Is limb immobilization a model of muscle disuse?

Limb immobilization is often used as a model of disuse-atrophy of skeletal muscle. Because the degree of atrophy is somewhat dependent on the length at which the muscle is immobilized, information regarding the effect of immobilization at various joint positions on the electrical activity of slow and fast muscles should provide a better understanding of the role of “use-disuse” in determining the degree of atrophy. In this study, the soleus muscle (SOL) and medial gastrocnemius muscle (MG) of adult female rats were chronically implanted with bipolar recording electrodes. The hind limbs were immobilized bilaterally using an external brace such that the muscles were fixed in a shortened, neutral, or lengthened position. Electromyograms (EMGs) of the SOL and MG were recorded simultaneously during a 15-min period each hour for 24 consecutive hours 3 and 9 days after electrode implantation, and 7, 17, and 28 days after immobilization. The weights of both muscles decreased significantly (P < 0.05) when fixed in a shortened or neutral, but not in a lengthened position after 28 days. As indicated by the 24-h integrated EMG (IEMG), the SOL, which was more active than the MG in unrestrained rats, showed the greatest decrease in postimmobilization activity. After 28 days of immobilization in a shortened position, the IEMG of the SOL and MG was significantly reduced (P < 0.05) by 77 and 50%, respectively. The IEMG of the SOL immobilized in a neutral position decreased 50% whereas no change was seen in the MG. There was no significant change in the IEMG for the SOL or MG fixed in a lengthened position. These data suggest that the EMG was dependent on the type of muscle and the length of fixation. Furthermore, it appeared that the degree of atrophy was not related closely to changes in the IEMG suggesting that limb immobilization was not necessarily a model of “disuse”.     

Quantitative evaluation of electromyogram activity in rat extensor and flexor muscles immobilized at different lengths

Because immobilization of muscles in the "long" position mitigates the effects of inactivity and rapid wasting occurs when muscles are immobilized in the "short" position, a study was made of the EMG activity in the soleus (SOL)--an extensor muscle--and the tibialis anterior (TA)--a flexor muscle--in order to clarify the possible role of muscle function in modifying the course of disuse atrophy. EMG activity was recorded in the SOL and TA muscles in adult rats in which the ankle had been immobilized in a plaster cast either in plantar flexion or dorsiflexion. The number of action potentials per minute in samples of the EMG activity from control and immobilized muscles was assessed before, for 10 days during immobilization, and up to 9 days after removal of the cast. Immobilization in the short position (plantar flexion) led to a dramatic reduction in the EMG activity of the SOL (to 10% of the control). On the other hand, fixation of the SOL in the long position was without effect upon resting EMG activity. In the TA, EMG activity was exclusively phasic in character and corresponded to about 3% of that of the SOL. Neither the fixation of the ankle in plantar flexion nor dorsiflexion had any appreciable effect upon EMG activity in the TA. We conclude that, because immobilization in the lengthened position does not increase EMG activity in either extensor or flexor muscles, passive stretch appears to be the factor mainly responsible for mitigating the effects of disuse in this situation. On the other hand, when a typical extensor muscle (SOL) is immobilized in the shortened position and undergoes rapid wasting, an accessory role of decreased activity cannot be excluded.     

Core myofibers and related alterations induced in rats' soleus muscle by immobilization in shortened position

Experimental induction of core myofibers by tenotomy or local tetanus suggests that mechanical factors such as muscle tension loss, shortening or immobilization may play a role in core fiber formation. To test this hypothesis, we investigated the morphologic alterations induced in soleus (SOL) and extensor digitorum longus (EDL) muscles following immobilization of rats' hindlimb in various positions. The SOL and EDL muscles were immobilized in either shortened or lengthened state by applying wire-meshed plaster cast for 1, 2 and 3 weeks. The muscles were dissected out, measured, weighed and examined by histochemistry and electron microscopy. Gross atrophy was noted in all muscles but was greatest in shortened SOL. The SOL atrophy was diffuse and associated with relative increase in type 2 fibers. In EDL, the atrophy selectively involved fibers with low oxidative enzyme activity. Core myofibers were seen mainly in shortened SOL and consisted of myofibrillar derangement, loss of myofilaments and streaming of Z bands. The preferential involvement of shortened SOL (tonic, fatigue-resistant, slow-twitch muscle) suggests that the functional length, loss of tension subsequent to shortening and intrinsic biochemical properties of the muscle are important in core fiber formation.     

Architectural alterations of rat hind-limb skeletal muscles immobilized at different lengths

Modifications in architectural profiles of the rat soleus (Sol), medial gastrocnemius (MG), and tibialis anterior (TA) muscles were studied after hind-limb immobilization with an external brace. The muscles were chronically lengthened, shortened, or maintained at approximately resting length (neutral) for 4 weeks, when muscle weight and length, and cross-sectional areas of fibers (F-XSA) and whole muscle were measured. Compared with control muscles, the Sol and MG immobilized in a lengthened position were able to maintain muscle weight better than when immobilized neutrally (approximately 85 versus 55% of control weight) due, in part, to elongation of muscle fibers of the Sol and MG (123 and 111% of control length, respectively). In addition, the F-XSAs of lengthened Sol and MG muscles were 72 and 20% greater, respectively, compared with neutrally fixed muscle fibers. Immobilization in a shortened position resulted in the most extreme muscle atrophy due to significantly shorter fibers (86% of control Sol; 74% of control MG) and reduced F-XSA (42% of control Sol; 42% of control MG). In contrast to the Sol and MG, muscle weight, fiber length, and F-XSA of the TA immobilized with the ankle in extreme plantarflexion were not significantly different from neurtrally fixed muscle. That the TA was unaffected in this position is due presumably to a difference in the anatomic attachment of the distal tendon of this muscle, compared with that of the Sol and MG. These results suggest that for muscle affected by immobilization at extreme joint angles, chronic lengthening is a potent stimulus for elongation of muscle fibers and more normally maintained F-XSA. In contrast, the removal of this stimulus leads to extreme muscle atrophy which is manifested by reductions in both muscle fiber length and F-XSA.     

Effects of immobilizing a single muscle on the morphology and the activation of its muscle fibers

A single muscle of Wistar female rats, either soleus or peroneus longus, was immobilized by fixing its cut distal tendon to the bone during 8 weeks. We observed a transitory weight loss in both muscles; the mean fiber cross-sectional area (CSA) showed a reduction at day 30, followed by an increase at day 60. The time course of the activation of the immobilized muscle was evaluated by recording the chronic electromyographic (EMG) activity during short periods (1 min every other day) of treadmill locomotion. During immobilization, the integrated EMG amplitude of the soleus increased, reaching a maximum at 4 weeks, but remained close to control values during 8 weeks for the peroneus. The median frequency (MF) of the power density spectrum of the soleus EMG was not statistically different between immobilized and control muscles, while MF of the immobilized peroneus EMG was permanently higher than that of control muscles. This suggests two different modes of adaptation in motor unit command, depending on the muscle profile, which could be concomitant with the restoration of muscle fibers CSA after 8 weeks.     

Role of nerve and tension in maturation of posthatching slow-tonic muscle in chicken

The role of motor innervation and muscle tension in the posthatching maturation of the slow-tonic anterior latissimus dorsi (ALD) muscle of the chicken has been investigated. Modification of the muscle tension was obtained either by maintaining ALD in a shortened state or by stretching, after or without denervation. In denervated as well as in innervated ALD, shortening resulted in atrophy and inhibition of developmental change in muscle fiber population. In contrast, stretch causes hypertrophy, transformation of all 3B fibers, increase in SM2 isomyosin expression, and decrease in Ca2+-activated myosin ATPase in innervated or denervated ALD. On the other hand oxidative activity in ALD fibers was strikingly reduced after denervation even in presence of stretch-induced hypertrophy. This study suggests that a passive stretch can be involved in some, but not all, changes in ALD characteristics occurring after denervation and may be also involved in normal posthatching development of the slow-tonic muscle. Possible clinical implications of these results in relation to treatments for preventing muscle atrophy resulting from immobilization or disuse are suggested.     

Effect of hind-limb suspension on young and adult skeletal muscle. II. Dystrophic mice

Disuse atrophy induced by limb immobilization reportedly protects dystrophic mouse muscle from histopathological changes. This study was conducted to determine whether disuse atrophy induced by hind-limb suspension (HS) limits the histopathology and contractile abnormalities typically observed in the dystrophic mouse. Two weeks of hind-limb suspension were applied to dystrophic mice (line 129B6F1) at two ages, 4 weeks (6 mice) and 12 weeks (8 mice). Thirty-one untreated dystrophics served as controls. In general, HS exaggerated the dystrophic signs, especially in the younger mice; it reduced animal weight, muscle weight, maximum tetanic and twitch tensions, and rates of tetanic and twitch tension development. HS further slowed the contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles, and increased their fatigue resistance. HS reduced the size of type I and IIA fibers in the 6-week SOL and EDL, but not in the 14-week muscles. HS produced a preferential atrophy of SOL type I fibers, with a parallel increase in type IIA fibers. However, it did not alleviate the fiber size variability, degree of necrosis, central nucleation, inflammation, or muscle fibrosis in dystrophic muscles. These data demonstrate that disuse by hind-limb suspension does not prevent the histopathological deterioration or loss of muscle function in 6- and 14-week dystrophic mice.     

Induction of spheroid cytoplasmic bodies in a rat muscle by local tetanus

The term "cytoplasmic body" or "spheroid body" myopathy refers to a heterogeneous group of familial or sporadic diseases characterized primarily by the presence of abundant spheroid or cytoplasmic bodies in the muscles. The morphogenesis of these inclusions remains unclear. This article describes the induction and evolution of spheroid cytoplasmic bodies (SCBs) in the rat plantaris muscle (PL) with local tetanus, which was induced in rats by the injection of a minute amount of tetanus toxin. In contrast to the tetanized soleus muscle (SOL), which developed core fibers (central cores, minicore, target fiber, targetoid fiber, and rods), the tetanized PL produced numerous SCBs with a predictable time course. They were induced in both type 1 and 2 fibers of PL, which is composed predominantly (95%) of type 2 fibers, in contrast to SOL (85% type 1 fibers). Factors inducing SCBs may include immobilization, shortening, intact innervation, and disuse atrophy.     

Effect of nerve extract on atrophy of denervated or immobilized muscles

Changes in denervated muscles are due to disuse caused by paralysis of the muscle and the loss of special neurotrophic substances. We determined the relative roles of these two factors in the production of atrophy in denervated rats' extensor digitorum longus (EDL) muscles. Muscles were denervated and/or immobilized (by fixation of the ankle) for 7 days. Some rats also received daily intramuscular injections of a saline extract of rats' sciatic nerves (2.0 mg protein/ml). Atrophy was assessed by measurement of wet weight, total protein, and cross-sectional areas of types IIA and IIB fibers (in sections stained for ATPase). Both denervation and immobilization produced significant decreases in weight, protein, and areas of fiber. The group of rats with denervated EDL muscles had significantly greater atrophy than the group with immobilized muscles. In another group, denervated EDL muscles had significantly greater atrophy than contralateral muscles which were immobilized. However, when denervated muscles were injected with nerve extract, they did not differ significantly from contralateral, noninjected, immobilized muscles. Comparisons of the group of rats in which one EDL was denervated with groups in which one muscle was immobilized or was denervated and injected with nerve extract, indicated that loss of trophic influence was responsible for about 40% of the decreases in wet weight, total protein, and cross-sectional area of type IIB fibers, and the remaining 60% was due to disuse. Loss of trophic influence was responsible for only about 5% of the atrophy of denervated type IIA fibers. Therefore, inactivity and loss of neurotrophic influence were responsible for the atrophy which occurred in denervated skeletal muscles, and these two factors influenced the two types of fiber differently. The component of denervation atrophy due to loss of trophic influence could be completely prevented by injection of substances extracted from peripheral nerves.     

Noninvasive monitoring of muscle damage during reloading following limb disuse

Cast immobilization causes skeletal muscle disuse atrophy and an increased susceptibility to muscle damage. The objective of this study was to explore the utility of noninvasive magnetic resonance (MR) imaging to monitor muscle damage in the lower hindlimb muscles of the mouse during reloading following cast immobilization and to compare the findings in different muscles. The hindlimbs of C57BL6 mice were immobilized for 2 weeks in plantarflexion using a bilateral casting model. Following immobilization the mice were allowed to reambulate and muscle damage was monitored at different times. Cage-restricted reloading following cast immobilization induced a significant shift (P < 0.0001) in the transverse (T2) relaxation characteristics of the postural slow-twitch soleus muscle, but not in the neighboring gastrocnemius. Soleus T2 values peaked at 2 days of reloading. Muscle-specific changes in MR T2 relaxation properties correlated with uptake of Evans blue dye, a histological marker of muscle damage. This study demonstrates that T2 MR imaging can be implemented to monitor noninvasively and sequentially muscle-specific damage during reloading following limb disuse.     

Preserving sarcomere number after tenotomy requires stretch and contraction

Introduction: Passive stretch therapy is utilized to improve the range of motion of chronically shortened muscles. However, human studies show conflicting results as whether passive stretch is clinically effective. Methods: The soleus muscles of adult rats were tenotomized to induce muscle shortening adaptation. Muscles included were non‐treated normal, subjected to daily static stretch, or lengthened and isometrically contracted for 20 min/day. Muscle fiber structure was analyzed histochemically. Sarcomeres per millimeter length were counted to assess the effect of treatment. Results: Passive stretch significantly reduced central core lesion formation, but sarcomere loss was not prevented. The addition of isometric contraction during static stretch significantly (P < 0.001) reduced sarcomere loss. Conclusions: Passive stretch alone does not prevent shortening adaptation. Contraction is required in combination with stretch to preserve the number of sarcomeres in series. The combination of stretch and contraction is necessary to maintain proper muscle fiber length. Muscle Nerve, 2012     

New mouse model of skeletal muscle atrophy using spiral wire immobilization

Introduction: Disuse‐induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. Methods: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. Results: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin‐1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse‐induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. Conclusions: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions. Muscle Nerve 54 : 788–791, 2016     

Apoptotic changes in a full-lengthened immobilization model of rat soleus muscle

Introduction: Lengthened immobilization may prevent muscle shortening, and help maintain normal muscle length. However, its apoptotic effects remain unclear. We evaluated the effects of long-term immobilization on apoptotic proteins. Methods: Rat soleus muscles were immobilized by casting in a neutral (NEUT) or lengthened (LENG) position for 21 days. We evaluated dynamic weight load and muscle atrophy following the 21-day period using hematoxylin and eosin staining. We measured Bax (pro-apoptotic Bcl-2 family member), MyoD (myogenic differentiation factor D), MYH (myosin heavy chain), and cleaved poly(ADP-ribose)polymerase levels and examined apoptotic nucleus expression. Results: Decreased dynamic weight load and muscle atrophy changes were observed in LENG. Both NEUT and LENG showed significantly reduced levels of MYH. LENG showed a significant increase in Bax and MyoD expression as well as in the number of apoptotic nuclei. Conclusions: Long-term lengthened immobilization may increase apoptotic changes and decrease muscle formation proteins in muscle. Muscle Nerve 59 :263–269, 2019     

Functional effects of uridine triphosphate on the atrophied soleus muscle of rat after unloading

The purposes of the study were to determine the effects of a pyrimidine nucleotide, the uridine triphosphate (UTP), on the contractile and histochemical properties of the soleus (SOL) muscle following disuse atrophy due to hindlimb unloading (HU) hypokinesia. UTP was injected either during the HU period (2 weeks) or later during the recovery period. In this latter condition, contractile and histochemical properties were studied after 5, 8, 11, and 15 days of spontaneous recovery. HU induced decreases in the SOL weight, force output (twitch and tetanic tensions), time to peak tension during the twitch, and the percentage of type I fibers. The injection of UTP during the HU period did not counteract the modification in speed-related properties, but the decrease in force output was partly counteracted and the proportion of type II C fibers was increased. When UTP was injected during the recovery periods, force-related properties recovered more rapidly. These results suggest that UTP may reduce the loss of force induced by atrophy. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 172–178, 1997.     

Effects of short- and long-term rat hind limb immobilization on spinal cord insulin-like growth factor-I and its receptor

In this study we investigated changes in the spinal cord insulin-like growth factor-I peptide (IGF-I) and its receptors (IGF-IR) after hind limb immobilization for 5 days, 2, 4, and 8 weeks. Moreover, effects on IGF-I and nicotinic cholinergic receptors (nAChRs) in two types of skeletal muscle were also investigated. IGF-I levels were measured by radioimmunoassay (RIA) whereas IGF-IR and nAChRs were measured by quantitative receptor autoradiography. Spinal cord IGF-I levels decreased significantly after 5 days, 2 and 4 weeks of immobilization, whereas IGF-IR increased significantly after 4 and 8 weeks compared to controls. In skeletal muscles, nAChRs increased significantly after 5 days and 2 weeks in the soleus (SOL) and tibialis anterior (TIB) muscles, respectively, and continued up to 8 weeks in both muscles. IGF-I concentration decrease significantly after 4 and 8 weeks in the SOL and TIB muscles, respectively. Despite the normal levels of IGF-I in both muscles at the early time points (5 days and 2 weeks), low levels of IGF-I were observed concurrently in the spinal cord ipsilateral to the immobilized limb. Our findings suggest that the early decrease in the IGF-I level and the late upregulation in the IGF-IR in the spinal cord might represent a nervous system response to disuse.     

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 nonsuspended 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.     

Repetitive stretch suppresses denervation‐induced atrophy of soleus muscle in rats

This study was conducted to examine whether stretch‐related mechanical loading on skeletal muscle can suppress denervation‐induced muscle atrophy, and if so, to depict the underlying molecular mechanism. Denervated rat soleus muscle was repetitively stretched (every 5 s for 15 min/day) for 2 weeks. Histochemical analysis showed that the cross‐sectional area of denervated soleus muscle fibers with repetitive stretching was significantly larger than that of control denervated muscle (P < 0.05). We then examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) cascade in the suppressive effects of repetitive stretching on muscle atrophy. Repetitive stretching significantly increased the Akt, p70S6K, and 4E‐BP1 phosphorylation in denervated soleus muscle compared to controls (P < 0.05). Furthermore, repetitive stretching‐induced suppression of muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These results indicate that denervation‐induced muscle atrophy is significantly suppressed by stretch‐related mechanical loading of the muscle through upregulation of the Akt/mTOR signal pathway. Muscle Nerve, 2009     

Lack of caspase‐3 attenuates immobilization‐induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

Introduction: Immobilization by casting induces disuse muscle atrophy (DMA). Methods: Using wild type (WT) and caspase‐3 knockout (KO) mice, we evaluated the effect of caspase‐3 on muscle mass, apoptosis, and inflammation during DMA. Results: Caspase‐3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase‐3 decreased immobilization‐induced increased apoptotic myonuclei (3.2‐fold) and macrophage infiltration (2.2‐fold) in soleus muscle and attenuated increased monocyte chemoattractant protein‐1 mRNA expression (2‐fold in KO vs. 18‐fold in WT) in gastrocnemius. Conclusions: Caspase‐3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. Muscle Nerve 47: 711–721, 2013     

Alteration of spinal cord IGF-I receptors and skeletal muscle IGF-I after hind-limb immobilization in the rat.

The effects of 4 weeks' hind-limb immobilization on the spinal cord insulin-like growth factor-I (IGF-I) receptors and skeletal muscle IGF-I level was investigated in rats. Quantitative receptor autoradiography using [125I]IGF-I as a ligand was performed to measure IGF-I receptors in cryosections from the lumbar region of the spinal cord. IGF-I receptor levels were significantly higher in all spinal cord laminae on the side ipsilateral to the immobilized limb than in the same spinal level of the controls. Using radioimmunoassay (RIA), IGF-I levels were significantly low in the soleus (SOL), but not the tibialis anterior (TIB) muscles, compared to the controls. The enhancement of the spinal cord IGF-I receptors after hind-limb immobilization may constitute part of the nervous system response to disuse.     

Clenbuterol reduces soleus muscle fatigue during disuse in aged rats

High levels of clenbuterol have been shown to preserve muscle mass and function during disuse. In this study we report that a low dose of clenbuterol (10 microg/kg per day) lessened the loss of in situ soleus muscle isometric force normalized to wet muscle weight (P(o)/g wet weight) by 8% and reduced isometric fatigue by approximately 30% in senescent rats after 21 days of hindlimb suspension (HS). Clenbuterol did not reduce the loss of relative force in the soleus of adult rats or the plantaris of old or adult rats. Furthermore, clenbuterol failed to improve muscle force or isometric fatigue in the soleus of adult rats or in the plantaris of either age group after HS. We conclude that low levels of clenbuterol reduce muscle fatigue in slow muscles during disuse and this beta-agonist may also have therapeutic value for reducing fatigue in slow muscles (e.g., postural muscles) in the elderly during disuse.