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

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

Improvement of Osteoporosis in Rats With Hind-Limb Unloading Treated With Pulsed Electromagnetic Field and Whole-Body Vibration.

OBJECTIVE: Physical factors have been used to address disuse osteoporosis, but their effects and mechanism remain unclear. The purpose of this study was to determine the effects of pulsed electromagnetic field (PEMF) and whole-body vibration (WBV) on disuse osteoporosis to increase knowledge about treating osteoporosis. METHODS: A disuse osteoporosis rat model was developed by hind-limb unloading (HU) for 6 weeks. Forty 4-month-old female Sprague-Dawley rats were divided into 5 groups and given the following interventions: HU, HU treated with PEMF (HUP), HU treated with WBV (HUW), HU treated with both PEMF and WBV (HUPW), and no intervention (controls). After 8 weeks of intervention, measurements were taken. RESULTS: HU induced a decrease in bone mineral density (BMD), whereas HUP, HUW, and HUPW increased it. Moreover, the bone resorption markers tartrate-resistant acid phosphatase (TRAP) and C-terminal peptide of type 1 collagen in the HU group significantly increased, whereas the osteogenesis markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly decreased. The markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly increased, but TRAP and C-terminal peptide of type 1 collagen significantly decreased in the HUPW, HUP, and HUW groups compared with the HU group. In particular, HUPW effectively increased osteocalcin and decreased TRAP compared with HUP and WBV. Microcomputed tomography analysis of the femur indicated that HUPW improved trabecular number, bone volume over total volume, bone surface over bone volume, trabecular separation, and the structure model index compared with HUP and that it improved bone surface over bone volume, trabecular separation, and structure model index compared with HUW. The HUPW group showed a significant increase in maximum load compared with the HUW group and a significant increase in elastic modulus compared with the HUP group. CONCLUSION: PEMF, WBV, and their combination all attenuated bone resorption and enhanced osteogenesis. WBV and the combination of treatments have great potential to improve osteogenesis compared with PEMF. In addition, HUPW significantly attenuated bone resorption compared with HUW and HUP. IMPACT: The results of this study indicated that HUPW could effectively improve disuse osteoporosis compared with HUP, given that trabecular number and bone volume over total volume are associated with disuse osteoporosis. Moreover, BMD recovered well with HUP, HUW, and HUPW but the bone structure-especially mechanical performance-did not, indicating that osteoporosis should be evaluated with BMD and mechanical performance, not with BMD in isolation.

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

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

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

Electroacupuncture stimulation at BL20, BL23 and SP6 prevents hind limb unloading-induced osteoporosis in rats.

BACKGROUND: Bone loss induced by microgravity is a serious problem in space flight. However, the effects of acupuncture stimulation on osteoporosis induced by microgravity have not been studied. With the goal of developing an effective countermeasure, our aim was to evaluate the effects of electroacupuncture (EA) stimulation at BL20, BL23, and SP6 on osteoporosis induced by simulated microgravity in rats. METHODS: Thirty male Wistar rats (aged 10 weeks) were randomly divided into three groups: healthy control group (CON, n = 10), hind limb unloading by tail-suspension group (T-S, n = 10), and EA treatment group (TRE, n = 10). Rats in the T-S and TRE groups were subjected to tail-suspension at -30° for 30 days, while the CON group experienced freedom of activity. In this period, the TRE group received EA treatment at BL20, BL23, and SP6 for 30 min every other day, which continued for 30 days. The microarchitecture of the proximal tibia and the biomechanical features of the femur in the rats were analyzed. In addition, the levels of serum biomarkers bone alkaline phosphatase (BALP) and osteocalcin (BGP) were measured. RESULTS: Compared with the CON group, the value of bone volume/total volume (BV/TV) and trabecular number (Tb.N) of the tibias in the TRE group remarkably decreased (p < 0.01). However, these changes were markedly less than those of the T-S group after 4 weeks of EA treatment (p < 0.05). Moreover, the serum concentration of BGP in the TRE group was also significantly higher than that of the T-S group (p < 0.05). CONCLUSIONS: These findings indicate that EA stimulation at BL20, BL23, and SP6 retards osteoporosis induced by hind limb unloading in rats.

Simultaneous exposure to chronic irradiation and simulated microgravity differentially alters immune cell phenotype in mouse thymus and spleen.

Deep-space missions may alter immune cell phenotype in the primary (e.g., thymus) and secondary (e.g., spleen) lymphoid organs contributing to the progression of a variety of diseases. In deep space missions, astronauts will be exposed to chronic low doses of HZE radiation while being in microgravity. Ground-based models of long-term uninterrupted exposures to HZE radiation are not yet available. To obtain insight in the effects of concurrent exposure to microgravity and chronic irradiation (CIR), mice received a cumulative dose of chronic 0.5 Gy gamma rays over one month ± simulated microgravity (SMG). To obtain insight in a dose rate effect, additional mice were exposed to single acute irradiation (AIR) at 0.5 Gy gamma rays. We measured proportions of immune cells relative to total number of live cells in the thymus and spleen, stress level markers in plasma, and change in body weight, food consumption, and water intake. CIR affected thymic CD3+/CD335+ natural killer T (NK-T) cells, CD25+ regulatory T (Treg) cells, CD27+/CD335- natural killer (NK1) cells and CD11c+/CD11b- dendritic cells (DCs) differently in mice subjected to SMG than in mice with normal loading. No such effects of CIR on SMG as compared to normal loading were observed in cell types from the spleen. Differences between CIR and AIR groups (both under normal loading) were found in thymic Treg and DCs. Food consumption, water intake, and body weight were less after coexposure than singular or no exposure. Compared to sham, all treatment groups exhibited elevated plasma levels of the stress marker catecholamines. These data suggest that microgravity and chronic irradiation may interact with each other to alter immune cell phenotypes in an organ-specific manner and appropriate strategies are required to reduce the health risk of crewmembers.

Tail suspension is useful as a sarcopenia model in rats.

BACKGROUND: Sarcopenia promotes skeletal muscle atrophy and exhibits a high mortality rate. Its elucidation is of the highest clinical importance, but an animal experimental model remains controversial. In this study, we investigated a simple method for studying sarcopenia in rats. RESULTS: Muscle atrophy was investigated in 24-week-old, male, tail-suspended (TS), Sprague Dawley and spontaneously hypertensive rats (SHR). Age-matched SD rats were used as a control group. The skeletal muscle mass weight, muscle contraction, whole body tension (WBT), cross-sectional area (CSA), and Muscle RING finger-1 (MuRF-1) were assessed. Enzyme-linked immunosorbent assay was used to evaluate the MuRF-1 levels. Two muscles, the extensor digitorum longus and soleus muscles, were selected for representing fast and slow muscles, respectively. All data, except CSA, were analyzed by a one-way analysis of variance, whereas CSA was analyzed using the Kruskal-Wallis test. Muscle mass weight, muscle contraction, WBT, and CSA were significantly lower in the SHR (n = 7) and TS (n = 7) groups than in the control group, whereas MuRF-1 expression was dominant. CONCLUSIONS: TS and SHR presented sarcopenic phenotypes in terms of muscle mass, muscle contraction and CSA. TS is a useful technique for providing muscle mass atrophy and weakness in an experimental model of sarcopenia in rats.

Protein Supplementation Enhances the Effects of Intermittent Loading on Skeletal Muscles by Activating the mTORC1 Signaling Pathway in a Rat Model of Disuse Atrophy.

Inactivity leads to skeletal muscle atrophy, whereas intermittent loading (IL) during hind limb unloading (HU) attenuates muscle atrophy. However, the combined effects of IL and protein supplementation on disuse muscle atrophy are unclear. Therefore, we investigated the effects of IL and a high-protein oral nutritional supplement (HP) during HU on skeletal muscle mass and protein synthesis/breakdown. Male F344 rats were assigned to the control (CON), 14-day HU (HU), IL during HU (HU + IL), and IL during HU followed by HP administration (2.6 g protein/kg/day; HU + IL + HP) groups. Soleus and gastrocnemius muscles were sampled 30 min after the last IL and HP supplementation. HU decreased relative soleus and gastrocnemius muscle masses. Relative muscle masses and p70 ribosomal protein S6 kinase/ribosomal protein S6 phosphorylation in soleus and gastrocnemius muscles were higher in the HU + IL group than the HU group and further higher in the HU + IL + HP group than the HU + IL group in gastrocnemius muscle. Therefore, protein administration plus IL effectively prevented skeletal muscle atrophy induced by disuse, potentially via enhanced activation of targets downstream of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway.

Intestinal microbiota contributes to altered glucose metabolism in simulated microgravity mouse model.

Exposure to space environment induces alterations in glucose and lipid metabolism that contribute to muscular atrophy, bone loss, and cardiovascular disorders. Intestinal microbiota is also changed, but its impact on spaceflight-related metabolic disorder is not clear. We investigated the relationship between glucose metabolic changes and gut dysbiosis in a hind limb-unloading (HU) mouse model, a well-accepted ground-based spaceflight analog. Impaired body weight gain, glucose intolerance, and peripheral insulin resistance were found in 2-4-wk HU mice. Reduced abundance of gut Bifidobacterium spp. and Akkermansia muciniphila was observed within 3 d of HU. The ground-based control (Ctrl) mice that were cohoused with HU mice showed similar patterns of dysbiosis and metabolic changes. Compared with the Ctrls, higher levels of plasma LPS-binding protein and altered transcription of Tnfa and glucose metabolism-related genes in the liver were observed in HU mice. The supplementation of Bifidobacterium spp. suppressed endotoxemia and liver inflammation and improved glucose tolerance in HU mice. The results indicate a close relationship between dysbiosis and altered glucose metabolism in the HU model and also emphasize the importance of evaluating intestinal microbiota in astronauts and its effect on glucose metabolism.-Wang, Y., Zhao, W., Shi, J., Wang, J., Hao, J., Pang, X., Huang, X., Chen, X., Li, Y., Jin, R., Ge, Q. Intestinal microbiota contributes to altered glucose metabolism in simulated microgravity mouse model.

Metabolomics Analyses of Muscle Atrophy Induced by Hind Limb Unloading.

Acute periods of contractile inactivity cause skeletal muscle atrophy along with profound alterations in tissue metabolism. Hind limb unloading via tail suspension is a commonly used rodent model of muscle atrophy. Here, we describe a sample preparation and LC-MS/MS approach for quantifying specific panels of acylcarnitines, amino acids, and organic acids in small (~8 mg) samples of atrophied mouse soleus following a period of hind limb unloading.

Temporary Loading Prevents Cancer Progression and Immune Organ Atrophy Induced by Hind-Limb Unloading in Mice.

Although the body's immune system is altered during spaceflight, the effects of microgravity (µG) on tumor growth and carcinogenesis are, as yet, unknown. To assess tumor proliferation and its effects on the immune system, we used a hind-limb unloading (HU) murine model to simulate µG during spaceflight. HU mice demonstrated significantly increased tumor growth, metastasis to the lung, and greater splenic and thymic atrophy compared with mice in constant orthostatic suspension and standard housing controls. In addition, mice undergoing temporary loading during HU (2 h per day) demonstrated no difference in cancer progression and immune organ atrophy compared with controls. Our findings suggest that temporary loading can prevent cancer progression and immune organ atrophy induced by HU. Further space experiment studies are warranted to elucidate the precise effects of µG on systemic immunity and cancer progression.

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

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

ß-Carotene prevents bone loss in hind limb unloading mice.

ß-Carotene has been reported to be useful to maintain a positive balance of bone turnover. However, the effects of ß-carotene on bone loss remain to be elucidated in mice with hind limb unloading. Therefore, we investigated whether ß-carotene prevented bone loss induced by skeletal hind limb unloading in mice. Female 8-week-old ddY mice were divided into six groups (n = 6-8 each) and subjected to: (1) normal housing, (2) sham unloading fed a control diet, (3) hind limb unloading fed a control diet, (4) hind limb unloading fed a 0.025% ß-carotene-containing diet, (5) hind limb unloading fed a 0.05% ß-carotene-containing diet, and (6) hind limb unloading fed a 0.25% ß-carotene-containing diet. After 3 weeks, bone mineral density of the tibia was markedly reduced by unloading, which was prevented by 0.025% ß-carotene. Histological analysis revealed a hind limb unloading-induced decrease in the calcified bone of the femur, which was slightly prevented by 0.025% ß-carotene. The 0.025% ß-carotene-containing diet increased the gene expression of osteoprotegerin in the bone marrow cells in unloading mice. These results suggest that a ß-carotene-containing diet may preserve bone health in subjects with disabilities as well as in astronauts.

In vivo hypobaric hypoxia performed during the remodeling process accelerates bone healing in mice.

We investigated the effects of respiratory hypobaric hypoxia on femoral bone-defect repair in mice because hypoxia is believed to influence both mesenchymal stromal cell (MSC) and hematopoietic stem cell mobilization, a process involved in the bone-healing mechanism. To mimic conditions of non-weight-bearing limb immobilization in patients suffering from bone trauma, our hypoxic mouse model was further subjected to hind-limb unloading. A hole was drilled in the right femur of adult male C57/BL6J mice. Four days after surgery, mice were subjected to hind-limb unloading for 1 week. Seven days after surgery, mice were either housed for 4 days in a hypobaric room (FiO2 at 10%) or kept under normoxic conditions. Unsuspended control mice were housed in either hypobaric or normoxic conditions. Animals were sacrificed on postsurgery day 11 to allow for collection of both contralateral and lesioned femurs, blood, and spleen. As assessed by microtomography, delayed hypoxia enhanced bone-healing efficiency by increasing the closing of the cortical defect and the newly synthesized bone volume in the cavity by +55% and +35%, respectively. Proteome analysis and histomorphometric data suggested that bone-repair improvement likely results from the acceleration of the natural bone-healing process rather than from extended mobilization of MSC-derived osteoprogenitors. Hind-limb unloading had hardly any effect beyond delayed hypoxia-enhanced bone-healing efficiency.

Suppression of mast cell activity contributes to the osteoprotective effect of an herbal formula containing Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae.

OBJECTIVES: Mast cells are believed to contribute to the pathogenesis of osteoporosis as their number is increased in osteoporotic bones. Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae are three Chinese herbs traditionally for tonifying the 'kidney system' and a herbal formula (ELP) containing the respective herbs at the weight ratio of 5 : 4 : 1 was shown to prevent osteoporosis. This study evaluated if suppression of mast cell accumulation and activity contribute to the anti-osteoporotic action of ELP. METHODS: The herbs were boiled under reflux to produce the aqueous extract that was further concentrated under reduced pressure and lyophilized. An in-vivo rat osteoporosis model using hind limb unloading was employed for studying the accumulation of mast cells. The human mast cell line, LAD2, was employed to evaluate the mast cell modulating action of ELP. KEY FINDINGS: Mast cell number in the tibiae of hind limb unloaded rats increased significantly during the course of osteoporosis. ELP treatment (10 g/kg/day) prevented both osteoporosis and mast cell accumulation in these rats. Furthermore, ELP significantly inhibited histamine and tumour necrosis factor-α release from LAD2 cells. CONCLUSION: Mast cells contributed to hormone independent osteoporosis. The suppression of mast cell accumulation and activation may contribute to the anti-osteoporotic action of ELP.