Denervated Muscle Fibers Research Articles

Age-related motor neuron degeneration in DNA repair-deficient Ercc1 mice

Degeneration of motor neurons contributes to senescence-associated loss of muscle function and underlies human neurodegenerative conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors contributing to motor neuron vulnerability and degenerative phenotypes in vivo are therefore important for our understanding of the neuromuscular system in health and disease. Here, we analyzed neurodegenerative abnormalities in the spinal cord of progeroid Ercc1 Δ/− mice that are impaired in several DNA repair systems, i.e. nucleotide excision repair, interstrand crosslink repair, and double strand break repair. Ercc1 Δ/− mice develop age-dependent motor abnormalities, and have a shortened life span of 6–7 months. Pathologically, Ercc1 Δ/− mice develop widespread astrocytosis and microgliosis, and motor neuron loss and denervation of skeletal muscle fibers. Degenerating motor neurons in many occasions expressed genotoxic-responsive transcription factors p53 or ATF3, and in addition, displayed a range of Golgi apparatus abnormalities. Furthermore, Ercc1 Δ/− motor neurons developed perikaryal and axonal intermediate filament abnormalities reminiscent of cytoskeletal pathology observed in aging spinal cord. Our findings support the notion that accumulation of DNA damage and genotoxic stress may contribute to neuronal aging and motor neuron vulnerability in human neuromuscular disorders.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-010-0715-9) contains supplementary material, which is available to authorized users.

Differential muscle‐driven synaptic remodeling in the neuromuscular junction after denervation

We used knock-in mice that express green fluorescent protein (GFP)-labeled embryonic-type acetylcholine receptors to investigate postsynaptic responses to denervation of fast-twitch and slow-twitch muscle fibers, and to visualize the integration of newly synthesized GFP-labeled embryonic-type receptors into adult synapses. The embryonic-type receptors are transiently expressed and incorporated into the denervated endplates. They replaced synaptic adult-type receptors in a directed fashion, starting from the endplate's periphery and proceeding to its central regions. The progress of embryonic-type receptor expression with respect to transcriptional control is a transient, short-term activation mechanism. The less pronounced increase in the expression levels of the GFP-labeled receptors revealed a differential shift in the integration and degradation processes that constitute the dynamic equilibrium of the synaptic receptor pool. Therefore, we were able to model the changes in the total receptor load of the neuromuscular endplate following denervation as a function of the abundance of available receptors and the initial receptor load of the endplate.

Modeling Needle Stimulation of Denervated Muscle Fibers: Voltage–Distance Relations and Fiber Polarization Effects

A finite-element model of the human thigh was coupled with a 1-D compartment model to simulate the excitation of denervated muscle fibers with a needle electrode. For short electrode-fiber distances, the specific characteristics of the needle geometry determined the areas of lowest threshold values. With increasing distance, these areas shifted toward the needle's center of charge. Comparison of the 1-D model with a 3-D fiber model showed that the assumption of rotational symmetry underlying the 1-D model leads to an overestimation of thresholds. For a 40- micro-diameter fiber stimulated with 50 micross pulses at electrode-fiber distances between 50 microm and 1 mm, the 1-D/3-D threshold ratios were between 1.14 and 1.35 for the muscle fiber model, and between 1.11 and 1.17 for Hodgkin-Huxley membrane properties at 20 degrees C. For both membrane models, the deviation was more pronounced for large fiber diameters and short stimulation pulses. Qualitative results of the 1-D model like voltage-distance relations and predictions of spike initiation sites were correct.

Immunohistochemical analysis of laryngeal muscles in normal horses and horses with subclinical recurrent laryngeal neuropathy.

We used immunohistochemistry to examine myosin heavy-chain (MyHC)-based fiber-type profiles of the right and left cricoarytenoideus dorsalis (CAD) and arytenoideus transversus (TrA) muscles of six horses without laryngoscopic evidence of recurrent laryngeal neuropathy (RLN). Results showed that CAD and TrA muscles have the same slow, 2a, and 2x fibers as equine limb muscles, but not the faster contracting fibers expressing extraocular and 2B MyHCs found in laryngeal muscles of small mammals. Muscles from three horses showed fiber-type grouping bilaterally in the TrA muscles, but only in the left CAD. Fiber-type grouping suggests that denervation and reinnervation of fibers had occurred, and that these horses had subclinical RLN. There was a virtual elimination of 2x fibers in these muscles, accompanied by a significant increase in the percentage of 2a and slow fibers, and hypertrophy of these fiber types. The results suggest that multiple pathophysiological mechanisms are at work in early RLN, including selective denervation and reinnervation of 2x muscle fibers, corruption of neural impulse traffic that regulates 2x and slow muscle fiber types, and compensatory hypertrophy of remaining fibers. We conclude that horses afflicted with mild RLN are able to remain subclinical by compensatory hypertrophy of surviving muscle fibers.

Early detection of denervated muscle fibers in hindlimb muscles after sciatic nerve transection in wild type mice and in the G93A mouse model of amyotrophic lateral sclerosis

The cell adhesion molecule N-CAM is localized to the adult neuromuscular junction but is also expressed in the extrajunctional membrane of denervated muscles concurrent with extrajunctional acetylcholine receptors. Here we used N-CAM immunohistochemistry to determine whether we could detect early denervation in hindlimb muscles of the G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). In denervated wild type mouse muscles, N-CAM immunoreactivity on the sarcolemma of all fiber types and within the sarcoplasm of only type IIA fibers was detected at day 2: ∼30% of the muscle fibers in cross-section were fully circumscribed by N-CAM immunoreactivity and ∼25% of fibers were incompletely circumscribed. The proportion of the latter fibers remained constant over the next 8 days as the proportions of the former fibers increased exponentially. Thereafter, fully circumscribed muscle fibers increased to a maximum by 30 days with a concomitant fall in the incompletely circumscribed fibers. Hence, early muscle denervation was detected by the incomplete circumscription of fiber membranes by N-CAM immunoreactivity with full circumscription and intracellular localization indicating more long-term denervation. In the G93A transgenic mouse, rapid denervation of fast-twitch muscles was readily detected by a corresponding proportion of muscle fibers in cross-section with positive N-CAM immunoreactivity. The proportions of incompletely and completely circumscribed muscle fibers corresponded well with the rate of decline in intact motor units and reduced muscle contractile forces. Progressively more fully circumscribed muscle fibers became evident with age. We conclude that the N-CAM immunoreactivity on muscle fiber membranes in muscle cross-sections provides a sensitive means of detecting early muscle fiber denervation.

Functional Electrical Stimulation of Long‐term Denervated, Degenerated Human Skeletal Muscle: Estimating Activation Using T2‐Parameter Magnetic Resonance Imaging Methods

Functional electrical stimulation (FES) of long-term denervated, degenerated human skeletal muscle has proven to be a suitable method for improving a number of physiological parameters. The underlying mechanisms of activation of a denervated muscle fiber can be described with suitably modified and extended Hodgin-Huxley type models, coupled with three-dimensional (3D) finite element models of the surrounding electrical field. Regions of activation within a muscle can be determined using a 3D computer model. However, simulation results have not yet been validated experimentally. During and immediately after exercise, muscle shows increased T2-relaxation times. It is thus possible to estimate muscle activation noninvasively and spatially resolved with the magnetic resonance imaging (MRI) method of T2 mapping, which was, therefore, chosen as a suitable validation approach. Six patients were scanned prior to FES training with a multislice multiecho MSME-sequence at 3 Tesla and then asked to perform one of their regular daily training-sessions (leg extensions). Subjects were then repositioned in the MR-scanner and two to five postexercise scans were recorded. Pre- and postexercise scans were coregistered and T2-parameter maps were calculated. Regions of interest (ROIs) were drawn manually around quadriceps femoris and its antagonists. Activation was detected in all patients. In well-trained patients, activation in the quadriceps was found to be considerably higher than in its antagonists. These experimental results will help further improve existing models of FES of denervated degenerated human skeletal muscle.

O efeito do intervalo da estimulação elétrica no músculo desnervado de rato

OBJETIVO: Comparar o efeito da estimulacao eletrica (EE) aplicada diariamente e em dias alternados na densidade de area do tecido conjuntivo (TC) e na area de seccao transversa (AST) das fibras do musculo desnervado. MATERIAIS E METODOS: Trinta e cinco ratos foram divididos em grupos controle (C), desnervado (D), desnervado + eletroestimulado diariamente (EED) e desnervado + eletroestimulado em dias alternados (EEA). A aplicacao da EE no musculo gastrocnemio teve inicio 24 horas apos lesao nervosa do tipo axoniotmese, sendo a mesma aplicada durante 20 e 30 dias. Cortes transversais foram corados com HE para mensuracoes da AST e densidade de area de TC. Analise estatistica: teste Shapiro Wilk, seguido pela analise de variância (ANOVA) F (one-way) e teste de Tukey (5%). RESULTADOS: Na analise da densidade de area do TC, observou-se que somente o Grupo EED apresentou valores similares ao Grupo C nos dois periodos analisados. No Grupo 20 dias, nao houve diferenca na AST quando comparados os grupos submetidos a EE com o Grupo D (p> 0,05), e apos 30 dias todos os grupos experimentais alcancaram valores similares ao Grupo C. CONCLUSOES: A EE nao foi eficiente para minimizar a atrofia das fibras musculares. Entretanto, o TC foi responsivo a EE, sendo a aplicacao diaria mais benefica ao musculo do que a aplicacao em dias alternados, sugerindo que o intervalo de aplicacao da EE em musculo desnervado e variavel importante para as adaptacoes do TC.

Muscle fiber conduction abnormalities in early diabetic polyneuropathy

Objective Diabetic polyneuropathy (PNP) has been proposed to be a primary disorder of sensory nerves. At an early stage motor nerve conduction velocity (MNCV) and muscle strength remain preserved due to compensatory mechanisms (axonal sprouting, reinnervation). We evaluated the use of invasive muscle fiber conduction velocity (MFCV) measurements as a method to detect muscle fiber denervation atrophy, as an early sign of motor axonal loss in diabetes mellitus (DM). Methods Twelve selected male patients (8 type 1, 4 type 2; mean age 35.8 years, SD 10.6), without any sign of micro- or macroangiopathy, were studied by systematic clinical and neurophysiological testing including MFCV estimation. Results Hand-held dynamometry was normal in all subjects. There were no signs of recent denervation by concentric needle EMG in any of the patients. Sensory nerve conduction velocity (SNCV) was abnormal in 6 subjects, MFCV in 6 subjects (5 had also low SNCV). The ratio of fastest/slowest muscle fibers in MFCV was correlated to SNCV of sural nerve (−.59, p < .05), but not to MNCV. Conclusions Half of the clinically asymptomatic DM subjects showed sensory involvement together with MFCV abnormalities, despite normal needle EMG and force. Significance MFCV estimation offers a sensitive method in detecting early signs of motor axonal dysfunction in DM.

Current Distance Relations for Fiber Stimulation With Pointsources

The following results are based on computer simulations and on activating function analysis. In the near field, denervated muscle fibers as well as nerve fibers with a sealed ending are easier to stimulate in the central region than with electrodes close to the end. When electrode-fiber distance is increased, the electrode location for optimal stimulation efficacy shifts from a central position to a region beyond the fiber end for cathodic stimulation and to a position above the terminating part of the fiber for anodic currents. The phenomenon becomes more pronounced with increasing distance between the electrode and fiber axis, because in the far field, the current-distance relation changes from quadratic to cubic, whereas stimulation at the fiber end obeys a rather constant quadratic law.

Embryonic neurons transplanted into the tibial nerve reinnervate muscle and reduce atrophy but NCAM expression persists

Objective: The aim of this study was to use the glycogen depletion technique to determine whether reinnervated muscle fibers could be distinguished from denervated muscle fibers by their size or by neural cell adhesion molecule (NCAM) expression.Methods: Medial gastrocnemius muscles of five adult Fischer rats were reinnervated from embryonic neurons transplanted into the distal stump of the tibial nerve. Ten weeks later, the transplants were stimulated repeatedly to deplete reinnervated muscle fibers of glycogen. Areas of reinnervated (glycogen-depleted) muscle fibers were measured and assessed for NCAM expression. The areas of muscle fibers from reinnervated, denervated (n=5) and unoperated control muscles (n=5) were compared.Results: Mean reinnervated muscle fiber area was significantly larger than the mean for denervated fibers (mean ± SE: 40 ± 6 and 10 ± 1% of unoperated control fibers, respectively). NCAM was expressed in 55 ± 7% of reinnervated fibers (mean ± SE; range: 42–77%). The mean areas of reinnervated fibers that did or did not express NCAM were similar. NCAM was only expressed in some fibers in completely denervated muscles.Discussion: Our data show that NCAM expression does not differentiate muscle denervation or reinnervation. Quantifying the area of large fibers did distinguish reinnervated muscle fibers from denervated fibers and showed that reinnervation of muscle from neurons placed in peripheral nerve is a strategy to rescue muscle from atrophy.

SMAC-expression in Denervated Human Skeletal Muscle as a Potential Inhibitor of Coexpressed Inhibitor-of-apoptosis Proteins

In multinucleated skeletal muscle fibers, apoptotic muscle fiber loss is mediated by a consecutive disassembly of single fiber segments. During this period of time, proapoptotic and antiapoptotic factors compete for promotion or inhibition of apoptotic fiber degradation. In 16 patients with a neurogenic muscular atrophy, we studied the immunohistochemical expression of the inhibitor-of-apoptosis proteins (IAP) survivin, cIAP1, and XIAP and also of second mitochondria-derived activator of caspase (SMAC), which is released during apoptosis and binds to IAPs to prevent them from inhibiting caspases. Although normal control muscle fibers show no expression of SMAC and IAPs, there was a distinct sarcoplasmic expression of SMAC (12.0%+/-3.5%), survivin (10.2%+/-4.0%), cIAP1 (9.0%+/-3.1%), and XIAP (11.0%+/-4.6%) in varying numbers of muscle fibers in neurogenic muscular atrophy. Coexpression of SMAC and IAPs varied. Some denervated muscle fibers displayed up-regulation of either SMAC or IAPs. Other groups of atrophic muscle fibers showed coexpression of SMAC and IAPs. All factors were exclusively up-regulated in atrophic muscle fibers. These findings indicate that IAPs may inhibit apoptotic degradation of denervated muscle fibers. However, IAPs are finally insufficient to counterbalance and prevent muscle fiber apoptosis, as up-regulated expression of SMAC can antagonize this antiapoptotic potential and promote apoptotic muscle fiber disassembly and loss. The interplay between IAPs and SMAC may represent a threshold for muscle fiber-degrading caspase activities. If this bears a therapeutic potential in the prevention of loss of denervated muscle fibers, it remains highly speculative.

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Ciliary neurotrophic factor is not required for terminal sprouting and compensatory reinnervation of neuromuscular synapses: Re-evaluation of CNTF null mice

Loss of synaptic activity or innervation induces sprouting of intact motor nerve terminals that adds or restores nerve–muscle connectivity. Ciliary neurotrophic factor (CNTF) and terminal Schwann cells (tSCs) have been implicated as molecular and cellular mediators of the compensatory process. We wondered if the previously reported lack of terminal sprouting in CNTF null mice was due to abnormal reactivity of tSCs. To this end, we examined nerve terminal and tSC responses in CNTF null mice using experimental systems that elicited extensive sprouting in wildtype mice. Contrary to the previous report, we found that motor nerve terminals in the null mice sprout extensively in response to major sprouting-stimuli such as exogenously applied CNTF per se, botulinum toxin-elicited paralysis, and partial denervation by L4 spinal root transection. In addition, the number, length and growth patterns of terminal sprouts, and the extent of reinnervation by terminal or nodal sprouts, were similar in wildtype and null mice. tSCs in the null mice were also reactive to the sprouting-stimuli, elaborating cellular processes that accompanied terminal sprouts or guided reinnervation of denervated muscle fibers. Lastly, CNTF was absent in quiescent tSCs in intact, wildtype muscles and little if any was detected in reactive tSCs in denervated muscles. Thus, CNTF is not required for induction of nerve terminal sprouting, for reactivation of tSCs, and for compensatory reinnervation after nerve injury. We interpret these results to support the notion that compensatory sprouting in adult muscles is induced primarily by contact-mediated mechanisms, rather than by diffusible factors.

Motor neuron targeting of IGF-1 attenuates age-related external Ca 2+-dependent skeletal muscle contraction in senescent mice

A population of fast muscle fibers from aging mice is dependent on external Ca 2+ to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca 2+-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca 2+-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca 2+-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca 2+ to maintain tetanic force in a population of muscle fibers from senescent mice.

Adaptive changes in structure of skeletal muscles from adult Sod1 homozygous knockout mice

Cu/Zn superoxide dismutase (SOD1), which is localized cytoplasmically and in the mitochondrial intermembrane space, is an enzyme that is critically important for superoxide free-radical elimination. Compared with age-matched wild-type littermates (Sod1 ( +/+ )), SOD1 homozygous knockout (Sod1 ( -/- )) mice have smaller body masses, heart and skeletal muscle masses, and muscle cross-sectional areas. At the light-microscopic level, cross sections of skeletal muscles from Sod1 ( -/- ) mice show no gross structural abnormalities. Following the staining of muscles of Sod1 ( -/- ) mice for succinate dehydrogenase (SDH) enzymatic activity, a grouping of SDH-positive fibers has been observed. Immunostaining for neural cell adhesion marker in the gastrocnemius muscle of Sod1 ( -/- ) mice has revealed a small number of atrophic denervated muscle fibers. No denervated fibers are observed in extensor digitorum longus (EDL), tibialis anterior, or plantaris muscles. An increase in mRNA expression levels of myogenin and acetylcholine receptor alpha has been detected in muscles in Sod1 ( -/- ) mice, but no changes in MyoD expression occur. Compared with fast oxidative fibers in EDL muscles of Sod1 ( +/+ ) mice, those of Sod1 ( -/- ) mice show increased accumulations of sub-sarcolemmal mitochondria. We conclude that the lack of SOD1 in adult Sod1 ( -/- ) mice does not result in extensive denervation of skeletal muscle fibers, although the distribution of fiber types is modified, and that fast oxidative fibers develop alterations in the amount and spatial distribution of sub-sarcolemmal mitochondria.

Incisional Hernia Effect on Abdominal Neuromuscular Properties in the Rat

Midline hernias complicate approximately 11% of abdominal surgeries. Maladaptive changes in the abdominal neuromusculature may contribute to hernia persistence or recurrence. Both injury and denervation negatively affect muscle contractile properties immediately following muscle lesion, but it is unclear whether abdominal muscles are affected once wound healing has stabilized (after 28 days). The authors studied the contractile properties of specific abdominal muscles and subsequently immunohistologically labeled the muscles for fiber denervation. They hypothesized that, compared with sham-operated animals, abdominal muscles from rats with an abdominal hernia have a reduced capacity to produce force and exhibit a population of denervated muscle fibers. Additionally, since skeletal muscle weakens and muscle fiber denervation increases with increased age, they tested the interaction effect of age with surgery.

P32.1 Distribution of regular and irregular firing patterns of denervation activity in completely denervated human muscle

Background: It has been claimed that spontaneous activity from denervated muscle fibers (MFs) has a regular firing pattern (FP), though there is considerable disagreement regarding this claim. Aim/objective: The objective was to determine the distribution of firing patterns of denervated MFs. Patients and method: 553 FPs of individual fibrillation potentials or positive sharp waves from 182 recordings in 33 patients with completely denervated muscles were analyzed. Our own decomposition program EMGTools, originally developed for motor unit analysis, was used for extracting the FPs. This semi-automatic template matching method along with powerful visual tools for verifying the result enabled us to classify the FPs as being either regular or irregular. Each recording had a duration of 20 s.Most signals had 1–10 distinct potentials resulting in the same number of FPs. In some muscles a greater number of potentials occurred which made the analysis more difficult. Only FPs that could be visually confirmed as being full were included. Results: The variation in FPs and mean inter potential interval (IPI) was large across signals and patients. Fifty-seven percentage of the FPs was found to be regular, the rest of the FPs were identified as irregular. Amixture of regular and irregular FPs within the same recording was also found. Conclusion: Our investigation showed that about half of denervation potentials have irregular FPs.

Normal distribution of presenilin-1 and nicastrin in skeletal muscle and the differential responses of these proteins after denervation

Presenilin-1 and nicastrin, two components of γ-secretase associated with Alzheimer's disease plaques, are present in the synapses of the brain and in various peripheral organs, including skeletal muscle. In the present study, we examined the expression pattern of presenilin-1 and nicastrin in normal and denervated hindlimb muscles of the rat. Using immunohistochemical approaches, we found that presenilin-1 and AChRα was co-localized at the neuromuscular junction in the normal skeletal muscles of rats. The immunoreactivities of both presenilin-1 and nicastrin were also observed at the sarcolemma of muscle fibers. We discovered that presenilin-1 mRNA and its protein are upregulated after denervation of the soleus and tibialis anterior muscles. Furthermore, clear co-localization between presenilin-1 and DAPI, but not nicastrin, was noted in several myonuclei in the denervated muscles. We recognized a few fibers possessing both ubiquitin and presenilin-1 protein in the cytosol. The amount of presenilin-1 in the nucleus and membrane fraction was more abundantly expressed in the denervated muscle fibers. In contrast, no significant difference in the nicastrin protein level was observed between normal and denervated muscle fibers. These data suggest that enhanced presenilin-1 protein may play a role in the degeneration and regeneration of skeletal muscle.

Terminal Schwann cell structure is altered in diaphragm of mdx mice

The diaphragm muscle of the mdx mouse is a model system of Duchenne muscular dystrophy, since it completely lacks dystrophin and shows severe fiber necrosis and loss of specific muscle force by 4-6 weeks of age. Changes in neuromuscular junction structure also become apparent around 4 weeks including postsynaptic acetylcholine receptor declustering, loss of postsynaptic junctional folds, abnormally complex presynaptic nerve terminals, and muscle fiber denervation. Normally, terminal Schwann cells (TSCs) cap both nerve terminals and acetylcholine receptors at the neuromuscular junction, and play a crucial role in regeneration of motor axons following muscle denervation by guiding axons to grow from innervated junctions to nearby denervated junctions. However, their role in restoring innervation in dystrophic muscle is unknown. We now show that TSCs fail to cap fully the neuromuscular junction in dystrophic muscle; TSCs extend processes, but the organization of these extensions is abnormal. TSC processes of dystrophic muscle do not form bridges from denervated fibers to nearby innervated endplates, but appear to be directed away from these endplates. Adequate signaling for TSC reactivity is present, since significant muscle fiber denervation and acetylcholine receptor declustering are present. Thus, significant structural denervation is present in the diaphragm of mdx mice and the ability of TSCs to form bridges between adjacent endplates to guide reinnervation of muscle fibers is impaired, possibly attenuating the ability of dystrophic muscle to recover from denervation and ultimately leading to muscle weakness.

Abstracts

Abstracts