137 Cs, 2 Gy). Treatment for 2 wk caused lipid peroxidation in the bones WT but not mCAT mice, showing that transgene expression mitigated oxidative stress. Ex vivo osteoblast colony growth rate was 95% greater in mCAT than WT mice and correlated with catalase activity levels ( P < 0.005, r = 0.67), although terminal osteoblast and osteoclast differentiation were unaffected. mCAT mice had lower cancellous bone volume and cortical size than WT mice. Ambulatory control mCAT animals also displayed reduced cancellous and cortical structural properties compared with control WT mice. In mCAT but not WT mice, treatment caused an unexpectedly rapid radial expansion (+8% cortical area, +22% moment of inertia), reminiscent of compensatory bone growth during advancing age. In contrast, treatment caused similar structural deficits in cancellous tissue of mCAT and WT mice. In sum, mitochondrial ROS signaling via H 2 O 2 was important for the acquisition of adult bone structure and catalase overexpression failed to protect cancellous tissue from treatment. In contrast, catabolic stimuli caused radial expansion in mCAT not WT mice, suggesting that mitochondrial ROS in skeletal cells act to suppress tissue turnover in response to remodeling challenges.

Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.

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.

A round-trip human mission to Mars is anticipated to last roughly three years. Spaceflight conditions are known to cause loss of bone mineral density (BMD) in astronauts, increasing bone fracture risk. There is an urgent need to understand BMD progression as a function of spaceflight time to minimize associated health implications and ensure mission success. Here we introduce a nonlinear mathematical model of BMD loss for candidate human missions to Mars: (i) Opposition class trajectory (400-600 days), and (ii) Conjunction class trajectory (1000-1200 days). Using femoral neck BMD data (N = 69) from astronauts after 132-day and 228-day spaceflight and the World Health Organization's fracture risk recommendation, we predicted post-mission risk and associated osteopathology. Our model predicts 62% opposition class astronauts and 100% conjunction class astronauts will develop osteopenia, with 33% being at risk for osteoporosis. This model can help in implementing countermeasure strategies and inform space agencies' choice of crew candidates., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 Tahimic, Paul, Schreurs, Torres, Rubinstein, Steczina, Lowe, Bhattacharya, Alwood, Ronca and Globus.)

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.

Spaceflight entails exposure to numerous environmental challenges with the potential to contribute to both musculoskeletal and vascular dysfunction. The purpose of this review is to describe current understanding of microgravity and radiation impacts on the mammalian skeleton and associated vasculature at the level of the whole organism. Recent experiments from spaceflight and ground-based models have provided fresh insights into how these environmental stresses influence mechanisms that are related to redox signaling, oxidative stress, and tissue dysfunction. Emerging mechanistic knowledge on cellular defenses to radiation and other environmental stressors, including microgravity, are useful for both screening and developing interventions against spaceflight-induced deficits in bone and vascular function., Competing Interests: The authors declare no conflict of interest.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET 56 Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA's Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either 56 Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; 56 Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or 56 Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose 56 Fe (200 cGy) increased Catalase and Gadd45 . The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET ( 137 Cs γ) when irradiated in vitro, but had limited protective effects on high-LET 56 Fe-exposed cells. In sum, either protons or 56 Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET 56 Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss., Competing Interests: The authors declare no conflict of interest.

Background: Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved., Objectives: The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C., Methods: Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH)., Results: Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6-7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months., Conclusion: These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts., Competing Interests: Authors SC, SL and HER received salaries from their respective companies (KBRwyle for SC and SL, ASRC Federal Space and Defense, Inc for HER). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

As multiple spacefaring nations contemplate extended manned missions to Mars and the Moon, health risks could be elevated as travel goes beyond the Earth's protective magnetosphere into the more intense deep space radiation environment. The primary purpose of this study was to determine whether mortality rates due to cardiovascular disease (CVD), cancer, accidents and all other causes of death differ in (1) astronauts who never flew orbital missions in space, (2) astronauts who flew only in low Earth orbit (LEO), and (3) Apollo lunar astronauts, the only humans to have traveled beyond Earth's magnetosphere. Results show there were no differences in CVD mortality rate between non-flight (9%) and LEO (11%) astronauts. However, the CVD mortality rate among Apollo lunar astronauts (43%) was 4-5 times higher than in non-flight and LEO astronauts. To test a possible mechanistic basis for these findings, a secondary purpose was to determine the long-term effects of simulated weightlessness and space-relevant total-body irradiation on vascular responsiveness in mice. The results demonstrate that space-relevant irradiation induces a sustained vascular endothelial cell dysfunction. Such impairment is known to lead to occlusive artery disease, and may be an important risk factor for CVD among astronauts exposed to deep space radiation.

Current knowledge of stem cell characteristics, maintenance and renewal, evolution with age, location in 'niches', and radiosensitivity to acute and protracted exposures is reviewed regarding haematopoietic tissue, mammary gland, thyroid, digestive tract, lung, skin, and bone. The identity of the target cells for carcinogenesis continues to point to the more primitive and mostly quiescent stem cell population (able to accumulate the protracted sequence of mutations necessary to result in malignancy), and, in a few tissues, to daughter progenitor cells. Several biological processes could contribute to the protection of stem cells from mutation accumulation: (1) accurate DNA repair; (2) rapid induced death of injured stem cells; (3) retention of the intact parental strand during divisions in some tissues so that mutations are passed to the daughter differentiating cells; and (4) stem cell competition, whereby undamaged stem cells outcompete damaged stem cells for residence in the vital niche. DNA repair mainly operates within a few days of irradiation, while stem cell replications and competition require weeks or many months depending on the tissue type. This foundation is used to provide a biological insight to protection issues including the linear-non-threshold and relative risk models, differences in cancer risk between tissues, dose-rate effects, and changes in the risk of radiation carcinogenesis by age at exposure and attained age., (© The International Society for Prosthetics and Orthotics.)

The rodent hindlimb unloading (HU) model was developed in the 1980s to make it possible to study mechanisms, responses, and treatments for the adverse consequences of spaceflight. Decades before development of the HU model, weightlessness was predicted to yield deficits in the principal tissues responsible for structure and movement on Earth, primarily muscle and bone. Indeed, results from early spaceflight and HU experiments confirmed the expected sensitivity of the musculoskeletal system to gravity loading. Results from human and animal spaceflight and HU experiments show that nearly all organ systems and tissues studied display some measurable changes, albeit sometimes minor and of uncertain relevance to astronaut health. The focus of this review is to examine key HU results for various organ systems including those related to stress; the immune, cardiovascular, and nervous systems; vision changes; and wound healing. Analysis of the validity of the HU model is important given its potential value for both hypothesis testing and countermeasure development.

Weightlessness during spaceflight leads to functional changes in resistance arteries and loss of cancellous bone, which may be potentiated by radiation exposure. The purpose of this study was to assess the effects of hindlimb unloading (HU) and total-body irradiation (TBI) on the vasomotor responses of skeletal muscle arteries. Male C57BL/6 mice were assigned to control, HU (13-16 days), TBI (1 Gy (56)Fe, 600 MeV, 10 cGy/min) and HU-TBI groups. Gastrocnemius muscle feed arteries were isolated for in vitro study. Endothelium-dependent (acetylcholine) and -independent (Dea-NONOate) vasodilator and vasoconstrictor (KCl, phenylephrine and myogenic) responses were evaluated. Arterial endothelial nitric oxide synthase (eNOS), superoxide dismutase-1 (SOD-1) and xanthine oxidase (XO) protein content and tibial cancellous bone microarchitecture were quantified. Endothelium-dependent and -independent vasodilator responses were impaired in all groups relative to control, and acetylcholine-induced vasodilation was lower in the HU-TBI group relative to that in the HU and TBI groups. Reductions in endothelium-dependent vasodilation correlated with a lower cancellous bone volume fraction. Nitric oxide synthase inhibition abolished all group differences in endothelium-dependent vasodilation. HU and HU-TBI resulted in decreases in eNOS protein levels, while TBI and HU-TBI produced lower SOD-1 and higher XO protein content. Vasoconstrictor responses were not altered. Reductions in NO bioavailability (eNOS), lower anti-oxidant capacity (SOD-1) and higher pro-oxidant capacity (XO) may contribute to the deficits in NOS signaling in skeletal muscle resistance arteries. These findings suggest that the combination of insults experienced in spaceflight leads to impairment of vasodilator function in resistance arteries that is mediated through deficits in NOS signaling.

Spaceflight has profound effects on vascular function as a result of weightlessness that may be further compounded by radiation exposure. The purpose of the present study was to assess the individual and combined effects of hindlimb unloading (HU) and radiation (Rad) on vasodilator responses in the skeletal muscle vasculature. Adult male C57BL/6J mice were randomized to one of four groups: control (Con), HU (tail suspension for 15 days), Rad (200 cGy of (137)Cs), and HU-Rad (15-day tail suspension and 200 cGy of (137)Cs). Endothelium-dependent vasodilation of gastrocnemius feed arteries was assessed in vitro using acetylcholine (ACh, 10(-9)-10(-4) M) and inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX). Endothelium-independent vasodilation was assessed using Dea-NONOate (10(-9)-10(-4) M). Endothelium-dependent and -independent vasodilator responses were impaired relative to Con responses in all treatment groups; however, there was no further impairment from the combination of treatments (HU-Rad) relative to that in the HU and Rad groups. The NOS-mediated contribution to endothelium-dependent vasodilation was depressed with HU and Rad. This impairment in NOS signaling may have been partially compensated for by an enhancement of PGI2-mediated dilation. Changes in endothelium-dependent vasodilation were also associated with decrements in trabecular bone volume in the proximal tibia metaphysis. These data demonstrate that the simulated space environment (i.e., radiation exposure and unloading of muscle and bone) significantly impairs skeletal muscle artery vasodilation, mediated through endothelium-dependent reductions in NOS signaling and decrements in vascular smooth muscle cell responsiveness to NO.

This report provides a review of stem cells/progenitor cells and their responses to ionising radiation in relation to issues relevant to stochastic effects of radiation that form a major part of the International Commission on Radiological Protection's system of radiological protection. Current information on stem cell characteristics, maintenance and renewal, evolution with age, location in stem cell 'niches', and radiosensitivity to acute and protracted exposures is presented in a series of substantial reviews as annexes concerning haematopoietic tissue, mammary gland, thyroid, digestive tract, lung, skin, and bone. This foundation of knowledge of stem cells is used in the main text of the report to provide a biological insight into issues such as the linear-no-threshold (LNT) model, cancer risk among tissues, dose-rate effects, and changes in the risk of radiation carcinogenesis by age at exposure and attained age. Knowledge of the biology and associated radiation biology of stem cells and progenitor cells is more developed in tissues that renew fairly rapidly, such as haematopoietic tissue, intestinal mucosa, and epidermis, although all the tissues considered here possess stem cell populations. Important features of stem cell maintenance, renewal, and response are the microenvironmental signals operating in the niche residence, for which a well-defined spatial location has been identified in some tissues. The identity of the target cell for carcinogenesis continues to point to the more primitive stem cell population that is mostly quiescent, and hence able to accumulate the protracted sequence of mutations necessary to result in malignancy. In addition, there is some potential for daughter progenitor cells to be target cells in particular cases, such as in haematopoietic tissue and in skin. Several biological processes could contribute to protecting stem cells from mutation accumulation: (a) accurate DNA repair; (b) rapidly induced death of injured stem cells; (c) retention of the DNA parental template strand during divisions in some tissue systems, so that mutations are passed to the daughter differentiating cells and not retained in the parental cell; and (d) stem cell competition, whereby undamaged stem cells outcompete damaged stem cells for residence in the niche. DNA repair mainly occurs within a few days of irradiation, while stem cell competition requires weeks or many months depending on the tissue type. The aforementioned processes may contribute to the differences in carcinogenic radiation risk values between tissues, and may help to explain why a rapidly replicating tissue such as small intestine is less prone to such risk. The processes also provide a mechanistic insight relevant to the LNT model, and the relative and absolute risk models. The radiobiological knowledge also provides a scientific insight into discussions of the dose and dose-rate effectiveness factor currently used in radiological protection guidelines. In addition, the biological information contributes potential reasons for the age-dependent sensitivity to radiation carcinogenesis, including the effects of in-utero exposure.

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16 weeks) were exposed to high atomic number, high energy (HZE) iron ions ((56)Fe, 2 Gy, 600 MeV/ion) (IR, n=5) or sham-irradiated (Sham, n=5). In vivo axial loading was initiated 5 months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60 cycles/day, 3 day/wk for 4 weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation., (Published by Elsevier Inc.)

Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth's gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth's gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth.

Exposure to ionizing radiation can cause rapid mineral loss and increase bone-resorbing osteoclasts within metabolically active, cancellous bone tissue leading to structural deficits. To better understand mechanisms involved in rapid, radiation-induced bone loss, we determined the influence of total body irradiation on expression of select cytokines known both to stimulate osteoclastogenesis and contribute to inflammatory bone disease. Adult (16 week), male C57BL/6J mice were exposed to either 2 Gy gamma rays ((137)Cs, 0.8 Gy/min) or heavy ions ((56)Fe, 600MeV, 0.50-1.1 Gy/min); this dose corresponds to either a single fraction of radiotherapy (typical total dose is ≥10 Gy) or accumulates over long-duration interplanetary missions. Serum, marrow, and mineralized tissue were harvested 4 h-7 days later. Gamma irradiation caused a prompt (2.6-fold within 4 h) and persistent (peaking at 4.1-fold within 1 day) rise in the expression of the obligate osteoclastogenic cytokine, receptor activator of nuclear factor kappa-B ligand (Rankl), within marrow cells over controls. Similarly, Rankl expression peaked in marrow cells within 3 days of iron exposure (9.2-fold). Changes in Rankl expression induced by gamma irradiation preceded and overlapped with a rise in expression of other pro-osteoclastic cytokines in marrow (eg, monocyte chemotactic protein-1 increased by 11.9-fold, and tumor necrosis factor-alpha increased by 1.7-fold over controls). The ratio, Rankl/Opg, in marrow increased by 1.8-fold, a net pro-resorption balance. In the marrow, expression of the antioxidant transcription factor, Nfe2l2, strongly correlated with expression levels of Nfatc1, Csf1, Tnf, and Rankl. Radiation exposure increased a serum marker of bone resorption (tartrate-resistant acid phosphatase) and led to cancellous bone loss (16% decrement after 1 week). We conclude that total body irradiation (gamma or heavy-ion) caused temporal elevations in the concentrations of specific genes expressed within marrow and mineralized tissue related to bone resorption, including select cytokines that lead to osteoclastogenesis and elevated resorption; this is likely to account for rapid and progressive deterioration of cancellous microarchitecture following exposure to ionizing radiation.

Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions., (Copyright © 2015 the American Physiological Society.)

Mechanical loading of mammalian tissues is a potent promoter of tissue growth and regeneration, whilst unloading in microgravity can cause reduced tissue regeneration, possibly through effects on stem cell tissue progenitors. To test the specific hypothesis that mechanical unloading alters differentiation of bone marrow mesenchymal and hematopoietic stem cell lineages, we studied cellular and molecular aspects of how bone marrow in the mouse proximal femur responds to unloading in microgravity. Trabecular and cortical endosteal bone surfaces in the femoral head underwent significant bone resorption in microgravity, enlarging the marrow cavity. Cells isolated from the femoral head marrow compartment showed significant down-regulation of gene expression markers for early mesenchymal and hematopoietic differentiation, including FUT1(-6.72), CSF2(-3.30), CD90(-3.33), PTPRC(-2.79), and GDF15(-2.45), but not stem cell markers, such as SOX2. At the cellular level, in situ histological analysis revealed decreased megakaryocyte numbers whilst erythrocytes were increased 2.33 fold. Furthermore, erythrocytes displayed elevated fucosylation and clustering adjacent to sinuses forming the marrow-blood barrier, possibly providing a mechanistic basis for explaining spaceflight anemia. Culture of isolated bone marrow cells immediately after microgravity exposure increased the marrow progenitor's potential for mesenchymal differentiation into in-vitro mineralized bone nodules, and hematopoietic differentiation into osteoclasts, suggesting an accumulation of undifferentiated progenitors during exposure to microgravity. These results support the idea that mechanical unloading of mammalian tissues in microgravity is a strong inhibitor of tissue growth and regeneration mechanisms, acting at the level of early mesenchymal and hematopoietic stem cell differentiation., (Published by Elsevier B.V.)

Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by µCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by -1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone loss in microgravity, and indicate that this loss is not limited to osteoclastic degradation. Therefore, this study offers new evidence for microgravity-induced osteocytic osteolysis, and CDKN1a/p21-mediated osteogenic cell cycle arrest.

To determine the chronic effects of heavy ion irradiation, an antibody based proteomic microarray technology was applied to monitor alterations in the serum proteome, six months after whole body irradiation of adult male C57Bl/6 mice with 0.5 Gray of (56)Fe. Out of 507 proteins, irradiation reduced expression of 25 proteins and enhanced expression of 12 proteins in serum (> 5% change relative to sham-irradiated controls). Of the 25 proteins found to be down-regulated, Poly ADP Ribose Polymerase (PARP) was 13% lower in the 0.5Gy mice and among the up-regulated proteins, beta-Tubulin was found to be 10% higher in the 0.5Gy group compared to the sham-irradiated 0Gy controls. Thus, irradiation with a relatively low dose of heavy ions caused persistent and selective changes in serum levels of proteins that are typically intracellular, suggesting chronic genotoxic damage.

Osteoporosis can profoundly affect the aged as a consequence of progressive bone loss; high-dose ionizing radiation can cause similar changes, although less is known about lower doses (≤100 cGy). We hypothesized that exposure to relatively low doses of gamma radiation accelerates structural changes characteristic of skeletal aging. Mice (C57BL/6J-10 wk old, male) were irradiated (total body; 0-sham, 1, 10 or 100 cGy (137)Cs) and tissues harvested on the day of irradiation, 1 or 4 months later. Microcomputed tomography was used to quantify microarchitecture of high turnover, cancellous bone. Irradiation at 100 cGy caused transient microarchitectural changes over one month that were only evident at longer times in controls (4 months). Ex vivo bone cell differentiation from the marrow was unaffected by gamma radiation. In conclusion, acute ionizing gamma irradiation at 100 cGy (but not at 1 cGy or 10 cGy) exacerbated microarchitectural changes normally found during progressive, postpubertal aging prior to the onset of age-related osteoporosis.

Astronauts are exposed to both musculoskeletal disuse and heavy ion radiation in space. Disuse alters the magnitude and direction of forces placed upon the skeleton causing bone remodeling, while energy deposited by ionizing radiation causes free radical formation and can lead to DNA strand breaks and oxidative damage to tissues. Radiation and disuse each result in a net loss of mineralized tissue in the adult, although the combined effects, subsequent consequences for mechanical properties and potential for recovery may differ. First, we examined how a high dose (2 Gy) of heavy ion radiation ((56)Fe) causes loss of mineralized tissue in the lumbar vertebrae of skeletally mature (4 months old), male, C57BL/6 mice using microcomputed tomography and determined the influence of structural changes on mechanical properties using whole bone compression tests and finite element analyses. Next, we tested if a low dose (0.5 Gy) of heavy particle radiation prevents skeletal recovery from a 14-day period of hindlimb unloading. Irradiation with a high dose of (56)Fe (2 Gy) caused bone loss (-14%) in the cancellous-rich centrum of the fourth lumbar vertebra (L4) 1 month later, increased trabecular stresses (+27%), increased the propensity for trabecular buckling and shifted stresses to the cortex. As expected, hindlimb unloading (14 days) alone adversely affected microarchitectural and mechanical stiffness of lumbar vertebrae, although the reduction in yield force was not statistically significant (-17%). Irradiation with a low dose of (56)Fe (0.5 Gy) did not affect vertebrae in normally loaded mice, but significantly reduced compressive yield force in vertebrae of unloaded mice relative to sham-irradiated controls (-24%). Irradiation did not impair the recovery of trabecular bone volume fraction that occurs after hindlimb unloaded mice are released to ambulate normally, although microarchitectural differences persisted 28 days later (96% increase in ratio of rod- to plate-like trabeculae). In summary, (56)Fe irradiation (0.5 Gy) of unloaded mice contributed to a reduction in compressive strength and partially prevented recovery of cancellous microarchitecture from adaptive responses of lumbar vertebrae to skeletal unloading. Thus, irradiation with heavy ions may accelerate or worsen the loss of skeletal integrity triggered by musculoskeletal disuse., (Published by Elsevier Inc.)

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to (56)Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to (56)Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP(+) osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.

Exposure of astronauts in space to radiation during weightlessness may contribute to subsequent bone loss. Gamma irradiation of postpubertal mice rapidly increases the number of bone-resorbing osteoclasts and causes bone loss in cancellous tissue; similar changes occur in skeletal diseases associated with oxidative stress. Therefore, we hypothesized that increased oxidative stress mediates radiation-induced bone loss and that musculoskeletal disuse changes the sensitivity of cancellous tissue to radiation exposure. Musculoskeletal disuse by hindlimb unloading (1 or 2 wk) or total body gamma irradiation (1 or 2 Gy of (137)Cs) of 4-mo-old, male C57BL/6 mice each decreased cancellous bone volume fraction in the proximal tibiae and lumbar vertebrae. The extent of radiation-induced acute cancellous bone loss in tibiae and lumbar vertebrae was similar in normally loaded and hindlimb-unloaded mice. Similarly, osteoclast surface in the tibiae increased 46% as a result of irradiation, 47% as a result of hindlimb unloading, and 64% as a result of irradiation + hindlimb unloading compared with normally loaded mice. Irradiation, but not hindlimb unloading, reduced viability and increased apoptosis of marrow cells and caused oxidative damage to lipids within mineralized tissue. Irradiation also stimulated generation of reactive oxygen species in marrow cells. Furthermore, injection of alpha-lipoic acid, an antioxidant, mitigated the acute bone loss caused by irradiation. Together, these results showed that disuse and gamma irradiation, alone or in combination, caused a similar degree of acute cancellous bone loss and shared a common cellular mechanism of increased bone resorption. Furthermore, irradiation, but not disuse, may increase the number of osteoclasts and the extent of acute bone loss via increased reactive oxygen species production and ensuing oxidative damage, implying different molecular mechanisms. The finding that alpha-lipoic acid protected cancellous tissue from the detrimental effects of irradiation has potential relevance to astronauts and radiotherapy patients.

Ionizing radiation can cause substantial tissue degeneration, which may threaten the long-term health of astronauts and radiotherapy patients. To determine whether a single dose of radiation acutely compromises structural integrity in the postpubertal skeleton, 18-week-old male mice were exposed to (137)Cs gamma radiation (1 or 2 Gy). The structure of high-turnover, cancellous bone was analyzed by microcomputed tomography (microCT) 3 or 10 days after irradiation and in basal controls (tissues harvested at the time of irradiation) and age-matched controls. Irradiation (2 Gy) caused a 20% decline in tibial cancellous bone volume fraction (BV/TV) within 3 days and a 43% decline within 10 days, while 1 Gy caused a 28% reduction 10 days later. The BV/TV decrement was due to increased spacing and decreased thickness of trabeculae. Radiation also increased ( approximately 150%) cancellous surfaces lined with tartrate-resistant, acid phosphatase-positive osteoclasts, an index of increased bone resorption. Radiation decreased lumbar vertebral BV/TV 1 month after irradiation, showing the persistence of cancellous bone loss, although mechanical properties in compression were unaffected. In sum, a single dose of gamma radiation rapidly increased osteoclast surface in cancellous tissue and compromised cancellous microarchitecture in the remodeling appendicular and axial skeleton of postpubertal mice.

The mammalian skeleton adjusts bone structure and strength in response to changes in mechanical loading, however the molecular and cellular mechanisms governing this process in vivo are unknown. Terminally differentiated osteoblasts, the osteocytes, are presumptive mechanosensory cells for bone, and cell culture studies demonstrate that beta1 integrins participate in mechanical signaling. To determine the role of beta1 integrins in osteoblasts in vivo, we used the Cre-lox system to delete beta1 integrin from cells committed to the osteoblast lineage. While pCol2.3 Cre-mediated recombination was widespread in bones from Colalpha1(I)-Cre+/beta1fl/fl conditional knockout mice (cKO), beta1 integrin protein was depleted from cortical osteocytes, but not from cancellous osteocytes or cells lining bone surfaces in adults. Bones from cKO mice that were normally loaded were similar in structure to WT littermates. However, hindlimb unloading of adult cKO mice for one week intended to cause bone loss (disuse osteopenia), resulted in unexpected, rapid changes in the geometry of cortical bone; hindlimb unloading increased the cross-sectional area, marrow area, and moments of inertia in cKO, but not WT mice. Furthermore, these hindlimb unloading-induced geometric changes in cortical bone of cKO mice resulted in increased whole bone bending stiffness and strength of the femur. Together, these results confirmed the concept that osteocytes are mechanosensory cells and showed beta1 integrins in cortical osteocytes limited changes in cortical geometry in response to disuse, thus providing the first in vivo evidence that beta1 integrins on osteocytes mediate specific aspects of mechanotransduction.

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.

Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E2 (PGE2). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE2 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5-50 g) increases in PGE2 (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE2 release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.

The hindlimb unloading rodent model is used extensively to study the response of many physiological systems to certain aspects of space flight, as well as to disuse and recovery from disuse for Earth benefits. This chapter describes the evolution of hindlimb unloading, and is divided into three sections. The first section examines the characteristics of 1064 articles using or reviewing the hindlimb unloading model, published between 1976 and April 1, 2004. The characteristics include number of publications, journals, countries, major physiological systems, method modifications, species, gender, genetic strains and ages of rodents, experiment duration, and countermeasures. The second section provides a comparison of results between space flown and hindlimb unloading animals from the 14-day Cosmos 2044 mission. The final section describes modifications to hindlimb unloading required by different experimental paradigms and a method to protect the tail harness for long duration studies. Hindlimb unloading in rodents has enabled improved understanding of the responses of the musculoskeletal, cardiovascular, immune, renal, neural, metabolic, and reproductive systems to unloading and/or to reloading on Earth with implications for both long-duration human space flight and disuse on Earth.

Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they demonstrated differences in osteoblast rather than osteoclast parameters. Together, these results suggest that integrin signals generated during growth enhance the acquisition of a skeletal mass, structure, and strength to withstand the mechanical loads generated by weight-bearing.

Since its inception at the National Aeronautics and Space Administration (NASA) Ames Research Center in the mid-1970s, many laboratories around the world have used the rat hindlimb unloading model to simulate weightlessness and to study various aspects of musculoskeletal loading. In this model, the hindlimbs of rodents are elevated to produce a 30 degrees head-down tilt, which results in a cephalad fluid shift and avoids weightbearing by the hindquarters. Although several reviews have described scientific results obtained with this model, this is the first review to focus on the technical aspects of hindlimb unloading. This review includes a history of the technique, a brief comparison with spaceflight data, technical details, extension of the model to mice, and other important technical considerations (e.g., housing, room temperature, unloading angle, the potential need for multiple control groups, age, body weight, the use of the forelimb tissues as internal controls, and when to remove animals from experiments). This paper is intended as a reference for researchers, reviewers of manuscripts, and institutional animal care and use committees. Over 800 references, related to the hindlimb unloading model, can be accessed via the electronic version of this article.

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

A model that uses hindlimb unloading of rats was developed to study the consequences of skeletal unloading and reloading as occurs during and following space flight. Studies using the model were initiated two decades ago and further developed at National Aeronautics and Space Administration (NASA)-Ames Research Center. The model mimics some aspects of exposure to microgravity by removing weightbearing loads from the hindquarters and producing a cephalic fluid shift. Unlike space flight, the forelimbs remain loaded in the model, providing a useful internal control to distinguish between the local and systemic effects of hindlimb unloading. Rats that are hindlimb unloaded by tail traction gain weight at the same rate as pairfed controls, and glucocorticoid levels are not different from controls, suggesting that systemic stress is minimal. Unloaded bones display reductions in cancellous osteoblast number, cancellous mineral apposition rate, trabecular bone volume, cortical periosteal mineralization rate, total bone mass, calcium content, and maturation of bone mineral relative to controls. Subsequent studies reveal that these changes also occur in rats exposed to space flight. In hindlimb unloaded rats, bone formation rates and masses of unloaded bones decline relative to controls, while loaded bones do not change despite a transient reduction in serum 1,25-dihydroxyvitamin D (1,25D) concentrations. Studies using the model to evaluate potential countermeasures show that 1,25D, growth hormone, dietary calcium, alendronate, and muscle stimulation modify, but do not completely correct, the suppression of bone growth caused by unloading, whereas continuous infusion of transforming growth factor-beta2 or insulin-like growth factor-1 appears to protect against some of the bone changes caused by unloading. These results emphasize the importance of local as opposed to systemic factors in the skeletal response to unloading, and reveal the pivotal role that osteoblasts play in the response to gravitational loading. The hindlimb unloading model provides a unique opportunity to evaluate in detail the physiological and cellular mechanisms of the skeletal response to weightbearing loads, and has proven to be an effective model for space flight.

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.