Hindlimb Unloading Model Research Articles

Ckip-1 3'-UTR Attenuates Simulated Microgravity-Induced Cardiac Atrophy.

Microgravity prominently affected cardiovascular health, which was the gravity-dependent physical factor. Deep space exploration had been increasing in frequency, but heart function was susceptible to conspicuous damage and cardiac mass declined in weightlessness. Understanding of the etiology of cardiac atrophy exposed to microgravity currently remains limited. The 3′-untranslated region (UTR) of casein kinase-2 interacting protein-1 (Ckip-1) was a pivotal mediator in pressure overload-induced cardiac remodeling. However, the role of Ckip-1 3′-UTR in the heart during microgravity was unknown. We analyzed Ckip-1 mRNA 3′-UTR and coding sequence (CDS) expression levels in ground-based analogs such as mice hindlimb unloading (HU) and rhesus monkey head-down bed rest model. Ckip-1 3′-UTR had transcribed levels in the opposite change trend with cognate CDS expression in the hearts. We then subjected wild-type (WT) mice and cardiac-specific Ckip-1 3′-UTR-overexpressing mice to hindlimb unloading for 28 days. Our results uncovered that Ckip-1 3′-UTR remarkably attenuated cardiac dysfunction and mass loss in simulated microgravity environments. Mechanistically, Ckip-1 3′-UTR inhibited lipid accumulation and elevated fatty acid oxidation-related gene expression in the hearts through targeting calcium/calmodulin-dependent kinase 2 (CaMKK2) and activation of the AMPK-PPARα-CPT1b signaling pathway. These findings demonstrated Ckip-1 3′-UTR was an important regulator in atrophic heart growth after simulated microgravity.

The Skeletal Cellular and Molecular Underpinning of the Murine Hindlimb Unloading Model.

Bone adaptation to spaceflight results in bone loss at weight bearing sites following the absence of the stimulus represented by ground force. The rodent hindlimb unloading model was designed to mimic the loss of mechanical loading experienced by astronauts in spaceflight to better understand the mechanisms causing this disuse-induced bone loss. The model has also been largely adopted to study disuse osteopenia and therefore to test drugs for its treatment. Loss of trabecular and cortical bone is observed in long bones of hindlimbs in tail-suspended rodents. Over the years, osteocytes have been shown to play a key role in sensing mechanical stress/stimulus via the ECM-integrin-cytoskeletal axis and to respond to it by regulating different cytokines such as SOST and RANKL. Colder experimental environments (~20–22°C) below thermoneutral temperatures (~28–32°C) exacerbate bone loss. Hence, it is important to consider the role of environmental temperatures on the experimental outcomes. We provide insights into the cellular and molecular pathways that have been shown to play a role in the hindlimb unloading and recommendations to minimize the effects of conditions that we refer to as confounding factors.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Accumulation of formaldehyde causes motor deficits in an in vivo model of hindlimb unloading

During duration spaceflight, or after their return to earth, astronauts have often suffered from gait instability and cerebellar ataxia. Here, we use a mouse model of hindlimb unloading (HU) to explore a mechanism of how reduced hindlimb burden may contribute to motor deficits. The results showed that these mice which have experienced HU for 2 weeks exhibit a rapid accumulation of formaldehyde in the gastrocnemius muscle and fastigial nucleus of cerebellum. The activation of semicarbazide-sensitive amine oxidase and sarcosine dehydrogenase induced by HU-stress contributed to formaldehyde generation and loss of the abilities to maintain balance and coordinate motor activities. Further, knockout of formaldehyde dehydrogenase (FDH-/-) in mice caused formaldehyde accumulation in the muscle and cerebellum that was associated with motor deficits. Remarkably, formaldehyde injection into the gastrocnemius muscle led to gait instability; especially, microinfusion of formaldehyde into the fastigial nucleus directly induced the same symptoms as HU-induced acute ataxia. Hence, excessive formaldehyde damages motor functions of the muscle and cerebellum.

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.

RTMS alleviates cognitive and neural oscillatory deficits induced by hindlimb unloading in mice via maintaining balance between glutamatergic and GABAergic systems

Microgravity, as a part of the stress of space flight, has several negative effects on cognitive functions. Repetitive transcranial magnetic stimulation (rTMS), as a novel non-invasive technique, could be an effective approach to alleviated cognitive decline, applied in both preclinical and clinical studies. Neural oscillations and their interactions are involved in cognitive functions and support the communication of neural information. The neural oscillation could be a window from which we may understand what happens in the brain. The current study aimed to explore if 15 Hz rTMS plays a neural modulation role in a mouse model of hindlimb unloading. We hypothezed that rTMS can improve the cognitive and neural oscillatory deficits induced by hindlimb unloading via maintaining the balance between glutamatergic and GABAergic systems. Our data show that rTMS can significantly alleviate behavior deficits, modulate theta oscillation, improve the disturbed power distribution of theta oscillation and the decreased strength of Cross-Frequency Coupling in the dentate gyrus region, and effectively mitigated the blocked communication of neural information in the perforant pathway (PP)―dentate gyrus (DG) neural pathway in Hu mice. Furthermore, biochemical analysis using high-performance liquid chromatography and Western blot assay confirmed that rTMS increases the low expression of glutamate (Glu) and N-Methyl d-Aspartate receptor subtype 2B (NR2B) and decreases the high expression of γ-aminobutyric acid (GABA), 67 KDa isoform of glutamate decarboxylase (GAD67), and GABA type A receptor subunit alpha1 (GABAARα1) in the hippocampus of Hu mice. Taken together, the results suggest that rTMS plays a significant neural modulation role in the hippocampal neural activity disorders induced by Hu, which possibly depends on rTMS maintaining the balance of glutamatergic and gamma-aminobutyric acidergic (GABAergic) systems.

Global alternative splicing landscape of skeletal muscle atrophy induced by hindlimb unloading.

BackgroundLong-term exposure to microgravity will cause skeletal muscle atrophy, which can cause serious harm to astronauts in space travel. Therefore, it is important to explore skeletal muscle atrophy’s molecular mechanism for its prevention and treatment. However, as an important regulatory approach of skeletal muscle physiology, the role of alternative splicing in skeletal muscle atrophy, especially skeletal muscle atrophy caused by disuse, is unclear.MethodsWe established a rat hindlimb unloading model and performed RNA sequencing on soleus muscle, which was seriously atrophied during unloading. Several bioinformatics methods were used to identify alternative splicing events and determine their gene functions.ResultsMany alternative splicing events were found to occur at different time points (12 h, 24 h, 36 h, 3 days, and 7 days) after hindlimb unloading. These differential alternative splicing events mainly occurred in the gene's coding domain sequence region, and 59% of the alternative splicing events caused open reading frameshift. Bioinformatics analysis results showed that genes with different alternative splicing events were enriched in multiple pathways related to muscle atrophy, including the insulin signaling pathway, endocytosis, mitophagy, and ubiquitin-proteasome pathway. Moreover, alternative splicing of several deubiquitinase genes persisted during skeletal muscle atrophy induced by unloading. Additionally, we identified 10 differentially expressed RNA binding proteins during skeletal muscle atrophy induced by unloading, mainly containing Xpo4, Eif4e2, P4ha1, Lrrfip1, Zc3h14, Emg1, Hnrnp h1, Mbnl2, RBfox1, and Mbnl1. Hnrnp h1 and Mbnl2 were significantly downregulated, and RBfox1 and Mbnl1 were significantly upregulated during skeletal muscle atrophy caused by unloading.ConclusionsTo the best of our knowledge, the present study is the first to propose alternative splicing alterations related to disuse-induced muscle atrophy, emphasizing that alternative splicing is a new focus of attention in the occurrence of muscle atrophy.

Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy.

BackgroundMuscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well‐established model of hindlimb unloading (HU).MethodsOne hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism).ResultsBoth males and females lost ~30% of tibialis anterior cross‐sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross‐sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24–48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4‐fold greater content in females compared with ~2‐fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes.ConclusionsThese results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Hindlimb unloading-induced reproductive suppression via Downregulation of hypothalamic Kiss-1 expression in adult male rats

BackgroundSpaceflights-induced microgravity can alter various physiological processes in human’s body including the functional status of the reproductive system. Rodent model of tail-suspension hindlimb unloading is extensively used to stimulate the organs responses to the microgravity condition. This study explores the potential effects of hindlimb unloading on testicular functions and spermatogenesis in adult male rats and the underlying mechanism/s.MethodsTwenty Sprague-Dawley rats were allotted into two groups: normally loaded group (control; all arms were in touch with the grid floor) and hindlimb unloaded group (HU; only the forearms were in contact with the grid floor).ResultsFollowing 30 days of exposure, the HU group saw a decline in body weight, testicular and epidydimal weights, and all semen parameters. The circulating concentrations of gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone significantly decreased, while levels of kisspeptin, corticosterone, inhibin, prolactin and estradiol (E2) increased in the HU group. Intratesticular levels of 5α-reductase enzyme and dihydrotestosterone (DHT) were suppressed, while the levels of aromatase and kisspeptin were significantly elevated in the HU group. Hypothalamic kisspeptin (Kiss1) mRNA expression levels were downregulated while its receptors (Kiss1R) were upregulated in the HU group. On the contrary, the mRNA expression levels of testicular Kiss1 were upregulated while Kiss1R were downregulated. The pituitary mRNA expression levels of FSHβ and LHβ decreased in the HU group. The levels of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and nitric oxide (NO) concentrations, and total antioxidant capacity (TAC) were elevated while malondialdehyde (MDA) concentrations declined in the testes of HU group. The testes of the HU rats showed positive immunostaining of caspase-3, heat shock protein 70 (HSP70) and Bcl2.ConclusionsAltogether, these results revealed an inhibitory effect of hindlimb unloading on kisspeptin signaling in the hypothalamic-pituitary-testicular axis with impaired spermatogenesis and steroidogenesis.

Abaloparatide treatment increases bone formation, bone density and bone strength without increasing bone resorption in a rat model of hindlimb unloading.

Disuse osteoporosis can result from prolonged bed rest, paralysis, casts, braces, fractures and other conditions. Abaloparatide (ABL) is a PTHrP analog that increases bone density and strength by stimulating osteogenesis with limited effects on bone resorption. We examined skeletal responses to abaloparatide in young adult male rats with normal weight-bearing and with hindlimb unloading via a pelvic harness. Rats were allocated to four groups (10-12 per group): normal weight-bearing plus vehicle treatment (CON-VEH), normal weight-bearing plus ABL treatment (CON-ABL), hindlimb-unloading plus vehicle (HLU-VEH), or hindlimb-unloading plus ABL (HLU-ABL). Rats received ABL (25μg/kg/day, s.c.) or vehicle throughout the 28-day unloading period and were then sacrificed, at which time HLU-VEH rats exhibited reduced bone formation and significant deficits in tibial, femoral, and vertebral bone mass compared with CON-VEH. ABL treatment increased serum osteocalcin in CON and HLU animals while having no effect on the osteoclast marker TRACP-5b. Longitudinal peripheral quantitative computed tomography (pQCT) indicated that ABL increased trabecular and cortical bone mass in the tibia. ABL was also associated with improved trabecular and cortical bone mass and architectural parameters at the femur, tibia, and vertebrae by μCT. Tibial histomorphometry indicated increased trabecular and endocortical bone formation with HLU-ABL versus HLU-VEH and with CON-ABL versus CON-VEH, and ABL was also associated with lower trabecular and endocortical osteoclast surfaces. Vertebral finite element analysis indicated higher ultimate load and stiffness for CON-ABL versus CON-VEH and for HLU-ABL versus HLU-VEH. In summary, ABL was associated with improved trabecular and cortical bone density and architecture in normal weight-bearing and hindlimb-unloaded rats, with higher bone formation and no difference in bone resorption. ABL was also associated with improved bone biomechanical parameters. These results provide rationale for investigating the ability of abaloparatide to prevent or treat disuse osteoporosis in humans.

Approaching Gravity as a Continuum Using the Rat Partial Weight-Bearing Model.

For decades, scientists have relied on animals to understand the risks and consequences of space travel. Animals remain key to study the physiological alterations during spaceflight and provide crucial information about microgravity-induced changes. While spaceflights may appear common, they remain costly and, coupled with limited cargo areas, do not allow for large sample sizes onboard. In 1979, a model of hindlimb unloading (HU) was successfully created to mimic microgravity and has been used extensively since its creation. Four decades later, the first model of mouse partial weight-bearing (PWB) was developed, aiming at mimicking partial gravity environments. Return to the Lunar surface for astronauts is now imminent and prompted the need for an animal model closer to human physiology; hence in 2018, our laboratory created a new model of PWB for adult rats. In this review, we will focus on the rat model of PWB, from its conception to the current state of knowledge. Additionally, we will address how this new model, used in conjunction with HU, will help implement new paradigms allowing scientists to anticipate the physiological alterations and needs of astronauts. Finally, we will discuss the outstanding questions and future perspectives in space research and propose potential solutions using the rat PWB model.

The Immunomodulatory Effect of Radiofrequency Electromagnetic Field on Serum Cytokine Levels in A Mouse Model of Hindlimb Unloading.

Objective Astronauts are exposed to a wide range of environmental stresses during spaceflights that reduce their immune responses and make them more susceptible to infections and malignancies. Exposure to a low dose of a certain stress induces an adaptive response, which leads to resistance to higher doses of the same or other types of stress. We designed this study to investigate the effect of radiofrequency electromagnetic field (RF-EMF)-induced adaptive response on immune system modulation in a mouse model of hindlimb unloading (HU) as a ground-based animal model of spaceflight conditions. Materials and Methods In this experimental study, serum levels of T helper (Th)-mediated cytokines were determined by the multiplex cytometric bead assay in four groups of mice (n=10 per group): HU mice, RF-EMF-treated mice, HU mice pre-exposed to RF-EMF; and untreated controls. Mice were exposed to 2450 MHz RF-EMF with SAR 0.478 W/ kg for 12 hours/day for three successive days. Results Tumor necrosis factor-alpha (TNF-α), interleukin-9 (IL-9) and IL-22 were significantly decreased in HU mice. Comparison between HU mice and RF-EMF-treated mice showed an opposite change in IL-6, while IL-9, IL-22, IFN-γ and TNF-α decreased in both groups. However, just interferon gamma (IFN-γ) was significantly decreased in HU mice that were pre-exposed to RF-EMF compared to the control group. ConclusionThe effect of RF-EMF in elevating IL-6 and reducing IL-9 in opposite directions in HU mice suggest a modulating effect of RF-EMF on HU-induced changes in these cytokines, as Th2 and Th9 eventually returned to normal levels and balances in cytokine ratios were also restored in HU mice pre-exposed to RF-EMF.

Recombinant Irisin Prevents the Reduction of Osteoblast Differentiation Induced by Stimulated Microgravity through Increasing β-Catenin Expression.

Background: Irisin, a novel exercise-induced myokine, was shown to mediate beneficial effects of exercise in osteoporosis. Microgravity is a major threat to bone homeostasis of astronauts during long-term spaceflight, which results in decreased bone formation. Methods: The hind-limb unloading mice model and a random position machine are respectively used to simulate microgravity in vivo and in vitro. Results: We demonstrate that not only are bone formation and osteoblast differentiation decreased, but the expression of fibronectin type III domain-containing 5 (Fdnc5; irisin precursor) is also downregulated under simulated microgravity. Moreover, a lower dose of recombinant irisin (r-irisin) (1 nM) promotes osteogenic marker gene (alkaline phosphatase (Alp), collagen type 1 alpha-1(ColIα1)) expressions, ALP activity, and calcium deposition in primary osteoblasts, with no significant effect on osteoblast proliferation. Furthermore, r-irisin could recover the decrease in osteoblast differentiation induced by simulated microgravity. We also find that r-irisin increases β-catenin expression and partly neutralizes the decrease in β-catenin expression induced by simulated microgravity. In addition, β-catenin overexpression could also in part attenuate osteoblast differentiation reduction induced by simulated microgravity. Conclusions: The present study is the first to show that r-irisin positively regulates osteoblast differentiation under simulated microgravity through increasing β-catenin expression, which may reveal a novel mechanism, and it provides a prevention strategy for bone loss and muscle atrophy induced by microgravity.

Targeted silencing of miRNA-132-3p expression rescues disuse osteopenia by promoting mesenchymal stem cell osteogenic differentiation and osteogenesis in mice

BackgroundSkeletal unloading can induce severe disuse osteopenia that often occurs in spaceflight astronauts or in patients subjected to prolonged bed-rest or immobility. Previously, we revealed a mechano-sensitive factor, miRNA-132-3p, that is closely related to the osteoblast function. The aim of this study was to investigate whether miRNA-132-3p could be an effective target for treating disuse osteopenia.MethodsThe 2D-clinostat device and the hindlimb-unloaded (HU) model were used to copy the mechanical unloading condition at the cellular and animal levels, respectively. Mimics or inhibitors of miRNA-132-3p were used to interfere with the expression of miRNA-132-3p in bone marrow-derived mesenchymal stem cells (BMSCs) in vitro for analyzing the effects on osteogenic differentiation. The special in vivo antagonists of miRNA-132-3p was delivered to the bone formation regions of HU mice for treating disuse osteopenia by a bone-targeted (AspSerSer)6-cationic liposome system. The bone mass, microstructure, and strength of the hindlimb bone tissue were analyzed for evaluating the therapeutic effect in vivo.ResultsmiRNA-132-3p expression was declined under normal conditions and increased under gravitational mechanical unloading conditions during osteogenic differentiation of BMSCs in vitro. The upregulation of miRNA-132-3p expression resulted in the inhibition of osteogenic differentiation, whereas the downregulation of miRNA-132-3p expression enhanced osteogenic differentiation. The inhibition of miRNA-132-3p expression was able to attenuate the negative effects of mechanical unloading on BMSC osteogenic differentiation. Most importantly, the targeted silencing of miRNA-132-3p expression in the bone tissues could effectively preserve bone mass, microstructure, and strength by promoting osteogenic differentiation and osteogenesis in HU mice.ConclusionThe overexpression of miRNA-132-3p induced by mechanical unloading is disadvantageous for BMSC osteogenic differentiation and osteogenesis. Targeted silencing of miRNA-132-3p expression presents a potential therapeutic target for the prevention and treatment of disuse osteoporosis.

Effects of Long-Term and Acute Hindlimb Unloading Model on Neuroelectrophysiological Signals of Hippocampal Interneurons and Pyramidal Cells Using Microelectrode Arrays

Microgravity is the key environment of weightlessness experienced by all astronauts during spaceflights that cause severe physiological alterations in the human body. The effect of microgravity on the nervous system has not been elucidated. In this study, hindlimb unloading rats were used as the simulated microgravity model, and self-designed novel microelectrode arrays were used as the essential tools. The special arrangement of detection sites and platinum nanoparticles modification made the electrodes have excellent performance at the neuron level. By comparing the changes of the neuroelectrophysiological signals in the CA1 regions of rats in different periods of long-term and short-term modeling, the following results were obtained. In the long-term model, the firing rates reached a peak after about seven days of modeling, and then kept decreasing till 28 days. The average firing rates and individual single cell firing rates of the acute model rose immediately after modeling. Analysis of power spectral density signals showed that the signals shifted to the low frequency band after both long-term and acute modeling. The neurons in the CA1 were classified into pyramidal cells and interneurons. The continuous observation of neurons under the acute modeling showed that the firing rates and amplitude of pyramidal cells have a greater increase than interneurons. The self-designed novel implantable microelectrode arrays provide an advanced tool for the detection of neurons in hindlimb unloading rats. The hippocampal nerve cells were impaired after modulation, and that pyramidal cells were more susceptible than interneurons.

Simulated Weightlessness Perturbs the Intestinal Metabolomic Profile of Rats.

Recently, disorders of intestinal homeostasis in the space environment have been extensively demonstrated. Accumulating evidence have suggested microgravity and simulated weightlessness could induce dysbiosis of intestinal microbiota, which may contribute to the bowel symptoms during spaceflight. However, the specific responses of intestinal metabolome under simulated weightlessness and its relationship with the intestinal microbiome and immune characteristics remain largely unknown. In the current study, 20 adult Sprague-Dawley (SD) rats were randomly divided into the control group and the simulated weightlessness group using a hindlimb unloading model. The metabolomic profiling of cecal contents from eight rats of each group was investigated by gas chromatography-time of flight/mass spectrometry. The significantly different metabolites, biomarkers, and related pathways were identified. Multivariate analysis, such as principal component analysis and orthogonal projections to latent structures-discriminant analysis, demonstrated an obvious separation between the control group and the simulated weightlessness group. Significantly different metabolites, such as xylose, sinapinic acid, indolelactate, and digalacturonic acid, were identified, which participate in mainly pyrimidine metabolism, pentose and glucuronate interconversions, and valine, leucine and isoleucine metabolism. Cytidine-5′-monophosphate, 4-hydroxypyridine, and phloretic acid were determined as pivotal biomarkers under simulated weightlessness. Moreover, the significantly different metabolites were remarkably correlated with dysbiosis of the intestinal microbiota and disturbance of immunological characteristics induced by simulated weightlessness. These metabolic features provide crucial candidates for therapeutic targets for metabolic disorders under weightlessness.

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

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

Overexpression of Catalase in Mitochondria Mitigates the Inflammatory Effects of Simulated Microgravity and Social Isolation in Mouse Hippocampus

Space flight missions are becoming longer and more common and evidence points to the physiological toll the missions have on the human body. Aging, sedentary lifestyle, and spaceflight have similar degenerative effects on almost every part of our body; both exposure to the space environment and aging result in cardiovascular deconditioning, bone loss, muscle atrophy, brain changes, and immune response impairment. We hypothesize that exposure to the space environment generates excessive Reactive Oxygen Species (ROS), which results in neuroinflammation and aging‐like degenerative symptoms in the brain. We used the hindlimb unloading (HU) model to mimic microgravity with either paired or single housed animals (social isolation). Responses to 30d of HU were compared in wildtype or transgenic MCAT mice, in which mitochondrial ROS is quenched by over‐expression of human catalase. Expression of 4‐Hydroxynonenal (4HNE) and Park7 (redox‐sensitive chaperone and sensor of oxidative stress) were measured by ELISA, a protein array quantified from the hippocampal cytokines and 8‐hydroxy‐2’‐deoxyguanosine in serum was measured by ELISA to assess oxidative DNA damage. Preliminary analysis of cage behavior patterns from video collected at the end of the study showed that MCAT HU mice (socially housed) were more active and conducted more exploratory activities compared to NL. Our biochemical results showed simulated microgravity and/or social isolation caused changes in levels of cytokines related to immune responses. Two‐way ANOVA revealed significant interaction effects of HU and genotype in expression levels of five cytokines (out of 35) in socially‐housed animals. Elevation of these generally pro‐inflammatory cytokines by HU in WT mice was mitigated in MCAT mice, suggesting a role for mitochondrial ROS signaling in inflammatory CNS responses to microgravity. Interestingly, some of these cytokines in the hippocampus displayed strong correlations to the 4HNE levels. We also found substantive cytokine responses to social isolation in the hippocampus; housing and genotype interaction effects were significant (by 2‐Factor ANOVA) for 15 cytokines, most of which were mitigated in MCAT mice.Taken together, our results showed that both simulated microgravity and social isolation influenced cytokine levels in the hippocampus and MCAT mice were at least partially protected from these changes. These findings implicate a potentially important role for mitochondrial ROS in CNS responses to the challenges posed by long duration spaceflight.Support or Funding InformationThis work is supported by a NASA Space Biology Grant to RKG (NNH14ZTT001N). LG was supported by a Space Biology NASA Postdoctoral Fellowship AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Age-dependent bone loss and recovery during hindlimb unloading and subsequent reloading in rats

BackgroundBone structure and strength are rapidly lost during conditions of decreased mechanical loading, and aged bones have a diminished ability to adapt to increased mechanical loading. This is a concern for older patients that experience periods of limited mobility or bed rest, but the acute effects of disuse on the bones of aged patients have not been thoroughly described. Previous animal studies have primarily examined the effect of mechanical unloading on young animals. Those that have studied aged animals have exclusively focused on bone loss during unloading and not bone recovery during subsequent reloading. In this study, we investigated the effect of decreased mechanical loading and subsequent reloading on bone using a hindlimb unloading model in Adult (9 month old) and Aged (28 month old) male rats.MethodsAnimals from both age groups were subjected to 14 days of hindlimb unloading followed by up to 7 days of reloading. Additional Aged rats were subjected to 7 days of forced treadmill exercise during reloading or a total of 28 days of reloading. Trabecular and cortical bone structure of the femur were quantified using ex vivo micro-computed tomography (μCT), and mechanical properties were quantified with mechanical testing.ResultsWe found that Adult rats had substantially decreased trabecular bone volume fraction (BV/TV) following unloading (− 27%) while Aged animals did not exhibit significant bone loss following unloading. However, Aged animals had lower trabecular BV/TV after 3 days of reloading (− 20% compared to baseline), while trabecular BV/TV of Adult rats was not different from baseline values after 3 days of reloading. Trabecular BV/TV of Aged animals remained lower than control animals even with exercise during 7 days of reloading and after 28 days of reloading.ConclusionsThese data suggest that aged bone is less responsive to both increased and decreased mechanical loading, and that acute periods of disuse may leave older subjects with a long-term deficit in trabecular bone mass. These finding indicate the need for therapeutic strategies to improve the skeletal health of elderly patients during periods of disuse.

Responses of Intestinal Mucosal Barrier Functions of Rats to Simulated Weightlessness.

Exposure to microgravity or weightlessness leads to various adaptive and pathophysiological alterations in digestive structures and physiology. The current study was carried out to investigate responses of intestinal mucosal barrier functions to simulated weightlessness, by using the hindlimb unloading rats model. Compared with normal controls, simulated weightlessness damaged the intestinal villi and structural integrity of tight junctions, up-regulated the expression of pro-apoptotic protein Bax while down-regulated the expression of anti-apoptotic protein Bcl-2, thus improved the intestinal permeability. It could also influence intestinal microbiota composition with the expansion of Bacteroidetes and decrease of Firmicutes. The predicted metagenomic analysis emphasized significant dysbiosis associated differences in genes involved in membrane transport, cofactors and vitamins metabolism, energy metabolism, and genetic information processing. Moreover, simulated weightlessness could modify the intestinal immune status characterized by the increase of proinflammatory cytokines, decrease of secretory immunoglobulin A, and activation of TLR4/MyD88/NF-κB signaling pathway in ileum. These results indicate the simulated weightlessness disrupts intestinal mucosal barrier functions in animal model. The data also emphasize the necessity of monitoring and regulating astronauts’ intestinal health during real space flights to prevent breakdowns in intestinal homeostasis of crewmembers.