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Abstract Men and women have different cardiovascular responses to spaceflight; however, few studies have focused on direct comparisons between sexes. We investigated the mechanisms of aortic stiffening in socially and sexually mature 20-week-old male and female Sprague Dawley (SD) rats exposed to hindlimb unloading (HLU) for 14 days. Pulse wave velocity (PWV) was greater in the aortic arch of females after HLU versus control females (n = 6–8). HLU had no effect on aortic PWV in males (n = 5–6). Aortic α smooth muscle actin, myosin, collagen, elastin, and collagen-to-elastin ratio were not different in rats of either sex following HLU. The levels of G protein-coupled estrogen receptor (GPER) were lower in the aorta of SD females exposed to HLU compared with female controls but were not altered in males. HLU females also had lower aortic PPARγ, increased oxidative stress markers, and diastolic dysfunction compared with control females. GPER agonist G1 prevented the increase in PWV and 8-hydroxy-2’-deoxyguanosine without altering PPARγ or p47phox in HLU females (n = 4 in each group) suggesting that lower GPER may contribute to arterial stiffening in the setting of simulated microgravity. This study highlights sex-specific vascular adaptations to the state of simulated microgravity.

Abstract Exposure to ionizing radiation for oncological therapy increases the risk for late-onset fractures in survivors. However, the effects of total body irradiation (TBI) on adult bone are not well-characterized. The primary aim of this study was to quantify the long-term effects of TBI on bone microstructure, material composition, and mechanical behavior in skeletally mature rhesus macaque (Macaca mulatta) non-human primates. Femora were obtained post-mortem from animals exposed to an acute dose of TBI (6.0–6.75 Gy) nearly a decade earlier, age-matched non-irradiated controls, and non-irradiated young animals. The microstructure of femoral trabecular and cortical bone was assessed via micro-computed tomography. Material composition was evaluated by measuring total fluorescent advanced glycation end products (fAGEs). Cortical bone mechanical behavior was quantified via four-point bending and cyclic reference point indentation (cRPI). Animals exposed to TBI had slightly worse cortical microstructure, including lower cortical thickness (-11%, p = 0.037) and cortical area (-24%, p = 0.049), but similar fAGE content and mechanical properties as age-matched controls. Aging did not influence cortical microstructure, fAGE content, or cRPI measures but diminished femoral cortical post-yield properties, including toughness to fracture (-32%, p = 0.032). Because TBI was administered after the acquisition of peak bone mass, these results suggest that the skeletons of long-term survivors of adulthood TBI may be resilient, retaining or recovering their mechanical integrity during the post-treatment period, despite radiation-induced architectural deficits. Further investigation is necessary to better understand radiation-induced skeletal fragility in mature and immature bone to improve care for radiation patients of all ages.

IntroductionEffects and interactions between different spaceflight stressors are expected to be experienced by crew on missions when exposed to microgravity and galactic cosmic rays (GCRs). One of the limitations of previous studies on simulated weightlessness using hindlimb unloading (HU) is that a control HU condition was not included.MethodsWe characterized the behavioral performance of male Fischer rats 2 months after sham or total body irradiation with a simplified 5-ion 6-mixed-beam exposure representative of GCRs in the absence or presence of HU. Six months later, the plasma, hippocampus, and cortex were processed to determine whether the behavioral effects were associated with long-term alterations in the metabolic pathways.ResultsIn the open field without and with objects, interactions were observed for radiation × HU. In the plasma of animals that were not under the HU or control HU condition, the riboflavin metabolic pathway was affected most for sham irradiation vs. 0.75 Gy exposure. Analysis of the effects of control HU on plasma in the sham-irradiated animals showed that the alanine, aspartate, glutamate, riboflavin, and glutamine metabolisms as well as arginine biosynthesis were affected. The effects of control HU on the hippocampus in the sham-irradiated animals showed that the phenylalanine, tyrosine, and tryptophan pathway was affected the most. Analysis of effects of 0.75 Gy irradiation on the cortex of control HU animals showed that the glutamine and glutamate metabolic pathway was affected similar to the hippocampus, while the riboflavin pathway was affected in animals that were not under the control HU condition. The effects of control HU on the cortex in sham-irradiated animals showed that the riboflavin metabolic pathway was affected. Animals receiving 0.75 Gy of irradiation showed impaired glutamine and glutamate metabolic pathway, whereas animals receiving 1.5 Gy of irradiation showed impaired riboflavin metabolic pathways. A total of 21 plasma metabolites were correlated with the behavioral measures, indicating that plasma and brain biomarkers associated with behavioral performance are dependent on the environmental conditions experienced.DiscussionPhenylalanine, tyrosine, and tryptophan metabolism as well as phenylalanine and tryptophan as plasma metabolites are biomarkers that can be considered for spaceflight as they were revealed in both Fischer and WAG/Rij rats exposed to simGCRsim and/or HU.

Introduction: The space environment astronauts experience during space missions consists of multiple environmental challenges, including microgravity. In this study, we assessed the behavioral and cognitive performances of male Fisher rats 2 months after sham irradiation or total body irradiation with photons in the absence or presence of simulated microgravity. We analyzed the plasma collected 9 months after sham irradiation or total body irradiation for distinct alterations in metabolic pathways and to determine whether changes to metabolic measures were associated with specific behavioral and cognitive measures.Methods: A total of 344 male Fischer rats were irradiated with photons (6 MeV; 3, 8, or 10 Gy) in the absence or presence of simulated weightlessness achieved using hindlimb unloading (HU). To identify potential plasma biomarkers of photon radiation exposure or the HU condition for behavioral or cognitive performance, we performed regression analyses.Results: The behavioral effects of HU on activity levels in an open field, measures of anxiety in an elevated plus maze, and anhedonia in the M&M consumption test were more pronounced than those of photon irradiation. Phenylalanine, tyrosine, and tryptophan metabolism, and phenylalanine metabolism and biosynthesis showed very strong pathway changes, following photon irradiation and HU in animals irradiated with 3 Gy. Here, 29 out of 101 plasma metabolites were associated with 1 out of 13 behavioral measures. In the absence of HU, 22 metabolites were related to behavioral and cognitive measures. In HU animals that were sham-irradiated or irradiated with 8 Gy, one metabolite was related to behavioral and cognitive measures. In HU animals irradiated with 3 Gy, six metabolites were related to behavioral and cognitive measures.Discussion: These data suggest that it will be possible to develop stable plasma biomarkers of behavioral and cognitive performance, following environmental challenges like HU and radiation exposure.

Abstract It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were collected for analysis. A novel digital color-coded barcode counting technology (NanoStringTM) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p

Abstract Alpha(α)Klotho, a soluble transmembrane protein, facilitates calcium-phosphorus homeostasis through feedback between bone and kidney and is a potential systemic biomarker for bone-kidney health during spaceflight. We determined if: (1) plasma αKlotho was reduced after both spaceflight aboard the ISS and hindlimb unloading (HU); and (2) deficiency could be reversed with exercise. Both spaceflight and HU lowered circulating plasma αKlotho: plasma αKlotho recovered with exercise after HU.

Abstract Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

A limitation of simulated space radiation studies is that radiation exposure is not the only environmental challenge astronauts face during missions. Therefore, we characterized behavioral and cognitive performance of male WAG/Rij rats 3 months after sham-irradiation or total body irradiation with a simplified 5-ion mixed beam exposure in the absence or presence of simulated weightlessness using hindlimb unloading (HU) alone. Six months following behavioral and cognitive testing or 9 months following sham-irradiation or total body irradiation, plasma and brain tissues (hippocampus and cortex) were processed to determine whether the behavioral and cognitive effects were associated with long-term alterations in metabolic pathways in plasma and brain. Sham HU, but not irradiated HU, rats were impaired in spatial habituation learning. Rats irradiated with 1.5 Gy showed increased depressive-like behaviors. This was seen in the absence but not presence of HU. Thus, HU has differential effects in sham-irradiated and irradiated animals and specific behavioral measures are associated with plasma levels of distinct metabolites 6 months later. The combined effects of HU and radiation on metabolic pathways in plasma and brain illustrate the complex interaction of environmental stressors and highlights the importance of assessing these interactions.

Background: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. Methods: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. Results: The scaffold was devoid of donor cellular material on histology and DNA sampling (p < 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. Conclusion: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.

Biomedical use of radiation is utilized in effective diagnostic and treatment tools, yet can introduce risks to healthy tissues. High energy photons used for diagnostic purposes have high penetration depth and can discriminate multiple tissues based on attenuation properties of different materials. Likewise, the ability to deposit energy at various targets within tumors make the use of photons effective treatment for cancer. Radiation focused on a tumor will deposit energy when it interacts with a biological structure (e.g. DNA), which will result in cell kill should repair capacity of the tissue be overwhelmed. Likewise, damage to normal, non-cancerous tissues is a consequence of radiation that can lead to acute or late, chronic toxicity profiles. Adipose derived stem cells (ADSCs) are mesenchymal stem cells that have been proven to have similar characteristics to bone marrow derived stem cells, except that they are much easier to obtain. Within the body, ADSCs act as immunomodulators and assist with the maintenance and repair of tissues. They have been shown to have excellent differentiation capability, making them an extremely viable option for stem cell therapies and regenerative medicine applications. Due to the tissue ADSCs are derived from, they are highly likely to be affected by radiation therapy, especially when treating tumors localized to structures with relatively high ADSC content (eg., breast cancer). For this reason, the purpose behind this research is to better understand how ADSCs are affected by doses of radiation comparable to a single fraction of radiation therapy. We also measured the response of ADSCs to exposure at different dose rates to determine if there is a significant difference in the response of ADSCs to radiation therapy relevant doses of ionizing radiation. Our findings indicate that ADSCs exposed to Cesium (Cs 137)-gamma rays at a moderate dose of 2Gy and either a low dose rate (1.40Gy/min) or a high dose rate (7.31Gy/min) slow proliferation rate, and with cell cycle arrest in some populations. These responses ADSCs were not as marked as previously measured in other stem cell types. In addition, our results indicate that differences in dose rate in the Gy/min range typically utilized in small animal or cell irradiation platforms have a minimal effect on the function of ADSCs. The potential ADSCs have in the space of regenerative medicine makes them an ideal candidate for study with ionizing radiation, as they are one of the main cell types to promote tissue healing.

Purpose: Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. Methods: Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. Results: There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level ( p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. Conclusion: The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. Clinical Relevance: The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor α subunits, collagen α1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss.