Hindlimb Unloading Rats Research Articles

Serum Metabolomics Associating With Circulating MicroRNA Profiles Reveal the Role of miR-383-5p in Rat Hippocampus Under Simulated Microgravity.

Microgravity impacts various aspects of human health. Yet the mechanisms of spaceflight-induced health problems are not elucidated. Here, we mapped the fusion systemic analysis of the serum metabolome and the circulating microRNAome in a hindlimb unloading rat model to simulate microgravity. The response of serum metabolites and microRNAs to simulated microgravity was striking. Integrated pathway analysis of altered serum metabolites and target genes of the significantly altered circulating miRNAs with Integrated Molecular Pathway-Level Analysis (IMPaLA) software was mainly suggestive of modulation of neurofunctional signaling pathways. Particularly, we revealed significantly increased miR-383-5p and decreased aquaporin 4 (AQP4) in the hippocampus. Using rabies virus glycoprotein–modified exosomes, delivery of miR-383-5p inhibited the expression of AQP4 not only in rat C6 glioma cells in vitro but also in the hippocampus in vivo. Using bioinformatics to map the crosstalk between the circulating metabolome and miRNAome could offer opportunities to understand complex biological systems under microgravity. Our present results suggested that the change of miR-383-5p level and its regulation of target gene AQP4 was one of the potential molecular mechanisms of microgravity-induced cognitive impairment in the hippocampus.

NO-Dependent Mechanisms of Myosin Heavy Chain Transcription Regulation in Rat Soleus Muscle After 7-Days Hindlimb Unloading.

It is known that nitric oxide (NO) may affect myosin heavy chain (MyHC) isoform mRNA transcription in skeletal muscles. The content of NO in soleus muscles decreases during rat hindlimb unloading as well as slow MyHC mRNA transcription. We aimed to detect which signaling pathways are involved in NO-dependent prevention of hindlimb-suspension (HS)-induced changes in MyHCs’ expression pattern. Male Wistar rats were divided into four groups: cage control group (C), hindlimb suspended for 7 days (7HS), hindlimb suspended for 7 days with L-arginine administration (7HS+A) (500 mg/kg body mass), and hindlimb suspended for 7 days with both L-arginine (500 mg/kg) and NO-synthase inhibitor L-NAME administration (50 mg/kg) (7HS+A+N). L-arginine treatment during 7 days of rat HS prevented HS-induced NO content decrease and slow MyHC mRNA transcription decrease and attenuated fast MyHC IIb mRNA transcription increase; it also prevented NFATc1 nuclear content decrease, calsarcin-2 expression increase, and GSK-3β Ser 9 phosphorylation decrease. Moreover, L-arginine administration prevented the HS-induced myh7b and PGC1α mRNAs content decreases and slow-type genes repressor SOX6 mRNA transcription increase. All these slow fiber-type protective effects of L-arginine were blocked in HS+A+N group, indicating that these effects were NO-dependent. Thus, NO decrease prevention during HS restores calcineurin/NFATc1 and myh7b/SOX6 signaling.

Simulated Microgravity Induces Regionally Distinct Neurovascular and Structural Remodeling of Skeletal Muscle and Cutaneous Arteries in the Rat.

Introduction: Mechanical forces and sympathetic influences are key determinants of vascular structure and function. This study tested the hypothesis that hindlimb unloading (HU) exerts diverse effects on forelimb and hindlimb small arteries of rats in functionally different regions of the skeletal muscle and skin. Methods: Male Wistar rats were subjected to HU for 2 weeks, then skeletal muscle arteries (deep brachial and sural) and skin arteries (median and saphenous) were examined in vitro using wire myography or isobaric perfusion and glyoxylic acid staining. Results: HU increased lumen diameter of both forelimb arteries but decreased diameter of the sural artery; the saphenous artery diameter was not affected. Following HU, maximal contractile responses to noradrenaline and serotonin increased in the forelimb but decreased in the hindlimb skeletal muscle feed arteries with no change in skin arteries; all region-specific alterations persisted after endothelium removal. HU increased the sensitivity to vasoconstrictors in the saphenous artery but not in the sural artery. In the saphenous artery, initially high sympathetic innervation density was reduced by HU, sparse innervation in the sural artery was not affected. Electrical stimulation of periarterial sympathetic nerves in isobarically perfused segments of the saphenous artery demonstrated a two-fold decrease of the contractile responses in HU rats compared to that of controls. Conclusion: HU induces contrasting structural and functional adaptations in forelimb and hindlimb skeletal muscle arteries. Additionally, HU had diverse effects in two hindlimb vascular regions. Hyper-sensitivity of the saphenous artery to vasoconstrictors appears to result from the shortage of trophic sympathetic influence. Importantly, HU impaired sympathetically induced arterial vasoconstriction, consistent with the decreased sympathetic constrictor response in humans following space flight.

SUN-335 Abaloparatide Prevents Unloading-Induced Bone Loss in Adult Rats

Disuse osteoporosis (bone loss resulted from a reduction in mechanical loading) occurs in patients due to prolonged bed rest, paralysis and application of braces. Abaloparatide (ABL) is a synthetic peptide analog of PTHrP that has been shown to promote bone formation with limited bone resorption. ABL was approved by the FDA in 2017 to treat osteoporosis in postmenopausal women at high fracture risk. Yet, the ability of ABL to prevent bone loss in disuse is unknown. We hypothesized that ABL would prevent bone loss in the hindlimb unloading (HLU) rat model of disuse osteoporosis.Adult male Wistar rats, 13–14 weeks of age, were assigned to 1 of 4 groups (10 rats/group): ambulatory + vehicle (CON-VEH), ambulatory + ABL (CON-ABL), HLU + vehicle (HLU-VEH) or HLU + ABL (HLU-ABL). The rats received a daily subcutaneous injection of ABL (25µg/kg/day) or vehicle for 28 days. Blood serum was collected on day 0, 7, 14 and 28 to examine the expression of bone markers such as osteocalcin (OCN) and TRAcP5b. pQCT scans were acquired at the proximal tibia at day 0 and 28 to measure changes in the total and trabecular vBMD. Following euthanasia, trabecular (Tb) and cortical (Ct) bone microarchitecture from femurs, tibias and L4 vertebrae were assessed using µCT. Femurs were mechanically tested to failure in 3-point bending to determine ultimate load (N) and stiffness (N/mm). Treatment effects were evaluated using 2-way ANOVA. Effects were considered significant at p < 0.05. Data reported as mean±SD.HLU led to loss of bone density and structure that were prevented by ABL. Longitudinal pQCT revealed significant increases in total vBMD in ABL-CON (52±17%) vs. VEH-CON (20±5%); and in HLU-ABL (24±6%) vs. HLU-VEH (-2±3%) (p<0.001 for both). Significant differences were observed in the µCT analysis of the distal femur: Tb.BV/TV, thickness and BMD were 43.7%, 12.9% and 27.4% lower, respectively, in HLU-VEH compared to CON-VEH (p<0.05 for all). ABL prevented these negative effects, such that Tb.BV/TV, thickness and BMD were 66.5%, 39% and 50.3% higher in HLU-ABL compared to HLU-VEH (p<0.01 for all). A positive impact of ABL on bone morphology was also seen in the CON-VEH rats. CON-ABL had greater femoral stiffness (+22.9%, p=0.03) and ultimate load (+20.5%, p=0.01) than CON-VEH. Vertebral and tibial trabecular parameters mimicked the distal femur parameters. Serum TRAcP5b did not differ among groups, yet both ABL groups had higher OCN levels than the VEH-treated control groups (+63%, p<0.05).We demonstrated positive effects of ABL on BMD, trabecular bone mass and structure in both ambulating and unloaded rats. These results are consistent with prior studies showing positive effects of ABL on bone mass, structure and strength in OVX and ORX rats. Limits include only male rats and 1 dose of ABL. However, the results observed in this study provide a strong rationale for investigating the ability of ABL to prevent disuse bone loss in humans.

Effects of mild hyperbaric oxygen on osteoporosis induced by hindlimb unloading in rats.

Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317hPa with 40% oxygen on unloading-induced osteoporosis. Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21days and recovery for 10days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21days and recovery for 10days with mild hyperbaric oxygen (HU + MHO, n = 6). The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.

Cardiovascular Deconditioning Enhances Cardiovascular and Autonomic Response to Arterial Chemoreflex Stimulation

Cardiovascular deconditioning (CVD) due to bedrest, inactivity or exposure to microgravity impairs cardiovascular regulation and circulatory homeostasis. Cardiovascular changes associated with CVD, including resting tachycardia, decreased exercise capacity and autonomic impairments are reproduced in an animal model of CVD, hindlimb unloaded (HU) rats. Autonomic dysfunction, including blunted baroreflex function, in CVD is associated with mechanisms involving the central nervous system. We hypothesized that CVD augments the arterial chemoreflex. Male Sprague‐Dawley rats were subjected to 2 weeks hindlimb unloading (HU) to simulate CVD, or maintained at normal postural position (cage control, CC). Under inactin anesthesia, baseline heart rate (HR) was elevated in HU vs CC (381±8 bpm vs 334±6 bpm). To examine the chemoreflex, HU and CC rats (n=5–6 each) were given bolus NaCN (25, 50, 75ug/kg, iv) or acute hypoxia (12% O2, 45 seconds). NaCN dose dependently increased HR and mean arterial pressure (MAP) in HU but not CC. At the highest NaCN concentration, HU increased MAP (22±4 mmHg) and HR (16±3 bpm) more than CC MAP (8±1 mmHg) and HR (6±2 bpm). NaCN also increased splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA) in both groups, and responses tended to be greater in HU rats. Likewise, the peak hypoxic response of SSNA and PhrNA were greater in HU than CC rats (SSNA, HU 143 ± 26% vs CC 111 ± 9%; PhrAmp%; HU 382 ± 208 vs CC 300 ± 103). Exposure to NaCN or hypoxia did not change PhrFreq in either group. These data suggest that HU enhances cardiovascular responses to chemoreflex activation.Support or Funding InformationFunding: HL132836

Mechanisms underlining excitatory synaptic plasticity in the Nucleus Tractus Solitarii following Cardiovascular Deconditioning

The nucleus tractus solitarii (nTS) is a critical central region for autonomic control and integration of cardiovascular reflexes. Neuroplasticity in nTS contributes to several cardiovascular dysfunctions. Our previous data showed that Hindlimb‐unloading (HU), a well‐established model of cardiovascular deconditioning in rats, increases glutamatergic neurotransmission yet reduces the excitability of nTS neurons. In this study we aimed to investigate the mechanisms underlining these alterations. For this purpose, stainless wire rings were placed in the tail of male Sprague‐Dawley rats (3 weeks). After one week of recovery the rings was connected to a suspension apparatus and rats were suspended at an angle of 30–35° for two weeks with free access to food and water. Control animals were maintained in normal postural conditions. After the HU protocol, coronal brainstem slices were generated and the whole‐cell patch clamp technique was performed to evaluate the synaptic neurotransmission and electrophysiological properties of nTS neurons. Compared to control rats, HU increased the amplitude of excitatory postsynaptic currents evoked by afferent tractus solitarii stimulus (TS‐EPSC). The addition of a NMDA receptor antagonist (AP5, 10 μM) significantly reduced TS‐EPSC amplitude in HU but not controls. Despite the increase in glutamatergic inputs, the neurons of HU vs control rats are hyperpolarized and have decreased excitability. The block of both NMDA and non‐NMDA (NBQX, 10 μM) glutamatergic receptors did not affect these parameters. However, in the presence of a GABAA receptor antagonist (Gabazine, 25 μM) the resting membrane potential depolarized and excitability of HU neurons were comparable to neurons from control rats. The results indicate that HU increases TS‐EPSC amplitude via a NMDA receptor component. Furthermore, the decreased excitability and hyperpolarized membrane is associated with enhanced GABAergic modulation to these neurons. This functional neuroplasticity in the nTS may contribute to the cardiovascular deconditioning induced by HU.Support or Funding InformationHL132836 AAS lundbeck fellowship

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.

Effects of astaxanthin supplementation and electrical stimulation on muscle atrophy and decreased oxidative capacity in soleus muscle during hindlimb unloading in rats.

The effects of a combination of the antioxidant astaxanthin (AX) and electrical stimulation (ES) on muscle mass and mitochondrial oxidative capacity were investigated in the soleus muscle of hindlimb unloaded rats. Five groups of male Sprague-Dawley rats were used; control, 1-week hindlimb unloading (HU), HU + AX, HU + ES, and HU + AX + ES. Respective rats in the AX groupsreceived 50-mg/kg AX twice daily during HU. Calf muscles of rats in the ES groups were electrically stimulated for 240s/day during HU. One-week HU decreased muscle mass along with decreased FoxO3a phosphorylation and increased ubiquitinated proteins expressions, decreased oxidative enzymatic activity accompanied with decline in PGC-1α protein expression, and increased reactive oxygen species production. However, the combination treatment could synergistically attenuate/suppress all HU-related changes, suggesting protective effects on muscle atrophy and decreased muscle oxidative capacity due to chronic neuromuscular inactivity.

UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy.

We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining. In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy. We demonstrated that UBR5 was epigenetically altered via DNA methylation during recovery from atrophy. We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy. UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation. Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes. We aimed to investigate a novel and uncharacterized E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 vs. well characterized E3-ligases, MuRF1/MAFbx, where, after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated, yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post TTX-induced atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, whereas a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, as well as after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in vitro, and after chronic electrical stimulation-induced hypertrophy in rats in vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 single nucleotide polymorphisms) demonstrated that the A alleles of rs10505025 and rs4734621 single nucleotide polymorphisms in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power vs. endurance/untrained phenotypes. Overall, we suggest that: (i) UBR5 comprises a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx; (ii) UBR5 is epigenetically regulated; and (iii) UBR5 is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.

Plantar mechanical stimulation prevents calcineurin-NFATc1 inactivation and slow-to-fast fiber type shift in rat soleus muscle under hindlimb unloading.

The prevailing myosin isoform [myosin heavy chain (MyHC)] in a skeletal muscle determines contractile properties of the muscle. Under actual or simulated microgravity conditions such as human bed rest or rat hindlimb unloading, decrease in expression of MyHC of the slow type [MyHC I(β)] has been observed. It was demonstrated that increasing sensory input by performing plantar mechanical stimulation (PMS) on the soles of the feet results in an increase in neuromuscular activation of the lower limb muscles and may prevent slow-to-fast fiber type shift. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway is the main cascade regulating MyHC I(β) expression. The present study was aimed to analyze the states of the calcineurin-NFATc1 signaling cascade under conditions of PMS during rat hindlimb unloading. Male Wistar rats were randomly assigned to vivarium control groups and 1-day unloading (1HS), 3-day unloading (3HS), 1HS+PMS, and 3HS+PMS groups. We found that both 1 day and 3 days of unloading caused decrease in MyHC I(β) mRNA expression and decrease in glycogen synthase kinase-3β phosphorylation (Ser 9) that brought about the kinase activation, and these effects of unloading were prevented by PMS. Three days of unloading also caused increase in expression of calsarcin-2 (myozenin-I), which was found to be the endogenous calcineurin inhibitor. The level of calsarcin-2 expression in the 3HS+PMS group did not differ from the control group. Therefore, we conclude that PMS upregulates the calcineurin-NFATc1 signaling pathway and prevents unloading-induced MyHC I(β) decrease. NEW & NOTEWORTHY It is widely accepted that changes in the myosin phenotype during functional unloading (disuse) are determined by a decreased expression of the myosin heavy chain (MyHC) I(β) gene, and this decrease leads to changes of contractile and fatigue characteristics of soleus muscle. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) pathway is one of the most important signaling cascades regulating slow MyHC isoform expression. The present study is the first to show that plantar mechanical stimulation upregulates calcineurin-NFATc1 signaling in soleus muscles of hindlimb-unloaded rats.

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats.

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been routinely employed since 1979 and the development of hind limb unloading (HLU). However, the next steps in space exploration now include to travel to Mars where the gravity is 38% of Earth's gravity. Since no human being has experienced this level of partial gravity, a sustainable ground-based model is necessary to investigate how the body, already impaired by the time spent in microgravity, would react to this partial load. Here, we used our innovative partial weight-bearing (PWB) model to mimic a short mission and stay on Mars to assess the physiological impairments in the hind limb muscles induced by two different levels of reduced gravity applied in sequential fashion. This could provide a safe, ground-based model to study the musculoskeletal adaptations to gravitational change and to establish effective countermeasures to preserve astronauts' health and function.

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

Cardiovascular Deconditioning Induces Changes in the Excitatory Neurotransmission and Active Properties of Nucleus Tractus Solitarii Neurons

Hindlimb‐unloading (HU) in rats induces cardiovascular deconditioning analogous to that observed in individuals exposed to microgravity or bed rest. Among other physiological changes, HU rats exhibit autonomic imbalance, increased baseline heart rate and altered baroreflex and chemoreflex. Considering that the nucleus tractus solitariis (nTS) is an important central region for autonomic control and integration of cardiovascular reflex function, in this study we aimed to investigate the effects of HU on glutamatergic synaptic transmission and intrinsic properties of nTS neurons. For this purpose, stainless steel wire rings were positioned in the tail of male Sprague‐Dawley rats (3 weeks) and after one week of recovery the rings was connected to a suspension apparatus. HU rats were suspended at an angle of 30–35° for two weeks with free access to food and water, while control animals were maintained under normal postural conditions. After this period, coronal brainstem slices were obtained and the whole‐cell patch clamp technique was performed to evaluate the electrophysiological properties of nTS neurons. The effectiveness of the HU protocol was confirmed by significant hindlimb muscle atrophy. Adrenal weight was comparable between HU and control rats. Resting membrane potential of nTS neurons from HU rats was comparable to neurons from controls. However, HU reduced the percentage of neurons that exhibited spontaneous firing activity (Control: 46% vs HU: 11%). In addition, HU induced a significant increase in the action potential rheobase and A‐type potassium current of nTS neurons, which may contribute to altered basal firing rate. Furthermore, the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) was significantly higher in the neurons of HU rats in comparison to the control. Taken together the data indicated that HU increases glutamatergic neurotransmission and reduces the excitability of NTS neurons. Therefore, the results suggest that changes in neuronal activity and synaptic transmission in the nTS may contribute to the cardiovascular reflex alterations observed in HU rats.Support or Funding InformationSupport: HL132836This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A modified protocol for generating the simulated weightlessness rat model

To introduce a modified protocol for generating the simulated weightlessness rat model by hindlimb unloading. Ninety male adult SD rats were randomly divided into three groups: the control group, classical suspension group and modified suspension group (n=30/group). In the classical suspension group, a strip of medical adhesive tape was attached to the tail, with horizontal filament tape wrapping. A piece of gauze was wrapped around the tail at the outermost layer and the tail was suspended for hindlimb unloading. In the modified suspension group, a layer of plastic net was added between the horizontal filament tape and the gauze to reduce the squeeze on the tail as a buffer zone and ensure proper circulation of the tail. After 4 weeks of suspension, damage to the tail and sheath detachment were observed. Meanwhile the body weight and right soleus wet weight of rats were measured. The ratio of right soleus wet weight to body weight was decreased significantly in both the classical suspension group and the modified suspension group compared with the control group, while there was no difference in body weight among the three different groups. Importantly, the incidence of tail ischemia and necrosis (13.3% vs 40.0% in the classical suspension group) and the incidence of sheath detachment from tail (3.3% vs 26.7% in the classical suspension group) were significantly lower whereas the success rates of model (33.3% vs 83.3% in classical suspension group) was significantly higher in the modified suspension group. The modified protocol decreases the incidence of tail necrosis and sheath detachment in the rat tail suspension and increases the success rate of the hindlimb unloading rat model, with improved simplicity and practicability.

Design and Experimental Validation of Hindlimb Unloading Rat Suspension Device with Adjustable Body Position

通过对大鼠尾吊模型进行改进，研制出一种新型可调节体位的大鼠后肢去负荷悬吊装置，研究模拟微重力效应下体液分布变化对大鼠骨代谢的影响.将36只SD大鼠均分为对照组（CON）、头低位后肢去负荷组（HDT）、水平位后肢去负荷组（HH）和头高位后肢去负荷组（HUT）4组，实验21天后，利用DXA检测大鼠的骨密度（BMD）.模拟微重力效应下的三组大鼠后肢均发生严重骨丢失，其中HH和HUT组后肢BMD显著大于HDT组.实验结果表明，体液分布变化可能在模拟微重力效应导致的骨丢失中起到重要作用，新型大鼠后肢去负荷悬吊装置能够调节大鼠体位（体液）进行模拟微重力效应研究.

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

Protective effects of Brazilian propolis supplementation on capillary regression in the soleus muscle of hindlimb-unloaded rats.

The protective effects of Brazilian propolis on capillary regression induced by chronically neuromuscular inactivity were investigated in rat soleus muscle. Four groups of male Wistar rat were used in this study; control (CON), control plus Brazilian propolis supplementation (CON + PP), 2-week hindlimb unloading (HU), and 2-week hindlimb unloading plus Brazilian propolis supplementation (HU + PP). The rats in the CON + PP and HU + PP groups received two oral doses of 500 mg/kg Brazilian propolis daily (total daily dose 1000mg/kg) for 2weeks. Unloading resulted in a decrease in capillary number, luminal diameter, and capillary volume, and an increase in the expression of anti-angiogenic factors, such as p53 and TSP-1, within the soleus muscle. Brazilian propolis supplementation, however, prevented these changes in capillary structure due to unloading through the stimulation of pro-angiogenic factors and suppression of anti-angiogenic factors. These results suggest that Brazilian propolis is a potential non-drug therapeutic agent against capillary regression induced by chronic unloading.

Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse.

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.

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.