Hindlimb Suspension Mice Research Articles

In vivo measurement of hindlimb neuromuscular function in mice

In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles. An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short- and long-latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice. PF did not change with one- and three-times-per-week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT. The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models.

Therapeutic potential of ghrelin treatment for unloading-induced muscle atrophy in mice

Ghrelin is a growth hormone (GH) secretagogue secreted mainly from the stomach that functions in controlling muscle volume and energy homeostasis. We here studied the effects of ghrelin on unloading-induced muscle atrophy using a mouse model of hindlimb suspension (HS). Ghrelin administration during 2-week HS alleviated reductions of muscle mass in the fast-twitch fiber-rich plantaris muscle and the slow-twitch fiber-rich soleus muscle of the hindlimb. Ghrelin administration during a 5-day recovery period following 2-week HS enhanced food intake and facilitated recovery from atrophy in both muscles. Ghrelin administration normalized hypercorticosteronemia in these studies. Ghrelin’s anti-muscle atrophy effect was found even under pair-feeding condition, but not in mice given des-acyl ghrelin. Insulin-like growth factor (IGF)-1 mRNA expression was significantly reduced in the atrophied plantaris muscle compared with control muscles. A single ghrelin administration to HS mice acutely increased plasma GH and also amplified phosphorylation of signal transducer and activator of transcription (STAT) 5 and increased IGF-1 mRNA expression in the plantaris muscle, but not in the soleus muscle. This study demonstrated that ghrelin stimulated the GH-STAT5-IGF-1 axis in the locally atrophied plantaris muscle, and its administration alleviated muscle atrophy and facilitated recovery from muscle atrophy. Ghrelin’s effects represent a novel therapeutic paradigm for the treatment of unloading-induced muscle atrophy induced by factors such as bed rest, injury, and joint immobilization.

Microfluidic enhancement of intramedullary pressure increases interstitial fluid flow and inhibits bone loss in hindlimb suspended mice

Interstitial fluid flow (IFF) has been widely hypothesized to mediate skeletal adaptation to mechanical loading. Although a large body of in vitro evidence has demonstrated that fluid flow stimulates osteogenic and antiresorptive responses in bone cells, there is much less in vivo evidence that IFF mediates loading-induced skeletal adaptation. This is due in large part to the challenges associated with decoupling IFF from matrix strain. In this study we describe a novel microfluidic system for generating dynamic intramedullary pressure (ImP) and IFF within the femurs of alert mice. By quantifying fluorescence recovery after photobleaching (FRAP) within individual lacunae, we show that microfluidic generation of dynamic ImP significantly increases IFF within the lacunocanalicular system. In addition, we demonstrate that dynamic pressure loading of the intramedullary compartment for 3 minutes per day significantly eliminates losses in trabecular and cortical bone mineral density in hindlimb suspended mice, enhances trabecular and cortical structural integrity, and increases endosteal bone formation rate. Unlike previously developed modalities for enhancing IFF in vivo, this is the first model that allows direct and dynamic modulation of ImP and skeletal IFF within mice. Given the large number of genetic tools for manipulating the mouse genome, this model is expected to serve as a powerful investigative tool in elucidating the role of IFF in skeletal adaptation to mechanical loading and molecular mechanisms mediating this process. © 2010 American Society for Bone and Mineral Research.

Quantification of Lacunar-Canalicular Interstitial Fluid Flow Through Computational Modeling of Fluorescence Recovery After Photobleaching.

Skeletal adaptation to mechanical loading has been widely hypothesized to involve the stimulation of osteocytes by interstitial fluid flow (IFF). However, direct investigation of this hypothesis has been difficult due in large part to the inability to directly measure IFF velocities within the lacunar-canalicular system. Measurements of fluorescence recovery after photobleaching (FRAP) within individual lacunae could be used to quantify lacunar-canalicular IFF when combined with mathematical modeling. In this study, we used a computational transport model to characterize the relationship between flow frequency (0.5-10 Hz), peak flow velocity (0-300 μm/s), tracer diffusion coefficient (100-300 μm(2)/s), and transport enhancement (i.e., (k/k(0)) - 1, where k and k(0) are the transport rates in the presence/absence of flow) during lacunar FRAP investigations. We show that this relationship is well described by a simple power law with frequency-dependent coefficients, and is relatively insensitive to variations in lacunar geometry. Using this power law relationship, we estimated peak IFF velocities in hindlimb mice subjected to intramedullary pressurization using values of k and k(0) previously obtained from ex vivo lacunar FRAP investigations. Together, our findings suggest that skeletal adaptation in hindlimb suspended mice subjected to dynamic intramedullary pressure occurred in the presence of IFF at levels associated with physiological loading.

Muscle atrophy and long bone osteopenia are attenuated with n‐3 PUFA in a mouse model of hindlimb suspension

Muscle atrophy and osteopenia are common issues associated with aging and disease. This study was conducted to evaluate the effects of long chain n‐3 PUFA [eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] on muscle and bone loss in a mouse model of induced hindlimb atrophy. Mouse hindlimb suspension (HS) is used as a disuse model to induce muscle atrophy and bone loss compared to weight bearing (WB). Six‐wk‐old male mice (ND4 Swiss Webster) were fed a control (n‐6 PUFA, n= 26), a moderate n‐3 PUFA (EPA + DHA 12 g/kg, n = 20), or a high n‐3 PUFA (19.4 g/kg, n= 21) diet for 2 wks. After pre‐feeding, mice were subjected to a baseline DEXA scan and then assigned to the WB or HS groups. Upon 14 days of HS or continued WB, mice were anesthetized for a repeated DEXA scan after which the mice were euthanized, and tissues were collected for analyses. Mice fed n‐3 PUFA demonstrated an elevation of EPA and DHA and reduced arachidonate in serum and tissues. HS resulted in fat, muscle mass and bone mineral content (BMC) losses. Bone ash was conserved in the HS n‐3 PUFA fed groups compared to control HS mice, which supports the protection of BMC/body wt also found in the n‐3 fed groups. There was no additional protection of bone in the high n‐3 diet group compared to the moderate group. Based on this study in mice and other investigations with rodents, the n‐3 PUFA appear to attenuate the physiological changes that occur with HS. Supported by USDA funds.

Chromium supplement inhibits skeletal muscle atrophy in hindlimb-suspended mice

Skeletal muscle atrophy and whole-body glucose intolerance are consequences of muscle disuse associated with conditions leading to prolonged bed rest. Nutritional supplementation with chromium has been shown to prevent weight loss and improve glucose tolerance in malnourished subjects on long-term total parenteral nutrition. The objective of this study was to evaluate the effect of oral supplementation with a novel chromium complex, chromium ( d-phenylalanine) 3 [Cr( d-phe) 3] at 45 μg/kg/day for 5 weeks, on skeletal muscle atrophy and glucose intolerance in a hindlimb suspension mouse model. Hindlimb-suspended mice exhibited reduced skeletal muscle fiber size and enhanced whole-body glucose intolerance, both of which were reversed by chromium treatment. The inhibition of skeletal muscle atrophy by chromium was associated with reductions in the ubiquitination ligase atrogin-1/muscle atrophy F-box, which is elevated in hindlimb-suspended mice. Neither hindlimb suspension nor chromium treatment altered the protein levels of the myostatin, phospho-Forkhead box O-1 and mammalian target of rapamycin. Chromium-treated animals exhibited elevated Akt ( Homo sapiens v-akt murine thymoma viral oncogene homolog) phosphorylation in their skeletal muscle, with no change observed in the levels of activated JNK (c-Jun N-terminal kinase). Thus, these data suggest that nutritional supplementation with chromium may have potential therapeutic benefits in minimizing skeletal muscle atrophy associated with long periods of muscle disuse.

Development of a low-dose anti-resorptive drug regimen reveals synergistic suppression of bone formation when coupled with disuse

Safe and effective countermeasures to spaceflight-induced osteoporosis are required to mitigate the potential for mission-critical fractures and ensure long-term bone health in astronauts. Two anti-resorptive drugs, the bisphosphonate zoledronic acid (ZOL) and the anti-receptor activator of NF-kappaB ligand protein osteoprotegerin (OPG), were investigated to find the minimum, comparable doses that yield a maximal increase in bone quality, while minimizing deleterious effects on turnover and mineralization. Through a series of five trials in normally loaded female mice (n = 56/trial), analysis of trabecular volume fraction and connectivity using microcomputed tomography, along with biomechanical testing, quantitative histomorphometry, and compositional analysis, was used to select 45 microg/kg ZOL and 500 microg/kg OPG as doses that satisfy these criteria. These doses were then examined for their ability to mitigate bone loss following short-term unloading through hindlimb suspension (HLS). Seventy-two mice were prophylactically administered ZOL, OPG, or PBS and assigned to loaded control or 2-wk HLS groups (n = 12 for each of 6 groups). Both anti-resorptives were able to preserve trabecular microarchitecture and femoral elastic and maximum force in HLS mice (+30-40% ZOL/OPG vs. PBS). In HLS mice, anti-resorptive dosing reduced resorption perimeter at the femoral endocortical surface by 30% vs. PBS. In loaded control mice, anti-resorptives produced no change in bone formation rate; however, reductions in bone formation rate brought about by HLS were exacerbated by anti-resorptive treatment, suggesting synergistic inhibition of osteoblasts during disuse. Refined anti-resorptive dosing will tend to target countermeasures to the period of disuse, resulting in faster recovery and less adverse effects for astronauts.

Evidences of apoptosis during the early phases of soleus muscle atrophy in hindlimb suspended mice

The purpose of this study was to investigate the occurrence and time-course of apoptosis in soleus skeletal muscle during the first 48 hours of unloading. Fifty Charles River mice were randomly divided into five groups (n=10 each) according to the time of hindlimb suspension (HS). Mice were suspended for 0 (Control), 6 (6HS), 12 (12HS), 24 (24HS), and 48 hours (48HS). Soleus muscle atrophy was confirmed by a significant decrease of 20 % in muscle-wet weight and of 5 % in the ratio protein concentration/muscle wet-weight observed after 48 hours of unloading. The apoptotic index, the AIF (apoptosis-inducing factor) and p53 expression presented their uppermost value (304 %, 241 % and 246 %, respectively) at 24HS, and were preceded by the highest activity of caspase-3 and -8 at 12HS (170 % and 218 %, respectively) and of Bax/Bcl-2 content at 6HS (160 %). There were no marked ultrastructural alterations until 24 hours of simulated weightlessness. Lysosomal autophagic activity and infiltration of phagocytic cells were observed at 24HS and 48HS and might have contributed to the degenerative changes noticed in both groups. Though not consistently supported by morphological evidences, the biochemical parameters sustain the concept that the occurrence of apoptosis parallels the soleus atrophic response in its early phase.

Mergla K + channel induces skeletal muscle atrophy by activating the ubiquitin proteasome pathway

Skeletal muscle atrophy results from an imbalance in protein degradation and protein synthesis and occurs in response to injury, various disease states, disuse, and normal aging. Current treatments for this debilitating condition are inadequate. More information about mechanisms involved in the onset and progression of muscle atrophy is necessary for development of more effective therapies. Here we show that expression of the mouse ether-a-go-go related gene (Merg1a) K+ channel is up-regulated in skeletal muscle of mice experiencing atrophy as a result of both malignant tumor expression and disuse. Further, ectopic expression of Merg1a in vivo induces atrophy in healthy wt-bearing mice, while expression of a dysfunctional Merg1a mutant suppresses atrophy in hindlimb-suspended mice. Treatment of hindlimb-suspended mice with astemizole, a known Merg1a channel blocker, inhibits atrophy in these animals. Importantly, in vivo expression of Merg1a in mouse skeletal muscle activates the ubiquitin proteasome pathway that is responsible for the majority of protein degradation that causes muscle atrophy, yet expression of a dysfunctional Merg1a mutant decreases levels of ubiquitin-proteasome proteolysis. Thus, expression of Merg1a likely initiates atrophy by activating ubiquitin-proteasome proteolysis. This gene and its product are potential targets for prevention and treatment of muscle atrophy.

COX-2 is necessary for venous ligation-mediated bone adaptation in mice

In osteoblasts, cyclooxygenase 2 (COX-2) is the major isozyme responsible for production of prostaglandins. Prostaglandins are local mediators of bone resorption and formation and are known to be involved in bone's adaptive response to fluid shear stress (FSS). We have previously described a model of trabecular bone loss in hindlimb-suspended mice and rats and demonstrated partial protection from osteopenia by ligation of the femoral vein. The increased FSS resulting from this ligation drove bone adaptation in the absence of mechanical loading. In this study, we investigated the role of COX-2 in this adaptive response to FSS by use of COX-2 knockout mice. COX-2 knockout (“KO”), COX-2 heterozygote (“HET”), and COX-2 wild-type (“WT”) animals all lost comparable amounts of trabecular bone from sham-operated limbs as a result of suspension. In WT mice, loss of trabecular BMD in the venous-ligated limb was significantly less than that of the sham-operated limb; this effect, however, was not seen in KO or HET mice. Percentage gain in femoral periosteal circumference was greater in the ligated limb than the sham-operated limb for WT mice. KO and HET mice already possess femora of larger periosteal circumference than their WT littermates and ligation in these bones did not result in an increase in perimeter relative to sham. Histomorphometry on embedded bones revealed thinner cortices and less mineralizing perimeter in KO femora than controls. In conclusion, this is the first in vivo study to show that fluid-flow-mediated bone adaptation, independent of mechanical strain, is COX-2 dependent.

Magnetic resonance microscopy of morphological alterations in mouse trabecular bone structure under conditions of simulated microgravity.

This work describes the use of magnetic resonance (MR) microscopy to examine changes in tibial trabecular bone structure in mice following 28 days of hindlimb suspension, a model simulating the effects of microgravity in rodents. In this first MR study involving mice, analysis of 3D images showed that apparent bone volume fraction, trabecular number, and trabecular thickness were decreased, and apparent trabecular spacing increased, significantly (P < 0.05) in hindlimb-suspended mice compared to controls. These changes agreed well with light microscopy measurements from an independent study and also with actual spaceflight experiments with rats.

Effect of hind-limb suspension on young and adult skeletal muscle: I. Normal mice

The purpose of this study was to determine the effect of hind-limb suspension (HS) on morphometric, histologic, and contractile characteristics of fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles in adult and immature mice. Hind-limb suspension for 2 weeks was used to produce atrophy in two groups of mice, ages 4 and 12 weeks, with nonsus-pended animals serving as controls. Young HS mice exhibited marked decreases in SOL weight, length, cross-sectional area (CSA), twitch and tetanic tensions, and rates of tension development and relaxation, with increases in fatigue resistance. HS reduced the diameter of both type I and IIA fibers, increased the percentage of type I fibers, and decreased the percentage of type IIA fibers in both young and adult SOL. Muscle weight, length, CSA, IIA and IIB fiber areas, and maximum rate of tetanic tension development were decreased in EDL of young HS mice; fatigue resistance and EDL half-relaxation times were increased. For most parameters evaluated, slow twitch muscle was more affected than fast twitch. HS affected contractile characteristics less than morphometric or histologic parameters. Rates of tension development and relaxation were the contractile parameters most affected by HS, and the time parameters of contraction were least affected. For all measurements young mice were more affected than adult mice.

Potential Nitric Oxide Treatment for Alleviating Muscle Atrophy in Hindlimb-Suspended Mice

Muscle atrophy is a debilitating condition that results from muscle disuse and aging. It is often experienced by people who are sedentary or elderly and cannot be effectively treated or prevented. Since nitric oxide (NO) activates muscle stem cells, this study aimed to test the hypothesis that treatment with a NO-donor drug, isosorbide dinitrate, during hindlimb suspension for 18 days, can alleviate muscle atrophy. Muscles in unloaded lower and weight-bearing upper limbs were weighed to study muscle loss. Western blots were used to measure protein levels in experimental (suspended and treated-suspended) and age-matched control groups of young (3 weeks old) and adult (6-8 weeks old) mice. Protein levels of atrogin-1, myostatin, and myosin heavy chain isoforms were analyzed (relative to ß-actin) for changes expected with muscle atrophy. Analysis of variance was used to determine if changes were statistically significant. The weight of unloaded muscles (quadriceps and soleus) in NO-treated-suspended mice tended to increase towards that of the control mice, compared to the atrophy after suspension without treatment. In the same muscles of young and adult suspended mice, NO treatment decreased atrogin-1, a negative regulator of muscle mass (p&lt;0.05 for quadriceps). A precursor protein of myostatin, an inhibitor of myogenesis, showed the same trend. Results suggest that NO treatment has the potential to attenuate muscle-disuse atrophy, with possible significance to preventing atrophy with aging or space travel. This research is supported by the University of Manitoba Research Grant Program (JEA) and NSERC Undergraduate Student Research Award (AZ).