Hindlimb Unloading Group Research Articles

Comparison of rectangular and sine waveforms on electrical stimulation to atrophy of rat soleus muscle

PURPOSEWe focused on the difference between rectangular and sine waves, investigated the effect of electrical stimulation on muscle atrophy.METHODSThe rats were randomly divided into hindlimb unloading (HU), hindlimb unloading plus electrical stimulation with rectangular waves (HU+RS), hindlimb unloading plus electrical stimulation with sine waves (HU+SS), and weightmatched control (Cont) groups. In the HU+RS and HU+SS groups, the stimulation was percutaneously provided to calf muscles. After 14 days, the soleus muscle located in deep layer of calf was removed.RESULTSThe value of muscle fiber cross‐sectional area was significantly less in the HU group than in the Cont group. Although there were no significant differences between the HU and HU+RS groups, the value in the HU+SS group was significantly larger than that in the HU and HU+RS groups. The expression level of ubiquitinated proteins was significantly higher in the HU group than in the Cont group, whereas the expression level was significantly less in the HU+SS group than in the HU and HU+RS groups.CONCLUSIONThe effect of electrical stimulation on muscle atrophy was depended on the waveform, and sine wave was effective in deep muscle. Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Low-intensity vibrations partially maintain intervertebral disc mechanics and spinal muscle area during deconditioning

Background contextReduced spinal loading degrades the intervertebral disc and alters the muscle size. PurposeTo determine the ability of high-frequency and low-intensity vibrations to maintain disc biomechanics and prevent muscle changes during hindlimb unloading. Study designThree groups of Sprague-Dawley rats were hindlimb unloaded for 4 weeks. In two hindlimb unloaded groups, unloading was interrupted for 15 min/d and the rats were positioned upright on a 90 Hz vertically oscillating plate or a sham control inactive plate. One author owns (provisional) patents regarding the application of vibrations to the musculoskeleton. MethodsThe motion segments L4–L5 were mechanically evaluated in compression-tension, axial creep, and torsion loading. In vivo microcomputed tomography was used to determine longitudinal psoas and paraspinal muscle area. This work was supported by National Institutes of Health, National Aeronautics and Space Administration (NASA), Alliance for Graduate Education and the Professoriate, and NASA-Harriett G. Jenkins Predoctoral and W. Burghardt Turner Fellowships. The author (SJ) holds (provisional) patents regarding the application of vibrations. ResultsThere were no differences between the discs of uninterrupted unloading and sham animals and these groups were pooled. Compared with normally ambulating age-matched controls, hindlimb unloaded discs had altered properties in every loading modality. Psoas area of the unloaded rats increased at L4 and L5 and the paraspinal area decreased at L4. Vibrations (90 Hz) maintained compression-tension properties, partially maintained creep properties, but did not mitigate torsional weakening because of unloading. Low-intensity vibrations prevented the increase in psoas area but did not abate paraspinal muscle loss. ConclusionsIn support of clinical studies, unloading deconditioned the rodent disc and altered the muscle area. Although brief exposures to upright posture provided only limited benefits, low-intensity vibrations superimposed on upright posture served to preserve disc mechanics during unloading.

Partial reductions in mechanical loading yield proportional changes in bone density, bone architecture, and muscle mass

Although the musculoskeletal system is known to be sensitive to changes in its mechanical environment, the relationship between functional adaptation and below-normal mechanical stimuli is not well defined. We investigated bone and muscle adaptation to a range of reduced loading using the partial weight suspension (PWS) system, in which a two-point harness is used to offload a tunable amount of body weight while maintaining quadrupedal locomotion. Skeletally mature female C57Bl/6 mice were exposed to partial weight bearing at 20%, 40%, 70%, or 100% of body weight for 21 days. A hindlimb unloaded (HLU) group was included for comparison in addition to age-matched controls in normal housing. Gait kinematics was measured across the full range of weight bearing, and some minor alterations in gait from PWS were identified. With PWS, bone and muscle changes were generally proportional to the degree of unloading. Specifically, total body and hindlimb bone mineral density, calf muscle mass, trabecular bone volume of the distal femur, and cortical area of the femur midshaft were all linearly related to the degree of unloading. Even a load reduction to 70% of normal weight bearing was associated with significant bone deterioration and muscle atrophy. Weight bearing at 20% did not lead to better bone outcomes than HLU despite less muscle atrophy and presumably greater mechanical stimulus, requiring further investigation. These data confirm that the PWS model is highly effective in applying controllable, reduced, long-term loading that produces predictable, discrete adaptive changes in muscle and bone of the hindlimb.

Discordant recovery of bone mass and mechanical properties during prolonged recovery from disuse

Profound bone loss at weight bearing sites is a primary effect of long-duration spaceflight. Moreover, a significant increase in estimated fracture risk remains even 1year after returning to Earth; hence, it is important to define how quickly bone integrity can recover following prolonged disuse. This study characterized the loss and recovery dynamics of bone following a period of rodent hindlimb unloading in three anatomic sites. We hypothesized that the rat femoral neck would exhibit a discordant recovery dynamic most similar to that observed in astronauts' proximal femur; that is, bone mineral content (absolute mass) at this site would recover faster and more completely than would bone density and cortical area, and they will all recover before bone strength does. We characterized loss and long-term recovery of densitometric properties at the femoral neck, proximal tibia metaphysis, and tibia diaphysis, and also mechanical properties at the femoral neck and tibia diaphysis for which mechanical testing is amenable. We assessed the relationship between calculated strength indices and measured mechanical properties.Adult male Sprague–Dawley rats (6months) were assigned to baseline, age-matched control (AC), and hindlimb unloaded (HU) groups. The HU group was unloaded for 28days and then returned to normal cage activity for 84days of weight bearing recovery (3 times the duration of HU). Fifteen animals were euthanized from each of the HU and AC groups on days 28, 56, 84, and 112 of the study. At baseline and then every 28days in vivo longitudinal pQCT scans were taken at proximal tibia metaphysis (PTM) and tibia diaphysis (TD); ex vivo pQCT scans were taken later at the femoral neck (FN). TD and FN were tested to failure to measure mechanical properties.The hypothesis that the femoral neck in rats will exhibit a discordant recovery dynamic most similar to that observed in astronauts' proximal femurs was not supported by our data. At the femoral neck, densitometric and geometric variables (total BMC, total vBMD, cancellous vBMD, and cortical area) recovered to age-matched control levels after a recovery period twice the duration of unloading. Contrary to our hypothesis, changes in densitometric variables at the PTM provided a better model for changes in the human femoral neck with prolonged weightlessness. Following 28days of HU, PTM total BMC recovered to age-matched control levels after roughly two times the duration of unloading; however, total vBMD did not recover even after three recovery periods. Cortical thinning occurred at the PTM following HU likely due to inhibition of periosteal growth; cortical shell thickness did not recover even after three recovery periods. Calculated strength indices suggested a loss in strength at the tibial diaphysis, which was not confirmed with direct testing of mechanical properties. HU had no effect on maximum fracture force at mid-tibia diaphysis; however, femoral neck experienced a significant loss of maximum force due to unloading that fully recovered after 28days. Estimated strength indices for the femoral neck suggested a recovery period of 56days in contrast to the 28-day recovery that was observed with mechanical testing. However, the inaccuracy of strength indices vs. directly measured mechanical properties highlights the continued importance of ground based animal models and mechanical testing. Our results demonstrate that the PTM in the rat better matches loss and recovery dynamics observed in astronauts' proximal femur than does the rat FN, at least in terms of densitometric variables. More complete utility of the rat PTM as a model in this case, however, depends upon meaningful characterization of changes in mechanical properties as well.

Whole-body vibration and resistance exercise prevent long-term hindlimb unloading-induced bone loss: independent and interactive effects

Skeletal unloading induced by disuse or immobilization causes a decrease in bone mass and strength. We investigated the relationship between whole-body vibration (WBV) and resistance exercise (RE) in preventing bone loss induced by 8-week hindlimb unloading in young male rats. Sixty male Wistar rats were assigned randomly to 6 groups: age-matched control group (CON, n=10), hindlimb unloading group (HU, n=10), hindlimb unloading+standing group (HU+ST, n=10), hindlimb unloading+WBV group (HU+WBV, n=10), hindlimb unloading+RE group (HU+RE, n=10) and hindlimb unloading+WBV+RE group (HU+WBV+RE, n=10). After 8-week hindlimb unloading, micro-CT scanning and three-point bending test were performed in the femur. Sera were collected for analysis of bone formation and resorption markers. Compared with HU group, WBV, RE and the combination of WBV and RE (WBV+RE) significantly improved (P<0.01) one repetition maximum (1RM) (expressed as the percentage change from baseline, HU: -23%, HU+WBV: 21%, HU+RE: 48%, HU+WBV+RE: 51%), and maintained (P<0.05) cancellous volumetric bone mineral density (vBMD) and trabecular structure. No difference of cortical vBMD was found among all groups (P>0.05). WBV had no effects on biomechanical properties of the femur diaphysis (P>0.05). RE and WBV+RE significantly increased maximum load and cross-sectional moment of inertia of the femur diaphysis in hindlimb unloading rats (P<0.05). There was an interaction between WBV and RE in improving cancellous bone. These results demonstrate that WBV and RE interactively maintain cancellous structure and vBMD, and independently partially mitigate the reduction of bone strength in long-term hindlimb unloading rats.

Simulated resistance training, but not alendronate, increases cortical bone formation and suppresses sclerostin during disuse

Mechanical loading modulates the osteocyte-derived protein sclerostin, a potent inhibitor of bone formation. We hypothesized that simulated resistance training (SRT), combined with alendronate (ALEN) treatment, during hindlimb unloading (HU) would most effectively mitigate disuse-induced decrements in cortical bone geometry and formation rate (BFR). Sixty male, Sprague-Dawley rats (6-mo-old) were randomly assigned to either cage control (CC), HU, HU plus either ALEN (HU+ALEN), or SRT (HU+SRT), or combined ALEN and SRT (HU+SRT/ALEN) for 28 days. Computed tomography scans on days -1 and 28 were taken at the middiaphyseal tibia. HU+SRT and HU+SRT/ALEN rats were subjected to muscle contractions once every 3 days during HU (4 sets of 5 repetitions; 1,000 ms isometric + 1,000 ms eccentric). The HU+ALEN and HU+SRT/ALEN rats received 10 μg/kg ALEN 3 times/wk. Compared with the CC animals, HU suppressed the normal slow growth-induced increases of cortical bone mineral content, cortical bone area, and polar cross-sectional moment of inertia; however, SRT during HU restored cortical bone growth. HU suppressed middiaphyseal tibia periosteal BFR by 56% vs. CC (P < 0.05). However, SRT during HU restored BFR at both periosteal (to 2.6-fold higher than CC) and endocortical (14-fold higher than CC) surfaces (P < 0.01). ALEN attenuated the SRT-induced BFR gains during HU. The proportion of sclerostin-positive osteocytes in cortical bone was significantly higher (+121% vs. CC) in the HU group; SRT during HU effectively suppressed the higher proportion of sclerostin-positive osteocytes. In conclusion, a minimum number of high-intensity muscle contractions, performed during disuse, restores cortical BFR and suppress unloading-induced increases in sclerostin-positive osteocytes.

Brief daily exposure to low-intensity vibration mitigates the degradation of the intervertebral disc in a frequency-specific manner

Hindlimb unloading of the rat causes rapid hypotrophy of the intervertebral disc (IVD) as well as reduced IVD height and glycosaminoglycan content. Here we tested the hypothesis that low-intensity mechanical vibrations (0.2 g), as a surrogate for exercise, will mitigate this degradation. Four groups of Sprague-Dawley rats (4.5 mo, n = 11/group) were hindlimb unloaded (HU) for 4 wk. In two of the HU groups, unloading was interrupted for 15 min/day by placing rats in an upright posture on a platform that was vertically oscillating at 45 or 90 Hz (HU+45, HU+90). Sham control rats stood upright on an inactive plate for 15 min/day (HU+SC). These three experimental groups were compared with HU uninterrupted by weightbearing (HU) and to normally ambulating age-matched controls. In the HU and HU+SC rats, 4 wk of unloading resulted in a 10% smaller IVD height, as well as less glycosaminoglycan in the whole IVD (7%) and nucleus pulposus (17%) and a greater collagen-to-glycosaminoglycan ratio in the whole IVD (17%). Brief daily exposure to 90 Hz mechanical oscillations mitigated this degradation; compared with HU ± SC, the IVD of HU+90 had an 8% larger height and greater glycosaminoglycan content in the whole IVD (12%) and nucleus pulposus (24%). In contrast, the 45 Hz signal failed to mitigate changes in height or glycosaminoglycan content brought with altered spinal loading, but normalized the collagen-to-glycosaminoglycan ratio to levels observed in age-matched controls. In summary, unloading caused marked phenotypic and biochemical changes in the IVD, a deterioration that was not slowed by brief weightbearing. However, low-intensity 90 Hz vibrations superimposed on weightbearing largely preserved the morphology and biochemistry of the IVD and suggest that these biomechanically based signals may help protect the IVD during long bouts of nonambulation.

The combined effect of electrical stimulation and resistance isometric contraction on muscle atrophy in rat tibialis anterior muscle

Electrical stimulation has been used to prevent muscle atrophy, but this method is different in many previous studies, appropriate stimulation protocol is still not decided. Although resistance exercise has also been shown to be an effective countermeasure on muscle atrophy, almost previous studies carried out an electrical stimulation without resistance. It was hypothesized that electrical stimulation without resistance is insufficient to contract skeletal muscle forcefully, and the combination of electrical stimulation and forceful resistance contraction is more effective than electrical stimulation without resistance to attenuate muscle atrophy. This study investigated the combined effects of electrical stimulation and resistance isometric contraction on muscle atrophy in the rat tibialis anterior muscle. The animals were divided into control, hindlimb unloading (HU), hindlimb unloading plus electrical stimulation (ES), and hindlimb unloading plus the combination of electrical stimulation and resistance isometric contraction (ES+IC). Electrical stimulation was applied to the tibialis anterior muscle percutaneously for total 240 sec per day. In the ES+IC group, the ankle joint was fixed to produce resistance isometric contraction during electrical stimulation. After 7 days, the cross-sectional areas of each muscle fiber type in the HU group decreased. Those were prevented in the ES+IC group rather than the ES group. The expression of heat shock protein 72 was enhanced in the ES and ES+IC groups. These results indicated that although electrical stimulation is effective to prevent muscle atrophy, the combination of electrical stimulation and isometric contraction have further effect.

Sex Differences in the Mechanical Properties of Rat Myocardium After Exposure to Simulated Microgravity

Female astronauts are more likely than male astronauts to develop presyncope and orthostatic intolerance when they return to normal gravity after space-flights. The underlying mechanisms for this are unclear but could include myocardial adaptations to micro-gravity conditions. This study was designed to test whether there are sex-specific changes in the mechanical properties of myocardial tissue in rats subjected to a period of simulated microgravity. Multicellular myocardial preparations were isolated from control male and female Sprague-Dawley rats and corresponding experimental animals that were hind-limb unloaded (HLU) for 14 days. Preparations were subjected to standard mechanical tests imposed under muscle length control in solutions with pCa values ranging from 9.0 to 4.5. The rates of tension recovery (ktr) measured in pCa 4.5 solution were significantly higher (p<0.002, 2-way ANOVA) in the preparations isolated from the HLU animals than in preparations isolated from the control groups (ktr mean values ± SEM measured in pCa 4.5 were: 7.45±0.37 s−1 for female control; 9.54±0.50 s−1 female HLU; 7.70±0.46 s−1 male control and 8.42±0.37 s−1 male HLU). The relative content of slower beta-Myosin Heavy Chain decreased in both HLU groups compared to the corresponding control groups (p<0.01, 2-way ANOVA). There was no interaction for this parameter between sex and unloading condition (p=0.93, 2-way ANOVA). The heart-to-body weight ratios increased significantly in the HLU groups (p<0.0001, 2-way ANOVA) but again there was no interaction with sex (p=0.84, 2-way ANOVA). If similar mechanisms operate in humans, female astronauts may be more likely to develop presyncope after space-flights than male astronauts because their hearts contain a greater relative content of the alpha-Myosin Heavy Chain when they return to Earth.

The Impact of Muscle Disuse on Muscle Atrophy in Severely Burned Rats

Severe burn induces a sustained hypermetabolic response, which causes long-term loss of muscle mass and decrease in muscle strength. In this study, we sought to determine whether muscle disuse has additional impact on muscle atrophy after severe burn using a rat model combining severe cutaneous burn and hindlimb unloading. Forty Sprague-Dawley rats (≈ 300 g) were randomly assigned to sham ambulatory (S/A), sham hindlimb unloading (S/HLU), burn ambulatory (B/A), or burn hindlimb unloading (B/HLU) groups. Rats received a 40% total body surface (TBSA) full thickness scald burn, and rats with hindlimb unloading were placed in a tail traction system. At d 14, lean body mass (LBM) was determined using DEXA scan, followed by measurement of the isometric mechanical properties in the predominantly fast-twitch plantaris muscle (PL) and the predominantly slow-twitch soleus muscle (SL). Muscle weight (wt), protein wt, and wet/dry wt were determined. At d 14, body weight had decreased significantly in all treatment groups; B/HLU resulted in significantly greater loss compared with the B/A, S/HLU, and S/A. The losses could be attributed to loss of LBM. PL muscle wt and Po were lowest in the B/HLU group (<0.05 versus S/A, S/HLU, or B/A). SL muscle wt and Po were significantly less in both S/HLU and B/HLU compared with that of S/A and B/A; no significant difference was found between S/HLU and B/HLU. Cutaneous burn and hindlimb unloading have an additive effect on muscle atrophy, characterized by loss of muscle mass and decrease in muscle strength in both fast (PL) and slow (SL) twitch muscles. Of the two, disuse appeared to be the dominant factor for continuous muscle wasting after acute burn in this model.

Influence of 14-day hind limb unloading on isolated muscle spindle activity in rats

During hind limb unloading (HU), the soleus is often in a shortened position and the natural physiological stimulus of muscle spindles is altered, such that muscle spindle activity also changes. Using isolated spindle conditions, the present study investigates the electrophysiological activity and ultrastructure of muscle spindles following HU. Results show that muscle spindle discharges fall into either of two main patterns, single spikes or spike clusters in shortened positions, with a steady frequency of 18-38 spikes/s (mean 29.08 +/- 2.45) in an extended position. Following 14-day HU, afferent discharge activity was significantly altered in soleus muscle spindles. Duration of individual spikes was significantly prolonged, from 0.54 +/- 0.05 ms for control rats to 1.53 +/- 0.25 ms for rats in the HU group. In a shortened position, regular rhythm afferent discharges were obviously depressed, and the majority of muscle spindles became silent, while in an extended position, the discharges remained continuous but with decreased frequency. Results also show that the ultrastructure of muscle spindles experience degenerative changes during HU. Altered muscle spindle afference could possibly modify the activity of motor neurons and further affect the activity of extrafusal fibers.

Simulated resistance training during hindlimb unloading abolishes disuse bone loss and maintains muscle strength

This study was designed to determine the effectiveness of simulated resistance training (SRT) without weight bearing in attenuating bone and muscle loss during 28 day hindlimb unloading (HU) in mature male rats. An ambulatory control group (CC) and four groups of HU rats were used: HU, HU + anesthesia (ANHU), HU + eccentric muscle contractions (HU + ECC), and HU + isometric and eccentric muscle contractions (HU + ISO/ECC). Animals in the two SRT groups were trained once every other day at 100% daily peak isometric torque (P(0)). HU resulted in significantly lower plantarflexor muscle mass (-33% versus CC) and reduced isometric strength (-10%), which reductions were partially attenuated in both training groups. Significantly reduced total and cancellous volumetric bone mineral density (vBMD) and total bone mineral content (BMC) at the proximal tibia metaphysis (PTM) also was evidenced in HU and ANHU groups compared with both SRT groups (p < .05). Training resulted in greater increases in cortical bone mass and area compared with all other groups (p < .05). Fourfold higher material properties of PTM cancellous bone were demonstrated in SRT animals versus HU or CC animals. A significant reduction in midshaft periosteal bone formation rate (BFR) in the HU group (-99% versus CC) was completely abolished in HU + ECC (+656% versus CC). These results demonstrate that high-intensity muscle contractions, independent of weight-bearing forces, can effectively mitigate losses in muscle strength and provide a potent stimulus to bone during prolonged disuse.

Implications Of Angiogenic Factors And Thrombospondin-1 Underlying Capillary Regression In Chronically Unloaded Rat Soleus Muscle

Capillary regression is induced by chronic unloading of skeletal muscles. The mechanisms involved, however, are not fully understood. At least two independent mechanisms can be considered, i.e., adaptations in the levels of specific angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietin, and of thrombospondin-1 (TSP-1), an inhibitor of angiogenesis that induces apoptosis in endothelial cells. PURPOSE: The aim of present study was to determine the responses of pro- and anti-angiogenic factors in rat soleus capillaries after 2 weeks of hindlimb unloading (HU). METHODS: Male Wistar rats (8-9 weeks of age) were assigned randomly into a control or HU group. Frozen transverse sections from the soleus mid-belly were stained for alkaline phosphatase which is present in the capillary endothelium. Apoptotic endothelial cells were identified by von Willebrand factor and DNA fragmentation (TUNEL) using immunofluorescent staining. VEGF, KDR, Flt-1, angiopoietin-1 (Ang-1), Ang-2, Tie-2, HIF 1 alpha, and TSP-1 mRNAs were determined by TaqMan probe-based real-time PCR reactions. RESULTS: The capillary-to-fiber ratio was 23% lower in HU than control rats. TUNEL-positive endothelial cells were observed only in HU rats and TSP levels were higher in HU than control rats, suggesting capillary regression with vascular endothelial cell apoptosis. mRNA levels of angiogenic factors VEGF and Ang-1, their receptors, and HIF 1 alpha were lower in HU than control rats whereas Ang-2 levels were unaffected. As a result, the Ang-2/Ang-1 ratio increased, suggesting that the vasculature was destabilized. Ang-2 expressed in the absence of VEGF also results in vessel regression. CONCLUSIONS: These data indicate that a decrease in angiogenic factors and an increase in TSP-1 expression may play an important role in capillary regression associated with chronic unloading. Supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, 19650148, and 19300196.

Short‐term hindlimb unloading induces translocation of nNOS from the sarcolemma to sarcoplasm in skeletal muscle

Long duration spaceflight missions cause skeletal muscle atrophy in astronauts. We hypothesize that neuronal Nitric Oxide Synthase (nNOS) and dystrophin translocate from the sarcolemma to the sarcoplasm following 48‐hrs of simulated microgravity as compared to ground controls. Eight Fisher‐344 rats underwent 48‐hour simulated microgravity using the well established hindlimb unloading model, and eight rats were used as ground controls. Hindlimb muscles were carefully dissected, snapped frozen, and stored for analysis. Cryostat sections of soleus muscle (10 micrometer thick), were prepared for immunohistochemical analysis of nNOS and dystrophin localization. Hindlimb unloading induced translocalization of nNOS from the sarcolemma to the sarcoplasm. However, dystrophin was localized at the sarcolemma in the hindlimb unloaded group. Both nNOS and dystrophin were localized to the sarcolemma in the ground control group. These data are the first to demonstrate that nNOS delocalizes from the sarcolema during short‐term hindlimb unloading (48‐hrs). Future studies should investigate the localization of other dystroglycan proteins such as B1‐syntrophin and alpha‐dystrobrevin. Supported by the Texas A&amp;M University Training Grant in Space Life Sciences with funding from NSBRI (NASA NCC 9‐58).

Influence of fixed muscle length and contractile properties on atrophy and subsequent recovery in the rat soleus and plantaris muscles

This study examined muscular atrophy and the recovery process induced by hindlimb unloading and joint immobilization in the rat soleus and plantaris muscles. Rats were divided into control, hindlimb unloading (HU), hindlimb unloading with ankle joint immobilization at the maximum dorsiflexion (HUD), and maximum plantarflexion (HUP) groups. The hindlimb was reloaded after fourteen days of unloading, and muscle atrophy and walking ability were assessed at 0, 3, and 7 days of reloading. A cross sectional area of muscle fibers in the soleus muscle on day 0 of reloading revealed sizes in order from the control, HUD, HUP down to the HU group, indicating that the HU group was the most atrophied among the four groups. These values in the plantaris muscle ranged in order from the control, HU, HUD, to HUP groups, the HUP group being the most atrophied among the four groups. These muscles recovered from atrophy in the same descending order, and the values in the HUD and HUP groups slowly recovered during the reloading periods. The HUD and HUP groups showed a central core lesion and reloading-induced lesions in some type I muscle fibers after the immobilization and reloading, one possible reason for the delayed recovery in these groups. The muscle atrophy in the HU, HUD, and HUP groups remained at day 7 although the walking ability appeared to be normal. Accordingly, further rehabilitation therapy might be necessary even if the functional ability appears to be normal.

Effects of exposure of mice to hindlimb unloading on leukocyte subsets and sympathetic nervous system activity

The hindlimb unloading (HU) rodent model was developed to simulate some of the aspects of spaceflight conditions. Our previous studies showed that exposure to HU for 48 h (h) followed by bacterial challenge, reduces the ability of mice to resist infection. The purpose of this study was to investigate the physiological changes in mice during the 48 h of exposure to HU to understand the mechanisms involved in the increased susceptibility to infection observed in mice subjected to these conditions. Female Swiss Webster mice were hindlimb-unloaded during 48 h. Blood samples, spleen and peritoneal cells were removed before and after 18 or 48 h of HU-exposure. Leukocyte subset analysis was performed in spleen and peritoneal cells by flow cytometry, and catecholamine levels were measured in plasma and whole spleen by a catecholamine enzyme immunoassay. Catecholamine levels measured in plasma and spleen were significantly greater in mice exposed to HU compared to control. This increase coincided with significant reductions in spleen size in the HU group. Flow cytometric analyses showed a significant reduction of splenic CD19 + B-cells and NK1.1+ cells in mice exposed to HU with a concomitant increase in T-cells. These results suggest that exposure to HU increases the activity of the sympathetic nervous system (SNS) and induces lymphocyte sub-population changes that may contribute to the deregulation of immunity seen in mice exposed to HU and, more importantly may predispose the otherwise healthy host to the subsequent reduced ability to resist infections.

Effects of L-Carnitine on Disuse Muscle Atrophy

Immobilization stress induces the formation of reactive oxygen species and leads to oxidative injury in various tissues. Lipid peroxidation is the most important mechanism in muscle atrophy. L-carnitine may prevent lipid peroxidation and may thus protect against muscle atrophy. PURPOSE: The aim of this study is to determine the effect of l-carnitine supplementation on disuse muscle atrophy. METHODS: Twenty-seven male Sprague Dawley rats were divided into 4 groups: control 21 (CONT), days of hindlimb unloading (HU), 21 days of hindlimb unloading + L-carnitine (HU + LC), and control L-carnitine (LC-CONT). The rats from the HU and HU + LC groups were suspended for 21 days according to the tail suspension model described by Morey-Holton and Globus. The soleus and extensor digitorum longus muscles from both hindlimbs were dissected, and the wet tissue weights were measured. The level of MDA, which is a marker for lipid peroxidation, was determined in the homogenates according to the thiobarbituric acid reaction described by Buege and Aust. The GSH levels were assayed in tissue homogenates according to the method of Ellman. All experimental procedures involving animals were approved by the ethical committee for animal research of the Baskent University Faculty of Medicine in Ankara, Turkey. RESULTS: Muscle unweighting caused a 33% decrease in the soleus muscle and a 20% decrease in the EDL mass. The GSH level was statistically significantly higher in the LC-CONT group than in the CONT, HU + LC, and HU groups. The GSH level in the HU group was statistically significantly lower than that in the HU + LC group. The GSH content was not found to be statistically significantly different in the HU group and the HU + LC group from that in the CONT group. MDA levels were statistically significantly lower in the HU + LC and LC-CONT groups than those in the CONT group and statistically significantly lower in the HU + LC group than those in the HU group (P < .0125). CONCLUSION: We concluded that L-carnitine reduced the degree of muscle atrophy and oxidative stress caused by 21 days of immobilization.

Intermittent Reloading Attenuates Muscle Atrophy through Modulating Akt/mTOR Pathway

The aim of this study is to investigate the effects of intermittent reloading during hindlimb unloading (HU) on the changes in intracellular signaling pathways in skeletal muscle. Male Wister rats were divided randomly into one of three experimental groups: 1) nonsuspended control, 2) HU for 7 d, and 3) HU with intermittent reloading (HU/IR) for 4 h.d(-1). After each experimental period, the antigravitational soleus muscle was analyzed. After 7 d of HU treatment, muscle fiber atrophy (decrease in relative muscle mass: 0.28 mg.g(-1) in the HU group vs 0.36 mg.g(-1) in the control group, P < 0.05; decrease in fiber CSA: 1682.6 microm2 in the HU group vs 2673.0 microm2 in the control group, P < 0.05) and a decrease in phosphorylation levels of anabolic signaling pathway (Akt and mTOR) were observed. Additionally, expressions of two types of muscle-specific E3 ubiquitin ligase mRNA (muscle atrophy F-box (MAFbx), and muscle ring finger 1 (MuRF1)) were upregulated during muscle atrophy. Increases in binding activities of nuclear factor kappa B (NFkappaB) were also determined. In contrast, IR treatment attenuated the muscle fiber atrophy (0.33 mg.g(-1) and 2067.5 microm2) and partially increased the phosphorylation levels of anabolic signaling molecules. Expression of MAFbx and MuRF1 mRNA were returned to the control level, and binding activities of nuclear NFkappaB was suppressed with the effects of IR. These results suggest that IR-induced attenuation of skeletal muscle atrophy is achieved by the synergy between increased anabolic and decreased catabolic signaling mechanisms.

Intermittent postural recovery during 14 days of hindlimb unloading (HU) is not sufficient to restore cardiac autonomic imbalance in rats

Previous data indicate that 14 days of continuous HU in rats produces cardiac autonomic imbalance. We hypothesized that a daily period of normal posture may resolve these effects. Male, Sprague‐Dawley rats were randomly assigned to control (CC; n=3), continuous HU (HUC; n=4) or intermittent HU (HUI; n= 5) groups. Control rats were maintained in the normal posture. HUC rats underwent continuous elevation of the hindlimbs for 14‐days while HUI rats were returned to the normal posture for 30 minutes each day during the HU procedure. On the 14th day, mean arterial pressure (MAP) and heart rate (HR) were measured followed by selective cardiac autonomic blockade administered via i.v. injection of methylatropine and propranolol. Both HU groups exhibited significant resting tachycardia compared to CC rats, but HR was lower in HUI vs. the HUC group (CC: 366±3.9; HUC 474±7.5; HUI: 434±1.6 bpm). However, both HU groups had significantly less parasympathetic (CC: 60±7.7; HUC: 25±10.9; HUI: 15±5.2 Δbpm) and greater sympathetic cardiac tone (CC: 16±7.9; HUC: 91±10.9; HUI: 77±9.2 Δbpm) than CC rats, with no difference between HU groups with respect to either autonomic parameter. Baseline MAP was not different among any of the groups. Data indicate that while daily recovery from HU may partially restore resting HR, it is not a sufficient stimulus to reverse the autonomic imbalance produced by HU‐induced deconditioning. (Supp. by: NIH HL14388 &amp; DK 066086).

Nutritional Interventions in Skeletal Muscle Atrophy

Skeletal muscle atrophy occurs with prolonged bed rest, limb immobilization, microgravity, physical inactivity, and advancing age. This study evaluated the efficacy of a carbohydrate‐protein (+ leucine) supplement (Pacific Health Laboratory) to reduce muscle mass loss and maintain muscle physiological function in a rat atrophy model of hindlimb unloading (HU). Rats were fed high (CPH) and low (CPL) doses of the supplement or vehicle (HOH) (n=10/group) and were suspended by their tails to remove all weight‐bearing function on their hindlimbs for 28 days. The supplement (5 ml/kg) or vehicle (H2O) was administered twice daily via gavage, excluding the naïve control (CON) group (n=10); all rats were fed rat chow ad libitum. On day 28 in‐situ force measurements of the left lateral gastrocnemius were recorded in all HU groups (CPL, CPH, and HOH). Statistical analyses utilized ANOVA and the Dunnett's method. Muscle weights of the soleus (−36%), plantaris (−16%), and gastrocnemius (−21%) were significantly reduced in HOH, CPL, and CPH rats after 28 days of HU (p&lt;0.05 compared to CON). The nutritional treatment (CPL and CPH) did not reduce muscle mass loss induced by HU. Specific in‐situ tetanic tension (maximal force/cross‐sectional area) of the left lateral gastrocnemius muscle was significantly less in the HOH group (17.2 N/cm2) compared to CPL (20.2 N/cm2, p&lt;0.05) and CON groups (20.4 N/cm2, p&lt;0.05). The specific tension of the CPL group did not differ significantly from the CON group. The CPH group's specific tension (19.2 N/cm2) did not significantly differ from either the HOH or CON groups. This carbohydrate‐protein (+ leucine) nutritional supplement was able to maintain muscle function (specific tension) but not muscle mass. Pacific Health Cooperation and The University of Texas at Austin supported this research.