Weeks Of Denervation Research Articles

EFFECT OF EXTREMELY LOW-FREQUENCY ELECTROMAGNETIC FIELDS ON PROMOTING MYOGENESIS IN THE EARLY PROCESS OF DENERVATED MUSCLEATROPHY

Objective: To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on myogenesis during the early stage of denervated muscle atrophy in Sprague-Dawley (SD) rats. Methods: a total of 36 female SD rats were fed in cages and divided into three groups: the normal control group, the muscle atrophy group and the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group. The rats in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group received a magnetic field intervention for 2[Formula: see text]h per day. The posterior muscle groups of the lower limbs of rats were sampled on the first, second, third and fourth weeks for evaluating the underlying changes. The harvested tissues were stained with hematoxylin–eosin (HE) dye to observe the morphology of the muscle atrophy and to measure the muscle fiber cross-sectional area ratio. A reverse transcription polymerase chain reaction was used to detect the expression of Myf5 mRNA in each group. The myogenesis-related proteins in the harvested muscle tissue were detected by western blot assay. Results: (1) HE staining showed that with the extension of denervation time, the structural disorder of muscle fibers was aggravated, and in the second week of the denervated muscle atrophy, muscle fiber cross-sectional area tended to increase in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group. Overall muscle atrophy was inevitable. (2) Results of RT-PCR showed that the expression of Myf5 mRNA in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly high in the second week of denervation, and the difference was statistically significant compared with the muscle atrophy group (*[Formula: see text]). (3) Western blot analysis showed that the expression of MYF5 proteins in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly higher than that in the muscle atrophy group (**[Formula: see text]) in the second week of denervation. Further, the expression of MYOG protein in the 10[Formula: see text]Hz/1.5[Formula: see text]mT magnetic field group was significantly higher than that in the muscle atrophy group (****[Formula: see text]). Conclusions: 10[Formula: see text]Hz/1.5[Formula: see text]mT ELF-EMF can significantly promote myogenesis during early denervated muscle atrophy in SD rats.

QS9: Determining the Critical Time of Chronic Schwann Cell Denervation on Functional Recovery and Rna Expression

Purpose:There is poor functional recovery following delayed peripheral nerve repair since both muscle and Schwann cells (SC) undergo denervation atrophy. We investigated the specific temporal effect of nerve/SC denervation on recovery as well as changes in RNA expression in the nerves that may elucidate changes in recovery potential. We hypothesized that functional recovery would be worse after prolonged nerve/SC denervation and that the expression profiles would differ.Methods:Our study was conducted using a forelimb model in adult Lewis rats. Each animal underwent unilateral forelimb denervation of 8, 12, 16, or 24 weeks duration. In the functional recovery arm of the study, the ulnar nerve was denervated proximally or a sham surgery was performed. After the denervation period had elapsed, an in situ nerve transfer of median to ulnar to median nerve was performed. Functional recovery was then measured by stimulated grip strength weekly for 12 weeks. In the RNA expression arm, median and ulnar nerves were denervated. The same time points were used with the addition of a 1-week denervation group. After the denervation period, the median and ulnar nerves were harvested bilaterally. To create a comprehensive RNA-Seq dataset, the median nerve, with an average length of 3 cm, was homogenized and RNA was purified. RNA-sequencing was carried out using TrueSeq RiboZero gold kit. Samples were analyzed through FastQC, aligned to reference genome using STAR and quantified as transcripts per million (TPM) using Salmon. Principle component analysis was performed, followed by differential gene analysis using a linear mixed effects model to control for the control nerves being from the same animals.Results:Functional recovery was statistically significantly different depending on the duration of nerve/SC denervation (P<0.01). Post-hoc tests were non-significant between the positive control and denervation periods of 8 (P=1.00) or 12 weeks (P=0.85). In contrast, when the ulnar nerve had been denervated for 16 or 24 weeks, final grip strength was significantly reduced compared to no denervation, 8, and 12 weeks of denervation (P<0.01). RNA sequence analysis showed significant differences in up- and downregulated genes depending on denervation status and duration of denervation. At a false-discovery rate >0.05, we identified 1624 genes differentially expressed, of which 327 genes were upregulated and rest (1297 genes) downregulated with denervation.Conclusions:Prolonged nerve/SC denervation of more than 12 weeks resulted in significantly worse functional recovery. RNA sequencing demonstrated that not only were there many genes differentially expressed, but these appear to vary with duration of denervation as well. Further investigation into the specific genes and their changes over time will allow us to know why recovery potential is decreased and targets for interventions to improve recovery.

Assessing the Dynamic Mitochondrial Fission and Fusion Events in Skeletal Muscle in vivo

Skeletal muscle plays an important role in systemic metabolism and overall health. Within skeletal muscle, there is a decline in mitochondrial function with age. Under normal healthy conditions, mitochondria remodel via two dynamic processes, mitochondrial protein turnover and mitochondrial dynamics, which includes mitochondrial fission and fusion events. While capturing the dynamic nature of mitochondrial protein turnover in skeletal muscle is now possible, assessing mitochondrial fission and fusion relies on snapshots of protein markers from muscle homogenate at distinct timepoints. Recent technical advancements allow for measurement of mitochondrial dynamics in skeletal muscle in vitro; however, rates of mitochondrial dynamics in other tissues are significantly slower in vivo compared to in vitro conditions. Therefore, the purpose of this study was to develop novel imaging methods to assess mitochondrial dynamics in vivo. Our hypothesis was that we could capture and assess in vivo mitochondrial fission/fusion events using a novel imaging approach. Using multiphoton microscopy, we activated a mitochondrial-targeted photo-activated GFP that was electroporated into the tibialis anterior (TA) muscle of 5 adult C57Bl6 mice to assess the rates of mitochondrial fission and fusion events. In vivo mitochondrial fission and fusion rates were about 50 times slower in comparison to previously reported rates in in vitro models. Due to the slower than expected rates of mitochondrial fission/fusion events in skeletal muscle in vivo, we used an additional imaging approach to explore changes mitochondrial morphology over more prolonged periods of time. To do so, we imaged control and denervated TA muscles of 5 adult C57Bl6 mice using an electroporated mitochondrial-targeted GFP (mitochondrial matrix) and TOM20_tdTomato (outer mitochondrial matrix). We determined that two weeks of denervation decreased the cross-sectional area that was co-stained in the denervated limb compared to a control limb, suggesting a decrease in cristae structure. In addition, two weeks of denervation decreased the cross-sectional area of the population of subsarcolemmal mitochondria in the denervated limb compared to a control limb. Together, our data emphasizes the importance of assessing mitochondrial dynamics in vivo. Additionally, changes to mitochondrial morphology in skeletal muscle fibers are regional, which stresses the importance of assessing mitochondrial subpopulations.

The effect of denervation on myosin chaperone UNC45B in mice skeletal muscle

I. Background UNC45B, a myosin chaperone, affects myosin expression in C.elegans and has been reported to decrease with age in rats and humans. Therefore, it is thought that the decrease in UNC45B in the elderly may lead to lowering myosin level and the inability to regulate muscle quality, resulting in sarcopenia, an age-related loss of skeletal muscle mass. However, the mechanism by which UNC45B decreases with age remains unclear. In sarcopenia, atrophy occurs mainly in fast-twitch muscle fibers. Similarly, denervation leads to muscle atrophy mainly in fast-twitch muscle fibers, and therefore, it has been used as a model for sarcopenia. If UNC45B is reduced by muscle inactivity due to aging, it would be also reduced by inactivity due to denervation. Thus, we examined UNC45B expression in denervated muscle. II. Methods The right hindlimb muscles of 13-week-old male C57BL/6J mice were denervated by cutting and removing a 2 mm segment of the sciatic nerve, while the left hindlimb muscles served as sham-operated control. Two weeks after denervation, the gastrocnemius muscle was removed and weighed. Then the solution containing the homogenized muscle was centrifuged at 10000 g and the supernatant was used for Western blotting. Ⅲ. Results The muscle weight of the gastrocnemius muscle was decreased by about 50% and that myosin level was also significantly decreased by denervation (p<0.01), but the amount of UNC45B protein did not differ from that of the control leg. Although muscle protein synthesis measured by the SUnSET method was not changed by denervation, the amount of ubiquitinated protein was increased (p<0.001). Total and phosphorylation levels of Akt (an upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) that facilitates protein synthesis), 4EBP1, and rpS6 (downstream targets of mTORC1) were increased by denervation (p<0.01). p62, LC3-I, and LC3-II, markers of autophagy, were increased in the denervated muscle (p62: p<0.05, LC3-I, LC3-II: p<0.01). Heat shock proteins that also act as chaperones for myosin, HSP90 and HSP73 were increased by denervation (HSP90: p<0.001, HSP73: p<0.05), while HSP70 was decreased (p<0.05). IV. Discussion Two weeks of denervation resulted in a significant gastrocnemius muscle weight decrease. However, no change had observed in UNC45B levels, indicating that UNC45B may not be involved in muscle atrophy caused by denervation, and the age-related decrease in UNC45B may also be not due to a decreased muscle activity. Denervation stimulated the protein degradation system while it did not affect muscle protein synthesis, suggesting that denervation-induced muscle atrophy may cause by enhancement of the degradation system. V. Conclusion In this study, there was no decrease in UNC45B after 2 weeks of denervation, suggesting that the decrease in UNC45B due to aging is not directly related to the decrease in muscle activity.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Effect of External Calcium and other Divalent Cations on K+ Contractures in Denervated Slow Skeletal Muscle Fibers of the Frog

The influence of Ca2+ and other divalent cations on contractile responses of slow skeletal muscle fibers of the frog (Rana pipiens) under conditions of chronic denervation was investigated.Isometric tension was recorded from slow bundles of normal and denervated cruralis muscle in normal solution and in solutions with free calcium concentration solution or in solutions where other divalent cations (Sr2+, Ni2+, Co2+ or Mn2+) substituted for calcium. In the second week after nerve section, in Ca2+-free solutions, we observed that contractures (evoked from 40 to 80 mM-K+) of non-denervated muscles showed significantly higher tensions (p&lt;0.05), than those from denervated bundles. Likewise, in solutions where calcium was substituted by all divalent cations tested, with exception of Mn2+, the denervated bundles displayed lower tension than non-denervated, also in the second week of denervation. In this case, the Ca2+ substitution by Sr2+ caused the higher decrease in tension, followed by Co2+ and Ni2+, which were different to non-denervated bundles, as the lowest tension was developed by Mn2+, followed by Co2+, and then Ni2+ and Sr2+. After the third week, we observed a recovery in tension. These results suggest that denervation altering the binding capacity to divalent cations of the voltage sensor.

Tendinosis-like changes in denervated rat Achilles tendon

BackgroundTendon disorders are common and lead to significant disability and pain. Our knowledge of the ‘tennis elbow’, the ‘jumpers knee’, and Achilles tendinosis has increased over the years, but changes in denervated tendons is yet to be described in detail. The aim of the present study was to investigate the morphological and biochemical changes in tendon tissue following two weeks of denervation using a unilateral sciatic nerve transection model in rat Achilles tendons.MethodsTendons were compared with respect to cell number, nuclear roundness, and fiber structure. The non-denervated contralateral tendon served as a control. Also, the expression of neuromodulators such as substance P and its preferred receptor neurokinin-1 receptor, NK-1R, was evaluated using real-time qRT-PCR.ResultsOur results showed that denervated tendons expressed morphological changes such as hypercellularity; disfigured cells; disorganization of the collagen network; increased production of type III collagen; and increased expression of NK-1R.ConclusionTaken together these data provide new insights into the histopathology of denervated tendons showing that denervation causes somewhat similar changes in the Achilles tendon as does tendinosis in rats.

Time course of denervation-induced changes in gastrocnemius muscles of adult and old rats

Denervation leads to significant muscle atrophy, but it is less clear whether 1) loss of capillaries, fibre size and oxidative capacity decline in parallel and 2) the time course of these changes differs between young and old animals. To investigate this, we denervated the left gastrocnemius muscle for 1, 2 or 4 weeks, while the right muscle served as an internal control, in rats that were 5 or 25 months old at the end of the experiment. In the fast part of the gastrocnemius muscle, almost all atrophy had occurred after two weeks (42%) of denervation. Even after 4 weeks of denervation, there was no significant reduction in the oxidative capacity of the muscle. Significant capillary loss occurred only after 4 weeks of denervation (P < 0.001) that lagged behind and was less than proportional to the decrease in fibre size. Consequently, the capillary density was elevated (P < 0.001). The time course of these morphological changes was similar in the 5- and 25-month-old rats. Comparing these data with those previously published in the soleus muscle from the same animals show that the decrease in oxidative capacity and capillary rarefaction were more pronounced and occurred earlier than in the gastrocnemius muscle, respectively. The time course of capillary loss lagged behind the decrease in fibre size, and combined with the absence of denervation-induced changes in oxidative capacity this resulted in a muscle capillary supply in excess of that expected by the metabolism and fibre size at least during the first 4 weeks after denervation.

An evaluation of common markers of muscle denervation in denervated young-adult and old rat gastrocnemius muscle

A large part of age-related muscle wasting is due to incomplete reinnervation of fibres that have become denervated following motoneuron loss. Neural cell adhesion molecule (NCAM) and sodium channel NaV1.5 are considered markers for denervation, but the time course of changes in their expression following denervation has never been systematically evaluated in young-adult and old muscle. To assess the time course of denervation-induced changes in their expression, the left gastrocnemius muscle in 15 young-adult (5-month) and 10 old (25-month) male Wistar rats was denervated for 1, 2 or 4 weeks, while the right muscle served as an internal control. Sections were stained for α-bungarotoxin, to visualise the neuromuscular junctions, combined with NCAM, polysialylated NCAM (PSA-NCAM) or NaV1.5.In young-adult animals, denervation induced a transient decrease in junctional and cytoplasmic NCAM expression, while in the old NCAM expression was increased after 2 weeks. Cytoplasmic PSA-NCAM was increased in both young-adult and old fibres after 2 weeks denervation with a further increase after 4 weeks in the young only. The junctional PSA-NCAM was transiently increased or decreased in the young and old muscles, respectively. NaV1.5 expression decreased after 1 and 2 weeks of denervation in NaV1.5 in young muscle fibres before returning to control levels, whereas old muscle fibres displayed a transient increase after 1 week followed by a decrease and a return to control levels after 2 and 4 weeks respectively.In conclusion, NCAM and NaV1.5 are not unequivocally elevated with denervation and consequently are not adequate markers of fibre denervation.

Tumor prevention facilitates delayed transplant of stem cell-derived motoneurons.

ObjectiveNerve injuries resulting in prolonged periods of denervation result in poor recovery of motor function. We have previously shown that embryonic stem cell‐derived motoneurons transplanted at the time of transection into a peripheral nerve can functionally reinnervate muscle. For clinical relevance, we now focused on delaying transplantation to assess reinnervation after prolonged denervation.MethodsEmbryonic stem cell‐derived motoneurons were transplanted into the distal segments of transected tibial nerves in adult mice after prolonged denervation of 1–8 weeks. Twitch and tetanic forces were measured ex vivo 3 months posttransplantation. Tissue was harvested from the transplants for culture and immunohistochemical analysis.ResultsIn this delayed reinnervation model, teratocarcinomas developed in about one half of transplants. A residual multipotent cell population (~ 6% of cells) was found despite neural differentiation. Exposure to the alkylating drug mitomycin C eliminated this multipotent population in vitro while preserving motoneurons. Treating neural differentiated stem cells prior to delayed transplantation prevented tumor formation and resulted in twitch and tetanic forces similar to those in animals transplanted acutely after denervation.InterpretationDespite a neural differentiation protocol, embryonic stem cell‐derived motoneurons still carry a risk of tumorigenicity. Pretreating with an antimitotic agent leads to survival and functional muscle reinnervation if performed within 4 weeks of denervation in the mouse.

α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization

Homozygosity for a common null polymorphism (R577X) in the ACTN3 gene results in the absence of the fast fibre-specific protein, α-actinin-3 in ∼16% of humans worldwide. α-Actinin-3 deficiency is detrimental to optimal sprint performance and benefits endurance performance in elite athletes. In the general population, α-actinin-3 deficiency is associated with reduced muscle mass, strength and fast muscle fibre area, and poorer muscle function with age. The Actn3 knock-out (KO) mouse model mimics the human phenotype, with fast fibres showing a shift towards slow/oxidative metabolism without a change in myosin heavy chain (MyHC) isoform. We have recently shown that these changes are attributable to increased activity of the calcineurin-dependent signalling pathway in α-actinin-3 deficient muscle, resulting in enhanced response to exercise training. This led us to hypothesize that the Actn3 genotype influences muscle adaptation to disuse, irrespective of neural innervation. Separate cohorts of KO and wild-type mice underwent 2 weeks immobilization and 2 and 8 weeks of denervation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse, as measured by a change in MyHC isoform. KO mice had a lower threshold to switch from the predominantly fast to a slower muscle phenotype (in response to immobilization) and a higher threshold to switch to a faster muscle phenotype (in response to denervation). We propose that this change is mediated through baseline alterations in the calcineurin signalling pathway of Actn3 KO muscle. Our findings have important implications for understanding individual responses to muscle disuse/disease and training in the general population.

Bidirectional axonal plasticity during reorganization of adult rat primary somatosensory cortex

Cortical sensory maps contain discrete functional subregions that are separated by borders that restrict tangential activity flow. Interestingly, the functional organization of border regions remains labile in adults, changing in an activity-dependent manner. Here, we investigated if axon remodeling contributes to this reorganization. We located the border between the forepaw and lower jaw representation (forepaw/lower jaw border, 1 1 Forepaw/lower jaw border (FP/LJ border). FP/LJ border) in SI of adult rats, and used a retrograde axonal tracer (cholera toxin subunit B 2 2 Cholera toxin subunit B (Ctb). , Ctb) to determine if horizontal axonal projections change after different durations of forelimb denervation or sham-denervation. In sham-denervated animals, neurons close to the border had axonal projections oriented away from the border (axonal bias). Forelimb denervation resulted in a sustained change in border location and a significant reduction in the axonal bias at the original border after 6 weeks of denervation, but not after 4 or 12 weeks. The change in axonal bias was due to an increase in axons that cross the border at 6 weeks, followed by an apparent loss of these axons by 12 weeks. This suggests that bidirectional axonal rearrangements are associated with relatively long durations of reorganization and could contribute transiently to the maintenance of cortical reorganization.

Time‐course of changes in the myonuclear domain during denervation in young‐adult and old rat gastrocnemius muscle

If myonuclear loss initiates muscle wasting, it should precede the loss of muscle mass. As aging affects muscle plasticity, the time-course of muscle atrophy during disuse may differ between young and old animals. To investigate this, gastrocnemius muscles of 5- and 25-month-old rats were exposed to 1, 2, or 4 weeks of denervation, whereas the contralateral gastrocnemius muscles served as controls. Muscle fibers of each type responded similarly to 4 weeks of denervation. For both ages most of the atrophy (36%; P < 0.001) occurred in the first 2 weeks. In young-adult muscles, the myonuclear number remained constant, but in old muscles it decreased to below control level after 4 weeks of denervation (P < 0.05). Despite this differential response, myonuclear domain size decreased similarly at both ages (P < 0.001). In both young-adult and old rats, denervation-induced atrophy was not preceded by a loss of myonuclei.

Low-intensity electrical stimulation ameliorates disruption of transverse tubules and neuromuscular junctional architecture in denervated rat skeletal muscle fibers

We determine the effects of direct electrical stimulation (ES) on the histological profiles in atrophied skeletal muscle fibers after denervation caused by nerve freezing. Direct ES was performed on the tibialis anterior (TA) muscle after denervation in 7-week-old rats divided into groups as follows: control (CON), denervation (DN), or denervation with direct ES (subdivided into a 4 mA (ES4), an 8 mA (ES8), or a 16 mA stimulus (ES16). The stimulation frequency was set at 10 Hz, and the voltage was set at 40 V (30 min/day, 6 days/week, for 3 weeks). Ultrastructural profiles of the membrane systems involved in excitation-contraction coupling, and four kinds of mRNA expression profiles were evaluated. Morphological disruptions occurred in transverse (t)-tubule networks following denervation: an apparent disruption of the transverse networks, and an increase in the longitudinal t-tubules spanning the gap between the two transverse networks, with the appearance of pentads and heptads. These membrane disruptions seemed to be ameliorated by relatively low intensity ES (4 mA and 8 mA), and the area of longitudinally oriented t-tubules and the number of pentads and heptads decreased significantly (P < 0.01) in ES4 and ES8 compared to the DN. The highest intensity (16 mA) did not improve the disruption of membrane systems. There were no significant differences in the (alpha1s)DHPR and RyR1 mRNA expression among CON, DN, and all ES groups. After 3 weeks of denervation all nerve terminals had disappeared from the neuromuscular junctions (NMJs) in the CON and ES16 groups. However, in the ES4 and ES8 groups, modified nerve terminals were seen in the NMJs. The relatively low-intensity ES ameliorates disruption of membrane system architecture in denervated skeletal muscle fibers, but that it is necessary to select the optimal stimulus intensities to preserve the structural integrity of denervated muscle fibers.

Changes of denervated skeletal muscle and motor endplate after nerve repair with different intervals of delay:an experimental study

Objective To investigate the changes of denervated skeletal muscle and motor endplate after nerve repair with various intervals of delay,and discuss the best timing for nerve repair. Methods Gastrocnemius muscle denervation was established in 36 SD rats which were randomly divided into 6 groups based on different intervals of delay for surgical nerve repair(0,2,4,6,8,10 weeks),with 6 each.All the manipulations were done on the right hind limbs while the unoperated left hind limbs served as control.Six weeks after nerve repair,muscles were harvested for evaluation.Results When nerve repair was carried our within 0 to 4 weeks of denervation,muscle wet weight continued to drop but showed to significant statistical difference between the groups(P>0.05).Muscle weight showed a remarkable drop when nerve repair was done after a 4week delay,and then maintained at a certain level.Diameter and cross sectional area of muscle fibers continued to decline.When the injured nerve was repaired within 2 weeks,the CCRP(calcitonin gene-related peptide)gray scale of motor endplate was not significantly different from that of the control side(P>0.05).The gray scale of CGRP of motor endplate in groups when nerve repair was done after 4 to 6 weeks was significantly lower than that in the group when nerve repair was done within 2 weeks(P<0.05).In the group of nerve repair with 8-week delay,CGRP gray scale further-declined,being significantly different(P<0.05)compared to that of the 6 week repair group.Ultrastruetural changes were in agreement with light microscopic changes.Conchlusion Nerve repair done within 2 to 4 weeks of injury lead to better results.Although muscle atrophy was less likely to be reversible when nerve repair was delayed for 6 weeks,there were still indications for nerve repair since some parameters still remained at a certain level. Key words: Motor endplate; Rats; Denervated muscle; Nerve repair

Comparison of Neurotization Versus Nerve Repair in an Animal Model of Chronically Denervated Muscle

PurposeReinnervation of chronically denervated muscle is clinically unpredictable and poorly understood. Current operative strategies include either direct nerve repair, nerve grafting, nerve transfer, or neurotization. The goal of this study is to compare muscle recovery using microneural repair versus neurotization in a rat model of chronic denervation.MethodsFifty-eight Sprague-Dawley rats had surgical denervation of the tibialis anterior muscle by transecting the common peroneal nerve. After 0, 8, 12, or 22 weeks of denervation, animals were assigned to either a direct repair or a neurotization cohort. An additional 7 animals were used for a sham cohort, and 7 of the 58 were used as controls. After a 12-week recovery period, animals had contractile strength and EMG testing of the tibialis anterior muscle. Peak force and characteristics were compared to the unoperated, contralateral limb. Tibialis anterior muscles were then harvested for mass and histologic evaluation.ResultsSixty-two animals completed testing. Denervated controls demonstrated a significant decrease in muscle mass, contractile strength, and peak motor nerve conduction amplitude compared to sham animals. In all groups, chronicity of denervation adversely affected functional recovery. On average, repair animals performed better than neurotization animals with respect to muscle mass, contractile strength, and peak motor amplitude. Differences in contractile force, however, were significant only at the 0 week denervation group (94% ± 30 vs 50% ± 20, repair vs neurotization). Neurotized muscles processed for histologic analysis demonstrated acetylcholinesterase activity at the nerve-muscle interface, confirming the formation of motor end plates de novo.ConclusionsWe demonstrated that neurotization is capable of reinnervating de novo end plates in chronically denervated muscle. Our data do not support the hypothesis that direct muscle neurotization is superior to nerve repair for functional restoration of chronically denervated muscle. However, as the duration of denervation increases, the difference between outcomes of the neurotization and repair group narrows, suggesting that neurotization may offer a viable surgical alternative in the setting of prolonged denervation.

The time course of denervation-induced changes is similar in soleus muscles of adult and old rats

Muscle denervation is accompanied by atrophy and a decline in oxidative capacity. We investigated whether the time course of adaptations following denervation of the soleus muscle differs in adult (5 months old) and older adult (25 months old) rats. We denervated the soleus muscle of the left leg, while the right leg served as an internal control. Two weeks after denervation, muscle mass was decreased both in adult and old animals to, respectively, 57% and 54% (p < 0.001) and capillary to fibre ratio (C:F) decreased to 51% and 50% (p < 0.01) of the control values. Yet, the capillary density was increased in older adult but not in adult muscles, indicating that the regression of the capillary bed during denervation lags behind the decrease in fibre size in the soleus muscle of the older rats. One week after denervation the optical density of sections stained for succinate dehydrogenase was 83% and 79% (p < 0.05) of control adult and older adult muscles, respectively, and then remained stable. This indicates that during the first week of denervation loss of oxidative capacity occurred at a relatively higher rate than that of muscle mass. No major changes occurred between 2 and 4 weeks of denervation, except for an increase in the proportion of hybrid (I/IIa) fibres in 4 week denervated muscles (adult 10% vs. 23%; old 1% vs. 13%; p < 0.05). Except for changes in capillarisation, the time course of atrophy and decrease in oxidative capacity following denervation was similar in soleus muscles from adult and old rats.

The effect of denervation on the molecular clock in skeletal muscle

We recently identified the circadian transcriptome in mouse skeletal muscle. The primary objective of this study was to test the hypothesis that innervation is required to maintain oscillation of the circadian molecular clock in muscle. A second objective was to determine if the phase of the molecular clock is the same in different muscles. Using the Period‐2:Luciferase mouse, the tibialas anterior was denervated (d‐TA) by sectioning the common peroneal nerve on one leg with the contralateral leg serving as a sham operated control. Following two weeks of denervation, various muscles and liver were collected from three animals every four hours for 24 hr. RT‐PCR analysis revealed that the expression pattern of components of the molecular clock (Bmal1, Per2 and Clock) as well as Myod1, a clock‐controlled gene, was altered in the d‐TA compared to i‐TA. Luciferase activity, reflecting Per2 protein expression, of the d‐TA was significantly elevated at each time point in comparison to inneravted‐TA (i‐TA), though the time of peak expression remained unchanged. In the heart, diaphragm and liver, peak luciferase activity peaked four hours later than what was observed in hindlimb muscles suggesting the phase of the clock is slightly different in these tissues. Collectively, these results indicate loss of neural input causes alterations in the molecular clock in adult skeletal muscle.KAE (AR045617) and JJM (AR053641)

Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice

Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at 2 weeks, hyperinnervation at 3 and 4 weeks, and restoration of a 1:1 axon to motor endplate relationship 6 weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by 6 weeks. SCs reveal reduced S100 expression within 2 weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.

Denervation does not change the ratio of collagen I and collagen III mRNA in the extracellular matrix of muscle

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type III and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 +/- 1.6%, 73 +/- 2.3%, and 83 +/- 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red confirmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity.