Simulated microgravity increases CD226+ Lin- CD117- Sca1+ mesenchymal stem cells in mice.

Microgravity is one of the most common causes counting for the bone loss. Mesenchymal stem cells (MSCs) contribute greatly to the differentiation and function of bone related cells. The development of novel MSCs biomarkers is critical for implementing effective therapies for microgravity induced bone loss. We aimed to find the new molecules involved in the differentiation and function of MSCs in mouse simulated microgravity model. We found CD226 was preferentially expressed on a subset of MSCs. Simulation of microgravity treatment significantly increased the proportion of CD226+ Lin- CD117- Sca1+ MSCs. The CD226+ MSCs produced higher IL-6, M-CSF, RANKL and lower CD200 expression, and promoted osteoclast differentiation. This study provides pivotal information to understand the role of CD226 in MSCs, and inspires new ideas for prevention of bone loss related diseases.

NASA SPRINT exercise program efficacy for vastus lateralis and soleus skeletal muscle health during 70 days of simulated microgravity.

The efficacy of the NASA SPRINT exercise countermeasures program for quadriceps (vastus lateralis) and triceps surae (soleus) skeletal muscle health was investigated during 70 days of simulated microgravity. Individuals completed 6° head-down-tilt bedrest (BR, n = 9), bedrest with resistance and aerobic exercise (BRE, n = 9), or bedrest with resistance and aerobic exercise and low-dose testosterone (BRE + T, n = 8). All groups were periodically tested for muscle (n = 9 times) and aerobic (n = 4 times) power during bedrest. In BR, surprisingly, the typical bedrest-induced decrements in vastus lateralis myofiber size and power were either blunted (myosin heavy chain, MHC I) or eliminated (MHC IIa), along with no change (P > 0.05) in %MHC distribution and blunted quadriceps atrophy. In BRE, MHC I (vastus lateralis and soleus) and IIa (vastus lateralis) contractile performance was maintained (P > 0.05) or increased (P < 0.05). Vastus lateralis hybrid fiber percentage was reduced (P < 0.05) and energy metabolism enzymes and capillarization were generally maintained (P > 0.05), while not all of these positive responses were observed in the soleus. Exercise offsets 100% of quadriceps and approximately two-thirds of soleus whole muscle mass loss. Testosterone (BRE + T) did not provide any benefit over exercise alone for either muscle and for some myocellular parameters appeared detrimental. In summary, the periodic testing likely provided a partial exercise countermeasure for the quadriceps in the bedrest group, which is a novel finding given the extremely low exercise dose. The SPRINT exercise program appears to be viable for the quadriceps; however, refinement is needed to completely protect triceps surae myocellular and whole muscle health for astronauts on long-duration spaceflights.NEW & NOTEWORTHY This study provides unique exercise countermeasures development information for astronauts on long-duration spaceflights. The NASA SPRINT program was protective for quadriceps myocellular and whole muscle health, whereas the triceps surae (soleus) was only partially protected as has been shown with other programs. The bedrest control group data may provide beneficial information for overall exercise dose and targeting fast-twitch muscle fibers. Other unique approaches for the triceps surae are needed to supplement existing exercise programs.

Adaptation to full weight-bearing following disuse in rats: The impact of biological sex on musculoskeletal recovery.

With the technological advances made to expand space exploration, astronauts will spend extended amounts of time in space before returning to Earth. This situation of unloading and reloading influences human physiology, and readaptation to full weight-bearing may significantly impact astronauts' health. On Earth, similar situations can be observed in patients who are bedridden or suffer from sport-related injuries. However, our knowledge of male physiology far exceeds our knowledge of female's, which creates an important gap that needs to be addressed to understand the sex-based differences regarding musculoskeletal adaptation to unloading and reloading, necessary to preserve health of both sexes. Using a ground-based model of total unloading for 14 days and reloading at full weight-bearing for 7 days rats, we aimed to compare the musculoskeletal adaptations between males and females. Our results reveal the existence of significant differences. Indeed, males experienced bone loss both during the unloading and the reloading period while females did not. During simulated microgravity, males and females showed comparable muscle deconditioning with a significant decline in rear paw grip strength. However, after 7 days of recovery, muscle strength improved. Additionally, sex-based differences in myofiber size existing at baseline are significantly reduced or eliminated following unloading and recovery.

Simulated space environmental factors of weightlessness, noise and low atmospheric pressure differentially affect the diurnal rhythm and the gut microbiome.

The circadian clock extensively regulates physiology and behavior. In space, astronauts encounter many environmental factors that are dramatically different from those on Earth; however, the effects of these factors on circadian rhythms and the mechanisms remain largely unknown. The present study aimed to investigate the changes in the mouse diurnal rhythm and gut microbiome under simulated space capsule conditions, including microgravity, noise and low atmospheric pressure (LAP). Noise and LAP were loaded in the capsule while the conditions in the animal room remained constant. The mice in the capsule showed disturbed locomotor rhythms and faster adaptation to a 6-h phase advance. RNA sequencing of hypothalamus samples containing the suprachiasmatic nucleus (SCN) revealed that microgravity simulated by hind limb unloading (HU) and exposure to noise and LAP led to decreases in the quantities of differentially expressed genes (DEGs), including circadian clock genes. Changes in the rhythmicity of genes implicated in pathways of cardiovascular deconditioning and more concentrated phases were found under HU or noise and LAP. Furthermore, 16S rRNA sequencing revealed dysbiosis in the gut microbiome, and noise and LAP may repress the temporal discrepancy in the microbiome community structure induced by microgravity. Changes in diurnal oscillations were observed in a number of gut bacteria with critical physiological consequences on metabolism and immunodefense. We also found that the superimposition of noise and LAP may repress normal changes in global gene expression and adaptation in the gut microbiome. Our data demonstrate that in addition to microgravity, exposure to noise and LAP affect the robustness of circadian rhythms and the community structure of the gut microbiome, and these factors may interfere with each other in their adaptation to respective conditions. These findings are important for furthering our understanding of the alterations in circadian rhythms in the complex environment of space.

Cardiovascular changes under the microgravity environment and the gut microbiome.

In view of the critical role the gut microbiome plays in human health, it has become clear that astronauts' gut microbiota composition changes after spending time in space. Astronauts are exposed to several risks in space, including a protracted period of microgravity, radiation, and mechanical unloading of the body. Several deleterious effects of such an environment are reported, including orthostatic intolerance, cardiovascular endothelial dysfunction, cellular and molecular changes, and changes in the composition of the gut microbiome. Herein, the correlation between the gut microbiome and cardiovascular disease in a microgravity environment is evaluated. Additionally, the relationship between orthostatic hypotension, cardiac shrinkage and arrhythmias during spaceflight, and cellular alterations during spaceflight is reviewed. Given its impact on human health in general, modifying the gut microbiota may significantly promote astronaut health and performance. This is merited, given the prospect of augmented human activities in future space missions.

Reorganization of the mouse oocyte' cytoskeleton after cultivation under simulated weightlessness.

Female germ cells provide the structural basis for the development of a new organism, while the main molecular mechanisms of the impact of weightlessness on the cell remain unknown. The aim of this work was to determine the relative content and distribution of the main proteins of microtubules and microfilaments, to assess the relative RNA content of genes in mouse oocytes after short-term exposure to simulated microgravity, and to determine the potential for embryo development up to the 3-cell stage. Before starting the study, BALB/c mice were divided into two groups. One group received water and standard food without any modifications. Before exposure to simulated microgravity, the oocytes of these animals were randomly divided into two groups - c and µg. The second group of animals additionally received essential phospholipids containing at least 80% phosphatidylcholines, per os for 6 weeks before the start of the experiment at a dosage of 350 mg/kg of the animal's body to modify the lipid composition of the oocyte membrane. The obtained oocytes of these animals were also randomly divided into two groups - ce and µge. To determine the protein distribution and its relative content, immunofluorescence analysis was performed, and the RNA content of genes was assessed using real-time PCR with reverse transcription. After cultivation under simulated microgravity, beta-actin and acetylated alpha-tubulin are redistributed from the cortical layer to the central part of the oocyte, and the relative content of acetylated alpha-tubulin and tubulin isoforms decreases. At the same time, the mRNA content of most genes encoding cytoskeletal proteins was significantly higher in comparison with the control level. The use of essential phospholipids led to a decrease in the content of cellular cholesterol in the oocyte and leveled changes in the content and redistribution of acetylated alpha-tubulin and beta-actin after cultivation under simulated microgravity. In addition, after in vitro fertilization and further cultivation under simulated weightlessness, we observed a decrease in the number of embryos that passed the stage of the 2-cell embryo, but while taking essential phospholipids, the number of embryos that reached the 3-cell stage did not differ from the control group. The results obtained show changes in the content and redistribution of cytoskeletal proteins in the oocyte, which may be involved in the process of pronucleus migration, the formation of the fission spindle and the contractile ring under simulated weightlessness, which may be important for normal fertilization and cleavage of the future embryo.

Atp6v1h Deficiency Blocks Bone Loss in Simulated Microgravity Mice through the Fos-Jun-Src-Integrin Pathway.

The microgravity conditions in outer space are widely acknowledged to induce significant bone loss. Recent studies have implicated the close relationship between Atp6v1h gene and bone loss. Despite this, the role of Atp6v1h in bone remodeling and its molecular mechanisms in microgravity have not been fully elucidated. To address this, we used a mouse tail suspension model to simulate microgravity. We categorized both wild-type and Atp6v1h knockout (Atp6v1h+/-) mice into two groups: regular feeding and tail-suspension feeding, ensuring uniform feeding conditions across all cohorts. Analysis via micro-CT scanning, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase assays indicated that wild-type mice underwent bone loss under simulated microgravity. Atp6v1h+/- mice exhibited bone loss due to Atp6v1h deficiency but did not present aggravated bone loss under the same simulated microgravity. Transcriptomic sequencing revealed the upregulation of genes, such as Fos, Src, Jun, and various integrin subunits in the context of simulated microgravity and Atp6v1h knockout. Real-time quantitative polymerase chain reaction (RT-qPCR) further validated the modulation of downstream osteoclast-related genes in response to interactions with ATP6V1H overexpression cell lines. Co-immunoprecipitation indicated potential interactions between ATP6V1H and integrin beta 1, beta 3, beta 5, alpha 2b, and alpha 5. Our results indicate that Atp6v1h level influences bone loss in simulated microgravity by modulating the Fos-Jun-Src-Integrin pathway, which, in turn, affects osteoclast activity and bone resorption, with implications for osteoporosis. Therefore, modulating Atp6v1h expression could mitigate bone loss in microgravity conditions. This study elucidates the molecular mechanism of Atp6v1h's role in osteoporosis and positions it as a potential therapeutic target against environmental bone loss. These findings open new possibilities for the treatment of multifactorial osteoporosis.

Differential Gene Expression in Human Fibroblasts Simultaneously Exposed to Ionizing Radiation and Simulated Microgravity.

During future space missions, astronauts will be exposed to cosmic radiation and microgravity (µG), which are known to be health risk factors. To examine the differentially expressed genes (DEG) and their prevalent biological processes and pathways as a response to these two risk factors simultaneously, 1BR-hTERT human fibroblast cells were cultured under 1 gravity (1G) or simulated µG for 48 h in total and collected at 0 (sham irradiated), 3 or 24 h after 1 Gy of X-ray or Carbon-ion (C-ion) irradiation. A three-dimensional clinostat was used for the simulation of µG and the simultaneous radiation exposure of the samples. The RNA-seq method was used to produce lists of differentially expressed genes between different environmental conditions. Over-representation analyses were performed and the enriched biological pathways and targeting transcription factors were identified. Comparing sham-irradiated cells under simulated µG and 1G conditions, terms related to response to oxygen levels and muscle contraction were identified. After irradiation with X-rays or C-ions under 1G, identified DEGs were found to be involved in DNA damage repair, signal transduction by p53 class mediator, cell cycle arrest and apoptosis pathways. The same enriched pathways emerged when cells were irradiated under simulated µG condition. Nevertheless, the combined effect attenuated the transcriptional response to irradiation which may pose a subtle risk in space flights.

Thrombotic triad in microgravity.

Thrombotic disease may be an underdiagnosed condition of prolonged exposure to microgravity and yet the underlying factors remain poorly defined. Recently, an internal jugular vein thrombosis was diagnosed in a low-risk female astronaut after an approximately 7-week space mission. Six of the additional 10 crew members demonstrated jugular venous flow risk factors, such as suspicious stagnation or retroversion. Fortunately, all were asymptomatic. Observations in space as well as clinical and in vitro microgravity studies on Earth, where experiments are designed to recapitulate the conditions of space, suggest effects on blood flow stasis, coagulation, and vascular function. In this article, the related literature on thrombotic disease in space is reviewed, with consideration of these elements of Virchow's triad.

Pathophysiologic Spine Adaptations and Countermeasures for Prolonged Spaceflight.

Low back pain due to spaceflight is a common complaint of returning astronauts. Alterations in musculoskeletal anatomy during spaceflight and the effects of microgravity (µg) have been well-studied; however, the mechanisms behind these changes remain unclear. The National Aeronautics and Space Administration has released the Human Research Roadmap to guide investigators in developing effective countermeasure strategies for the Artemis Program, as well as commercial low-orbit spaceflight. Based on the Human Research Roadmap, the existing literature was examined to determine the current understanding of the effects of microgravity on the musculoskeletal components of the spinal column. In addition, countermeasure strategies will be required to mitigate these effects for long-duration spaceflight. Current pharmacologic and nonpharmacologic countermeasure strategies are suboptimal, as evidenced by continued muscle and bone loss, alterations in muscle phenotype, and bone metabolism. However, studies incorporating the use of ultrasound, beta-blockers, and other pharmacologic agents have shown some promise. Understanding these mechanisms will not only benefit space technology but likely lead to a return on investment for the management of Earth-bound diseases.

Microgravity Effects and Aging Physiology: Similar Changes or Common Mechanisms?

Despite the use of countermeasures (including intense physical activity), cosmonauts and astronauts develop muscle atony and atrophy, cardiovascular system failure, osteopenia, etc. All these changes, reminiscent of age-related physiological changes, occur in a healthy person in microgravity quite quickly - within a few months. Adaptation to the lost of gravity leads to the symptoms of aging, which are compensated after returning to Earth. The prospect of interplanetary flights raises the question of gravity thresholds, below which the main physiological systems will decrease their functional potential, similar to aging, and affect life expectancy. An important role in the aging process belongs to the body's cellular reserve - progenitor cells, which are involved in physiological remodeling and regenerative/reparative processes of all physiological systems. With age, progenitor cell count and their regenerative potential decreases. Moreover, their paracrine profile becomes pro-inflammatory during replicative senescence, disrupting tissue homeostasis. Mesenchymal stem/stromal cells (MSCs) are mechanosensitive, and therefore deprivation of gravitational stimulus causes serious changes in their functional status. The review compares the cellular effects of microgravity and changes developing in senescent cells, including stromal precursors.

Lymphatic circulation in astronauts: basic knowledge, challenges and perspectives.

Space missions expose the astronauts' bodies to various stressors, including microgravity. While numerous studies have investigated the effects of this stressor, research on its impact on the lymphatic system remains confidential. This review highlights the importance of scientific research into the human lymphatic system exposed to long-duration space missions. The safety of astronauts is a major issue. Chronic slowing of lymphatic drainage disrupts the balance of fluid and macromolecule exchange within poorly drained anatomical areas. Their extracellular matrix gradually becomes the site of dispersed deposits of degraded proteins and increased local water content. The interaction between these two phenomena leads to mutual amplification, resulting in a slow, gradual increase in pressure within the impacted tissue, which undergoes an expansion known as edema. The speed at which these pathophysiological processes take hold includes the extent of the lymphatic insufficiency and any compensatory measures that may or may not be put in place. Lymphatics are present everywhere in the body where tissues receive blood. Organs such as the brain, heart, and intestines, among others, as well as local immune function, can be damaged over time when their lymphatic system becomes chronically insufficient. The human clinical experience of lymphatic insufficiency tells us that the onset of edema takes time and is an insidious but inevitable phenomenon if adequate compensation does not occur. The time required for the pathophysiological consequences of lymphatic insufficiency to become established does not coincide with the time allocated to bed rest experiments or current space missions. With the prospect of longer space missions, lymphatic insufficiency linked to microgravity could unexpectedly become a major obstacle to human life in space.

Neurovascular dysfunction associated with erectile dysfunction persists after long-term recovery from simulations of weightlessness and deep space irradiation.

There has been growing interest within the space industry for long-duration manned expeditions to the Moon and Mars. During deep space missions, astronauts are exposed to high levels of galactic cosmic radiation (GCR) and microgravity which are associated with increased risk of oxidative stress and endothelial dysfunction. Oxidative stress and endothelial dysfunction are causative factors in the pathogenesis of erectile dysfunction, although the effects of spaceflight on erectile function have been unexplored. Therefore, the purpose of this study was to investigate the effects of simulated spaceflight and long-term recovery on tissues critical for erectile function, the distal internal pudendal artery (dIPA), and the corpus cavernosum (CC). Eighty-six adult male Fisher-344 rats were randomized into six groups and exposed to 4-weeks of hindlimb unloading (HLU) or weight-bearing control, and sham (0Gy), 0.75 Gy, or 1.5 Gy of simulated GCR at the ground-based GCR simulator at the NASA Space Radiation Laboratory. Following a 12-13-month recovery, ex vivo physiological analysis of the dIPA and CC tissue segments revealed differential impacts of HLU and GCR on endothelium-dependent and -independent relaxation that was tissue type specific. GCR impaired non-adrenergic non-cholinergic (NANC) nerve-mediated relaxation in the dIPA and CC, while follow-up experiments of the CC showed restoration of NANC-mediated relaxation of GCR tissues following acute incubation with the antioxidants mito-TEMPO and TEMPOL, as well as inhibitors of xanthine oxidase and arginase. These findings indicate that simulated spaceflight exerts a long-term impairment of neurovascular erectile function, which exposes a new health risk to consider with deep space exploration.

The Impact of Microgravity on Immunological States.

As we explore other planetary bodies, astronauts will face unique environmental and physiological challenges. The human immune system has evolved under Earth's gravitational force. Consequently, in the microgravity environment of space, immune function is altered. This can pose problematic consequences for astronauts on deep space missions where medical intervention will be limited. Studying the unique environment of microgravity has its challenges, yet current research has uncovered immunological states that are probable during exploration missions. As microgravity-induced immune states are uncovered, novel countermeasure developments and personalized mitigation programs can be designed to improve astronaut health. This can also benefit immune-related monitoring programs for disorders on Earth. This is a comprehensive review, including gaps in knowledge, of simulated and spaceflight microgravity studies in human and rodent models.

Comprehensive assessment of physiological responses in women during the ESA dry immersion VIVALDI microgravity simulation.

Astronauts in microgravity experience multi-system deconditioning, impacting their inflight efficiency and inducing dysfunctions upon return to Earth gravity. To fill the sex gap of knowledge in the health impact of spaceflights, we simulate microgravity with a 5-day dry immersion in 18 healthy women (ClinicalTrials.gov Identifier: NCT05043974). Here we show that dry immersion rapidly induces a sedentarily-like metabolism shift mimicking the beginning of a metabolic syndrome with a drop in glucose tolerance, an increase in the atherogenic index of plasma, and an impaired lipid profile. Bone remodeling markers suggest a decreased bone formation coupled with an increased bone resorption. Fluid shifts and muscular unloading participate to a marked cardiovascular and sensorimotor deconditioning with decreased orthostatic tolerance, aerobic capacity, and postural balance. Collected datasets provide a comprehensive multi-systemic assessment of dry immersion effects in women and pave the way for future sex-based evaluations of countermeasures.

Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.

Frizzled-9 triggers actin polymerization and activates mechano-transducer YAP to rescue simulated microgravity-induced osteoblast dysfunction.

Long-term spaceflight can result in bone loss and osteoblast dysfunction. Frizzled-9 (Fzd9) is a Wnt receptor of the frizzled family that is vital for osteoblast differentiation and bone formation. In the present study, we elucidated whether Fzd9 plays a role in osteoblast dysfunction induced by simulated microgravity (SMG). After 1-7 days of SMG, osteogenic markers such as alkaline phosphatase (ALP), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2) were decreased, accompanied by a decrease in Fzd9 expression. Furthermore, Fzd9 expression decreased in the rat femur after 3 weeks of hindlimb unloading. In contrast, Fzd9 overexpression counteracted the decrease in ALP, OPN, and RUNX2 induced by SMG in osteoblasts. Moreover, SMG regulated phosphorylated glycogen synthase kinase-3ß (pGSK3ß) and ß-catenin expression or sublocalization. However, Fzd9 overexpression did not affect pGSK3ß and ß-catenin expression or sublocalization induced by SMG. In addition, Fzd9 overexpression regulated protein kinase B also known as Akt and extracellular signal-regulated kinase (ERK) phosphorylation and induced F-actin polymerization to form the actin cap, press the nuclei, and increase nuclear pore size, thereby promoting the nuclear translocation of Yes-associated protein (YAP). Our study findings provide mechanistic insights into the role of Fzd9 in triggering actin polymerization and activating YAP to rescue SMG-induced osteoblast dysfunction and suggest that Fzd9 is a potential target to restore osteoblast function in individuals with bone diseases and after spaceflight.

Regulation of Bone by Mechanical Loading, Sex Hormones, and Nerves: Integration of Such Regulatory Complexity and Implications for Bone Loss during Space Flight and Post-Menopausal Osteoporosis.

During evolution, the development of bone was critical for many species to thrive and function in the boundary conditions of Earth. Furthermore, bone also became a storehouse for calcium that could be mobilized for reproductive purposes in mammals and other species. The critical nature of bone for both function and reproductive needs during evolution in the context of the boundary conditions of Earth has led to complex regulatory mechanisms that require integration for optimization of this tissue across the lifespan. Three important regulatory variables include mechanical loading, sex hormones, and innervation/neuroregulation. The importance of mechanical loading has been the target of much research as bone appears to subscribe to the "use it or lose it" paradigm. Furthermore, because of the importance of post-menopausal osteoporosis in the risk for fractures and loss of function, this aspect of bone regulation has also focused research on sex differences in bone regulation. The advent of space flight and exposure to microgravity has also led to renewed interest in this unique environment, which could not have been anticipated by evolution, to expose new insights into bone regulation. Finally, a body of evidence has also emerged indicating that the neuroregulation of bone is also central to maintaining function. However, there is still more that is needed to understand regarding how such variables are integrated across the lifespan to maintain function, particularly in a species that walks upright. This review will attempt to discuss these regulatory elements for bone integrity and propose how further study is needed to delineate the details to better understand how to improve treatments for those at risk for loss of bone integrity, such as in the post-menopausal state or during prolonged space flight.

Pulmonary Function in Human Spaceflight.

Human spaceflight is entering a time of markedly increased activity fueled by collaboration between governmental and private industry entities. This has resulted in successful mission planning for destinations in low Earth orbit, lunar destinations (Artemis program, Gateway station) as well as exploration to Mars. The planned construction of additional commercial space stations will ensure continued low Earth orbit presence and destinations for science but also commercial spaceflight participants. The human in the journey to space is exposed to numerous environmental challenges including increased gravitational forces, microgravity, altered human physiology during adaptation to weightlessness in space, altered ambient pressure, as well as other important stressors contingent on the type of mission and destination. This chapter will cover clinically important aspects relevant to lung function in a normally proceeding mission; emergency scenarios such as decompression, fire, etc., will not be covered as these are beyond the scope of this review. To date, participation in commercial spaceflight by those with pre-existing chronic medical conditions is very limited, and hence, close collaboration between practicing pulmonary specialists and aerospace medicine specialists is of critical importance to guarantee safety, proper clinical management, and hence success in these important endeavors.