Hindlimb-unloaded Mice Research Articles

Mesenchymal stem cell transplant as an intervention to ameliorate disuse-induced muscle atrophy in a mouse model of simulated microgravity

BackgroundThe hindlimb unloaded (HU) mouse model exhibits disuse-induced muscle atrophy. However, effective interventions remain elusive. We investigated the therapeutic potential of mesenchymal stem cells (MSC) transplant on muscle decline in HU mice. MethodsWe divided 4-month-old male c57BL/6j mice into controls and HU mice treated with PBS as placebo (HU-PBS) or MSCs (HU-MSC; one million cells/100 μl PBS into gastrocnemius muscles once a week) for three weeks. We measured muscle mass, grip strength, and an unbiased transcriptome analysis of gastrocnemius muscles. ResultsMSC treatment prevented muscle atrophy and improved grip strength in HU mice. Transcriptome analysis revealed MSC-induced unique (557 genes) and differential (1,214 genes) expressions of several genes compared to the HU-PBS group. GO and KEGG term analysis revealed an HU-induced downregulation of pathways associated with the regulation of contractile apparatus, neuromuscular junction, and satellite cell function, which were partly reversed with MSC treatment. Lastly, MSC treatment also upregulated the pathways controlling muscle differentiation and growth in the HU mice. ConclusionAltogether, we report the therapeutic potential of MSCs in treating disuse-induced muscle atrophy and weakness. Our study may help unravel novel molecular mechanisms involved in MSCs-induced muscle restoration.

Angelicae dahuricae radix alleviates simulated microgravity induced bone loss by promoting osteoblast differentiation

Bone loss caused by long-duration spaceflight seriously affects the skeletal health of astronauts. There are many shortcomings in currently available treatments for weightlessness-induced bone loss. The aim of this study was to evaluate the preventive effect of Angelica dahuricae Radix (AR) on simulated microgravity-induced bone loss. Here, we established a hind limb unloading (HLU) mouse model and treated HLU mice with AR (2 g/kg) for 4 weeks. Results indicated that AR significantly inhibited simulated microgravity-induced bone loss. In addition, the components in AR were analyzed using UPLC-MS/MS; results showed that a total of 224 compounds were detected in AR, which mainly contained 7 classes of components. Moreover, the network pharmacological predictions suggested that active ingredients of AR might act on PTGS2 to prevent bone loss. These results elucidate the efficacy of AR in preventing microgravity-induced bone loss and its potential for use in protecting the bone health of astronauts.

Inhibiting histone deacetylase 6 suppresses the proliferation of microvascular endothelial cells by epigenetically activating miR-375-3p, potentially contributing to bone loss during mechanical unloading

BackgroundMechanical unloading-induced bone loss threatens prolonged spaceflight and human health. Recent studies have confirmed that osteoporosis is associated with a significant reduction in bone microvessels, but the relationship between them and the underlying mechanism under mechanical unloading are still unclear.MethodsWe established a 2D clinostat and hindlimb-unloaded (HLU) mouse model to simulate unloading in vitro and in vivo. Micro-CT scanning was performed to assess changes in the bone microstructure and mass of the tibia. The levels of CD31, Endomucin (EMCN) and histone deacetylase 6 (HDAC6) in tibial microvessels were detected by immunofluorescence (IF) staining. In addition, we established a coculture system of microvascular endothelial cells (MVECs) and osteoblasts, and qRT‒PCR or western blotting was used to detect RNA and protein expression; cell proliferation was detected by CCK‒8 and EdU assays. ChIP was used to detect whether HDAC6 binds to the miRNA promoter region.ResultsBone mass and bone microvessels were simultaneously significantly reduced in HLU mice. Furthermore, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. Mechanistically, we found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p by reducing histone acetylation in the miR-375 promoter region in MVECs. miR-375-3p was upregulated under unloading and it could inhibit MVEC proliferation by directly targeting low-density lipoprotein-related receptor 5 (LRP5) expression. In addition, silencing HDAC6 promoted the miR-375-3p/LRP5 pathway to suppress MVEC proliferation under mechanical unloading, and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions.ConclusionOur study revealed that disuse-induced bone loss may be closely related to a reduction in the number of bone microvessels and that the modulation of MVEC function could improve bone loss induced by unloading. Mechanistically, the HDAC6/miR-375-3p/LRP5 pathway in MVECs might be a promising strategy for the clinical treatment of unloading-induced bone loss.

Effects of Ginsenoside Rb1 on the Crosstalk between Intestinal Stem Cells and Microbiota in a Simulated Weightlessness Mouse Model.

Exposure to the space microenvironment has been found to disrupt the homeostasis of intestinal epithelial cells and alter the composition of the microbiota. To investigate this in more detail and to examine the impact of ginsenoside Rb1, we utilized a mouse model of hindlimb unloading (HU) for four weeks to simulate the effects of microgravity. Our findings revealed that HU mice had ileum epithelial injury with a decrease in the number of intestinal stem cells (ISCs) and the level of cell proliferation. The niche functions for ISCs were also impaired in HU mice, including a reduction in Paneth cells and Wnt signaling, along with an increase in oxidative stress. The administration of Rb1 during the entire duration of HU alleviated the observed intestinal defects, suggesting its beneficial influence on epithelial cell homeostasis. Hindlimb unloading also resulted in gut dysbiosis. The supplementation of Rb1 in the HU mice or the addition of Rb1 derivative compound K in bacterial culture in vitro promoted the growth of beneficial probiotic species such as Akkermansia. The co-housing experiment further showed that Rb1 treatment in ground control mice alone could alleviate the defects in HU mice that were co-housed with Rb1-treated ground mice. Together, these results underscore a close relationship between dysbiosis and impaired ISC functions in the HU mouse model. It also highlights the beneficial effects of Rb1 in mitigating HU-induced epithelial injury by promoting the expansion of intestinal probiotics. These animal-based insights provide valuable knowledge for the development of improved approaches to maintaining ISC homeostasis in astronauts.

Effects of N-Acetyl-Carnosine Treatment on Aging and Carbonyl Stress

By 2050 the number of individuals over the age of 80y will triple to reach 426 million and the rate of age-related comorbidities has also increased precipitously as our global population has continued to age. Currently, there is no clinically approved treatment for age-related functional decline creating demand for the development of such treatments. Our lab previously demonstrated that carbonyl stress is exacerbated in muscles during a hindlimb unloading (HU) model of inactivity. Importantly, the accumulation of proteins modified by the reactive lipid carbonyl 4HNE, as well as muscle dysfunction and atrophy, were attenuated in HU mice treated with the carbonyl-scavenging dipeptide N-acetyl-carnosine (N-Ac-Carn). Therefore, we hypothesized that N-Ac-Carn could prevent carbonyl stress in aging muscles and other tissues. 17-month-old male and female C57Bl6J mice from the NIA colony were treated for 6-months with 80 mM N-Ac-Carn or normal (CON) water. Before and after the 6-month intervention we assessed several markers of health including glucose tolerance, kidney, muscle, bone, and cognitive function. N-Ac-Carn preserved ex vivo muscle force production in soleus ( p=0.0015) and EDL ( p=0.0008) muscles from female but not male mice. N-Ac-Carn treated mice also possessed superior bone volume compared to CON ( p=0.0046). Following terminal tissue collection, we also assessed muscles for carbonyl stress by probing for proteins modified by total carbonyls, 4HNE, MDA, and methylglyoxal. N-Ac-Carn did not attenuate 4HNE, MDA, or total carbonyls in gastrocnemius. However, N-Ac-Carn showed strong trends for attenuating 4HNE burden in both soleus and EDL muscles. N-Ac-Carn is an endogenously produced dipeptide that is safe for human consumption and affordable making it a promising therapeutic strategy for the mitigation of age-related functional decline. AG074535-02S2. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Carbon monoxide-loaded cell therapy as an exercise mimetic for sarcopenia treatment

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia. Treatment of C2C12 myoblasts with the CO-donor increased the protein levels of PGC-1α which enhanced mitochondrial biogenesis and energy production. The CO-donor treatment also activated Akt, indicating that CO promotes muscle synthesis. CO levels were significantly elevated in the skeletal muscle of normal mice after intravenous administration of CO-RBCs. Furthermore, CO-RBCs restored the mRNA expression levels of PGC-1α in the skeletal muscle of two experimental sarcopenia mouse models, denervated (Den) and hindlimb unloading (HU) models. CO-RBCs also restored muscle mass in Den mice by activating Akt signaling and suppressing the muscle atrophy factors myostatin and atrogin-1, and oxidative stress. Treadmill tests further showed that the reduced running distance in HU mice was significantly restored by CO-RBC administration. These findings suggest that CO-RBCs have potential as an exercise mimetic for sarcopenia treatment.

Different impact of short-term and long-term hindlimb disuse on bone homeostasis

Disuse osteoporosis is one of the major problems of bone health which commonly occurs in astronauts during long-term spaceflight and bedridden patients. However, the mechanisms underlying such mechanical unloading induced bone loss have not been fully understood. In this study, we employed hindlimb-unloading mice models with different length of tail suspension to investigate if the bone loss was regulated by distinct factors under different duration of disuse. Our micro-CT results showed more significant decrease of bone mass in 6W (6-week) tail-suspension mice compared to the 1W (1-week) tail-suspension ones, as indicated by greater reduction of BV/TV, Tb.N, B.Ar/T.Ar and Ct.Th. RNA-sequencing results showed significant effects of hindlimb disuse on cell locomotion and immune system process which could cause bone loss.Real-time quantitative PCR results indicated a greater number of bone formation related genes that were downregulated in short-term tail-suspension mice compared to the long-term ones. It is, thus, suggested while sustained hindlimb unloading continuously contributes to bone loss, molecular regulation of bone homeostasis tends to reach a balance during this process.

Long non-coding RNA-NONMMMUT004552.2 regulates the unloading-induced bone loss through the miRNA-15b-5p/Syne1 in mice

Exercise-induced mechanical loading can increase bone strength whilst mechanical unloading enhances bone-loss. Here, we investigated the role of lncRNA NONMMUT004552.2 in unloading-induced bone-loss. Knockout of lncRNA NONMMUT004552.2 in hindlimb-unloaded mice caused an increase in the bone formation and osteoblast activity. The silencing of lncRNA NONMMUT004552.2 also decreased the osteoblast apoptosis and expression of Bax and cleaved caspase-3, increased Bcl-2 protein expression in MC3T3-E1 cells. Mechanistic investigations demonstrated that NONMMUT004552.2 functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of spectrin repeat containing, nuclear envelope 1 (Syne1) by competitively binding miR-15b-5p and subsequently inhibits the osteoblast differentiation and bone formation in the microgravity unloading environment. These data highlight the importance of the lncRNA NONMMUT004552.2/miR-15b-5p/Syne1 axis for the treatment of osteoporosis.

Identification of gravity-responsive serum proteins in spaceflight mice using a quantitative proteomic approach with data-independent acquisition mass spectrometry.

Physical inactivity associated with gravity unloading, such as microgravity during spaceflight and hindlimb unloading (HU), can cause various physiological changes. In this study, we attempted to identify serum proteins whose levels fluctuated in response to gravity unloading. First, we quantitatively assessed changes in the serum proteome profiles of spaceflight mice using mass spectrometry with data-independent acquisition. The serum levels of several proteins involved in the responses to estrogen and glucocorticoid, blood vessel maturation, osteoblast differentiation, and ossification were changed by microgravity exposure. Furthermore, a collective evaluation of serum proteomic data from spaceflight and HU mice identified 30 serum proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, whose levels varied to a similar extent in both gravity unloading models. These changes in serum levels could be involved in the physiological changes induced by gravity unloading. A collective evaluation of serum, femur, and soleus muscle proteome data of spaceflight mice also showed 24 serum proteins, including Igfbp5, Igfbp3, and Postn, whose levels could be associated with biological changes induced by microgravity. This study examined serum proteome profiles in response to gravity unloading, and may help deepen our understanding of microgravity adaptation mechanisms during prolonged spaceflight missions.

Plasminogen deficiency exacerbates skeletal muscle loss during mechanical unloading in developing mice.

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. In addition, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.NEW & NOTEWORTHY The expression levels of fibrinolysis-related proteins, including plasminogen, were significantly elevated in the gastrocnemius (Gast) and soleus muscles of mice following 30-day microgravity exposure. Plasminogen deficiency exacerbated atrophy of the Gast, but not the soleus, muscles in mice following 21-day hindlimb suspension. It was also suggested that protein breakdown via the autophagy-lysosome system was accelerated in the Gast muscles. Plasminogen might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal–regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

LLLT accelerates experimental wound healing under microgravity conditions via PI3K/AKT-CCR2 signal axis.

The risk of skin injuries in space is increasing with longer space missions and a growing astronaut population. This highlights the importance of understanding the adverse effects of weightlessness on wound healing. The objective of this research was to examine the therapeutic potential of Low-Level Light Therapy (LLLT) on skin healing processes under simulated microgravity (SMG) conditions and uncover the underlying molecular mechanisms, thus providing innovative solutions and a sound theoretical basis for space skin injuries. Hindlimb unloading (HU) mice models were used to simulate weightlessness conditions, with or without a complete management of LLLT for 14 days. A systematic testing consisting of HE, Masson and immunohistochemical staining was performed against the standardized mouse tissue specimens. In vitro assessment of cellular biological functions under SMG conditions was carried out in the rotation system of culture (RSOC) using HaCaT and NIH3T3 cell-lines. Under SMG conditions, LLLT significantly reduced skin wound area in HU mice, especially on Days 10 (p < 0.001), accompanied by increased collagen deposition and elevated levels of Ki67 and CD31. Moreover, LLLT showed impressive anti-inflammatory effects represented by the reduced in pro-inflammatory markers including LY6G, F4/80 and CD86, as well as the decreased levels of IL-1β, IL-6 and TNF-α. Conversely, an elevation in the anti-inflammatory marker CD206 was observed. By employing bioinformatics technology, we further found the PI3K/AKT signaling was prominent in the KEGG pathway analysis and CCR2 acted as a hub gene in the interaction network. Therefore, we demonstrated that LLLT could enhance the phosphorylation of PI3K/AKT and reduce CCR2 expression under SMG conditions, while CCR2 knockdown promoted the phosphorylation of PI3K/AKT, suggesting an important role of CCR2/PI3K/AKT signal axis in LLLT-accelerated wound healing under SMG conditions. LLLT induced activation of the PI3K/AKT signaling pathway through suppression of CCR2 expression, which significantly enhanced skin wound healing under SMG conditions.s.

Serum multi-omics analysis in hindlimb unloading mice model: Insights into systemic molecular changes and potential diagnostic and therapeutic biomarkers

Microgravity, in space travel and prolonged bed rest conditions, induces cardiovascular deconditioning along with skeletal muscle mass loss and weakness. The findings of microgravity research may also aid in the understanding and treatment of human health conditions on Earth such as muscle atrophy, and cardiovascular diseases. Due to the paucity of biomarkers and the unknown underlying mechanisms of cardiovascular and skeletal muscle deconditioning in these environments, there are insufficient diagnostic and preventative measures. In this study, we employed hindlimb unloading (HU) mouse model, which mimics astronauts in space and bedridden patients, to first evaluate cardiovascular and skeletal muscle function, followed by proteomics and metabolomics LC-MS/MS-based analysis using serum samples. Three weeks of unloading caused changes in the function of the cardiovascular system in c57/Bl6 mice, as seen by a decrease in mean arterial pressure and heart weight. Unloading for three weeks also changed skeletal muscle function, causing a loss in grip strength in HU mice and atrophy of skeletal muscle indicated by a reduction in muscle mass. These modifications were partially reversed by a two-week recovery period of reloading condition, emphasizing the significance of the recovery process. Proteomics analysis revealed 12 dysregulated proteins among the groups, such as phospholipid transfer protein, Carbonic anhydrase 3, Parvalbumin alpha, Major urinary protein 20 (Mup20), Thrombospondin-1, and Apolipoprotein C-IV. On the other hand, metabolomics analysis showed altered metabolites among the groups such as inosine, hypoxanthine, xanthosine, sphinganine, l-valine, 3,4-Dihydroxyphenylglycol, and l-Glutamic acid. The joint data analysis revealed that HU conditions mainly impacted pathways such as ABC transporters, complement and coagulation cascades, nitrogen metabolism, and purine metabolism. Overall, our results indicate that microgravity environment induces significant alterations in the function, proteins, and metabolites of these mice. These observations suggest the potential utilization of these proteins and metabolites as novel biomarkers for assessing and mitigating cardiovascular and skeletal muscle deconditioning associated with such conditions.

Simulated spaceflight-induced cardiac remodeling is modulated by gut microbial-derived trimethylamine N-oxide

Spaceflight is physically demanding and can negatively affect astronauts' health. It has been shown that the human gut microbiota and cardiac function are affected by spaceflight and simulated spaceflight. This study investigated the effects of the gut microbiota on simulated spaceflight-induced cardiac remodeling using 10° of head-down bed rest (HDBR) in rhesus macaques and 30° of hindlimb unloading (HU) in mice. The gut microbiota, fecal metabolites, and cardiac remodeling were markedly affected by HDBR in macaques and HU in mice, cardiac remodeling in control mice was affected by the gut microbiota of HU mice and that of HU mice was protected by the gut microbiota of control mice, and there was a correlation between cardiac remodeling and the gut microbial-derived metabolite trimethylamine N-oxide. These findings suggest that spaceflight can affect cardiac remodeling by modulating the gut microbiota and fecal metabolites.

Identification of mouse soleus muscle proteins altered in response to changes in gravity loading

Gravity-dependent physical processes strongly affect the ability of elderly people to maintain musculoskeletal health by reducing muscle atrophy and increasing bone mineral density, thereby increasing quality of life. A need therefore exists to identify molecules in the musculoskeletal system that are responsive to gravitational loading and to establish an objective indicator for the maintenance of healthy musculoskeletal systems. Here, we performed an integrated assessment of the results of soleus muscle proteomic analyses in three model mouse experiments under different gravity environments (hypergravity, hindlimb unloading, and spaceflight). Myl6b, Gpd1, Fbp2, Pvalb, and Actn3 were shown to be gravity-responsive muscle proteins, and alterations in the levels of these proteins indicated changes in muscle fiber type to slow-twitch type due to gravity loading. In addition, immunoblotting and enzyme-linked immunosorbent assays revealed that Pvalb levels in the sera of hindlimb-unloaded mice and osteoporosis patients were higher than in control subjects, suggesting that Pvalb levels might be useful to objectively evaluate soleus muscle atrophy and bone loss.

Transcranial photobiomodulation mitigates learning and memory impairments induced by hindlimb unloading in a mouse model of microgravity exposure by suppression of oxidative stress and neuroinflammation signaling pathways

Prolonged microgravity exposure causes cognitive impairment. Evidence shows that oxidative stress and neuroinflammation are involved in the causation. Here, we explore the effectiveness of transcranial near-infrared photobiomodulation (PBM) on cognitive deficits in a mouse model of simulated microgravity.24 adult male C57BL/6 mice were assigned into three groups (8 in each); control, hindlimb unloading (HU), and HU + PBM groups. After surgery to fit the suspension fixing, the animals were housed either in HU cages or in their normal cage for 14 days. The mice in the HU + PBM group received PBM (810 nm laser, 10 Hz, 8 J/cm2) once per day for 14 days. Spatial learning and memory were assessed in the Lashley III maze and hippocampus tissue samples were collected to assess oxidative stress markers and protein expression of brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2), Sirtuin 1 (Sirt1), and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).Behavioral testing showed that the PBM-treated animals had a shorter latency time to find the target and fewer errors than the HU group. PBM decreased hippocampal lipid peroxidation while increasing antioxidant defense systems (glutathione peroxidase, superoxide dismutase, and total antioxidant capacity) compared to HU mice. PBM increased protein expression of Sirt1, Nrf2, and BDNF while decreasing NF-κB compared to HU mice.Our findings suggested that the protective effect of PBM against HU-induced cognitive impairment involved the activation of the Sirt1/Nrf2 signaling pathway, up-regulation of BDNF, and reduction of neuroinflammation and oxidative stress in the hippocampus.

Enhancing microbial diversity as well as multi-organ health in hind-limb unloaded mice

During space travel, the gut microbiota is changed which can lead to health-related issues. Previously, we utilized the hind-limb unloaded (HU) mouse, which is an established ground-based in-vivo model of microgravity and observed altered gut microbiota. In this study, we evaluated the beneficial effects of novel bacterial conditioned media in HU mice to understand if they can offset the effects of unloading in the HU mouse model. We aimed to explore the influence of bacterial conditioned media on diversity and quantity of intestinal microbes in HU mice, and investigated the microarchitecture of mice retinas and kidneys to evaluate the potential systemic effects of bacterial conditioned media in HU mice. Four-month-old, male C57/Bl6 mice were separated into groups: including the ground-based control group, the HU group mice fed with vehicle as placebo (HU-placebo mice), and the HU group fed with bacterial conditioned media (HU-CP mice) and kept under controlled environmental conditions for three weeks. Next, mice were sacrificed; gut dissections were conducted, and metagenomic analysis of bacterial species was performed via DNA extraction and 16S rRNA analysis. The results revealed an HU-induced reduction in intestinal microbial diversity, and an increase in pathogenic bacteria dominated by Firmicutes (45%). In contrast, supplementation with bacterial conditioned media for three weeks led to a significant increase in gut microbial diversity with noticeable changes in the OTUs abundance in the HU mice. Additionally, HU-induced muscle weakness and structural abnormalities in the retina and kidney were partially prevented with bacterial conditioned media. Moreover, a greater diversity of several bacteria in the HU-CP was observed including, Bacteriodota, Firmicutes, Proteobacteria, Actionobacteriota, Verrucomicorbiota, Cyanobacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Myxococcota, and others. Prospective research involving molecular mechanistic studies are needed to comprehend the systemic effects of bacterial metabolites conditioned media on experimental animal models under chronic stress.

Low-intensity pulsed ultrasound mitigates cognitive impairment by inhibiting muscle atrophy in hindlimb unloaded mice.

Microgravity leads to muscle loss, usually accompanied by cognitive impairment. Muscle reduction was associated with the decline of cognitive ability. Our previous studies showed that low-intensity pulsed ultrasound (LIPUS) promoted muscle hypertrophy and prevented muscle atrophy. This study aims to verify whether LIPUS can improve cognitive impairment by preventing muscle atrophy in hindlimb unloaded mice. In this study, mice were randomly divided into normal control (NC), hindlimb unloading (HU), hindlimb unloading + LIPUS (HU+LIPUS) groups. The mice in the HU+LIPUS group received a 30 mW/cm2 LIPUS irradiation on gastrocnemius for 20 min/d. After 21 days, LIPUS significantly prevented the decrease in muscle mass and strength caused by tail suspension. The HU+LIPUS mice showed an enhanced desire to explore unfamiliar environments and their spatial learning and memory abilities, enabling them to quickly identify differences between different objects, as well as their social discrimination abilities. MSTN is a negative regulator of muscle growth and also plays a role in regulating cognition. LIPUS significantly inhibited MSTN expression in skeletal muscle and serum and its receptor ActRIIB expression in brain, upregulated AKT and BDNF expression in brain. Taken together, LIPUS may improve the cognitive dysfunction in hindlimb unloaded rats by inhibiting muscle atrophy through MSTN/AKT/BDNF pathway.

In search of stress: analysis of stress-related markers in mice after hindlimb unloading and social isolation

ABSTRACT Objectives Hindlimb unloading (HU), widely used to simulate microgravity effects, is known to induce a stress response. However, as single-housed animals are usually used in such experiments, social isolation (SI) stress can affect experimental results. In the present study, we aimed to delineate stressful effects of 3-day HU and SI in mice. Methods Three animal groups, HU, SI, and group-housed (GH) control mice, were recruited. A comprehensive analysis of stress-related markers was performed using ELISA, western blotting, and immunohistochemistry. Results Our results showed that blood corticosterone and activity of glucocorticoid receptors and cAMP response element-binding protein (CREB) in the hippocampus of SI and HU animals did not differ from GH control. However, SI mice demonstrated upregulation of the hippocampal corticotropin-releasing hormone (CRH), inducible NO synthase (iNOS), vesicular glutamate transporter 1 (VGLUT1), and glutamate decarboxylases 65/67 (GAD65/67) along with activation of Fos-related antigen 1 (Fra-1) in the amygdala confirming the expression of stress. In HU mice, the same increase in GAD65/67 and Fra-1 indicated the contribution of SI. The special HU effect was expressed only in neurogenesis attenuation. Discussion Thus, our data indicated that 3-day HU could not be characterized as physiological stress, but SI stress contributed to the negative effects of HU.

Suppression of endoplasmic reticulum stress reverses hindlimb unloading-induced hepatic cellular processes in mice

BackgroundThe Hindlimb unloaded mouse, an animal model of simulated microgravity demonstrates significant metabolic and hepatic derangements. However, cellular and molecular mechanisms driving liver dysfunction in Hindlimb unloaded mice are poorly characterized. MethodsWe investigated the possible contribution of dysregulated protein homeostasis by endoplasmic reticulum, endoplasmic reticulum stress, to liver dysfunction during HU. C57BL/6j male mice were grouped into ground-based controls or Hindlimb unloaded groups treated daily with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of endoplasmic reticulum stress. Following three weeks of HU, mice were sacrificed, and liver tissues were dissected for further analysis. ResultsHindlimb unloaded was associated with hepatic atrophy and elevated endoplasmic reticulum stress, which was restored by 4-PBA treatment. The Gene Ontology analysis revealed the downregulation of genes primarily involved in liver metabolic and Wingless-related integration site (WNT) signaling pathways, while those related to cytochrome P450, and liver fibrosis were upregulated. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of several genes involved in metabolic pathways following treatment with 4-PBA, induced by HU. ConclusionsWe report several differential and uniquely expressed genes associated with microgravity-induced elevated ER stress and liver injury. Our data has translational potential in unraveling novel molecular targets for pharmaceutical therapies of liver diseases. General significanceOur novel findings show a pathogenic role for elevated ER stress in liver injury in microgravity conditions.