Reinnervation Process Research Articles

The Spinal Transcriptome after Cortical Stroke: In Search of Molecular Factors Regulating Spontaneous Recovery in the Spinal Cord.

In response to cortical stroke and unilateral corticospinal tract degeneration, compensatory sprouting of spared corticospinal fibers is associated with recovery of skilled movement in rodents. To date, little is known about the molecular mechanisms orchestrating this spontaneous rewiring. In this study, we provide insights into the molecular changes in the spinal cord tissue after large ischemic cortical injury in adult female mice, with a focus on factors that might influence the reinnervation process by contralesional corticospinal neurons. We mapped the area of cervical gray matter reinnervation by sprouting contralesional corticospinal axons after unilateral photothrombotic stroke of the motor cortex in mice using anterograde tracing. The mRNA profile of this reinnervation area was analyzed using whole-genome sequencing to identify differentially expressed genes at selected time points during the recovery process. Bioinformatic analysis revealed two phases of processes: early after stroke (4–7 d post-injury), the spinal transcriptome is characterized by inflammatory processes, including phagocytic processes as well as complement cascade activation. Microglia are specifically activated in the denervated corticospinal projection fields in this early phase. In a later phase (28–42 d post-injury), biological processes include tissue repair pathways with upregulated genes related to neurite outgrowth. Thus, the stroke-denervated spinal gray matter, in particular its intermediate laminae, represents a growth-promoting environment for sprouting corticospinal fibers originating from the contralesional motor cortex. This dataset provides a solid starting point for future studies addressing key elements of the post-stroke recovery process, with the goal to improve neuroregenerative treatment options for stroke patients.SIGNIFICANCE STATEMENT We show that the molecular changes in the spinal cord target tissue of the stroke-affected corticospinal tract are mainly defined by two phases: an early inflammatory phase during which microglia are specifically activated in the target area of reinnervating corticospinal motor neurons; and a late phase during which growth-promoting factors are upregulated which can influence the sprouting response, arborization, and synapse formation. By defining for the first time the endogenous molecular machinery in the stroke-denervated cervical spinal gray matter with a focus on promotors of axon growth through the growth-inhibitory adult CNS, this study will serve as a basis to address novel neuroregenerative treatment options for chronic stroke patients.

MyomiRNAs Dysregulation in ALS Rehabilitation.

Amyotrophic lateral sclerosis (ALS) is a rare, progressive, neurodegenerative disorder caused by degeneration of upper and lower motor neurons. The disease process leads, because of lower motor neuron involvement, to progressive muscle atrophy, weakness, and fasciculations and for the upper motor neuron involvement leads to spasticity. Muscle atrophy in ALS is caused by a neural dysregulation in the molecular network controlling fast and slow muscle fibers. Denervation and reinnervation processes in skeletal muscle occur in the course of ALS and are modulated by rehabilitation. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in different biological functions under various pathophysiological conditions. MiRNAs can be secreted by various cell types and they are markedly stable in body fluids. MiR-1, miR-133 a miR-133b, and miR-206 are called “myomiRs” and are considered markers of myogenesis during muscle regeneration and contribute to neuromuscular junction stabilization or sprouting. We observed a positive effect of a standard aerobic exercise rehabilitative protocol conducted for six weeks in 18 ALS patients during hospitalization in our center. This is a preliminary study, in which we correlated clinical scales with molecular data on myomiRs. After six weeks of moderate aerobic exercise, we found lower levels in serum of myomiRNAs. Our data suggest that circulating miRNAs changed during skeletal muscle recovery in response to physical rehabilitation in ALS. However, no firm conclusions can be made on the ALS-specific effect of exercise on miRNA levels.

In vivo Evaluation of Nanostructured Fibrin-Agarose Hydrogels With Mesenchymal Stem Cells for Peripheral Nerve Repair.

The regenerative capability of peripheral nerves is very limited, and several strategies have been proposed to increase nerve regeneration. In the present work, we have analyzed the in vivo usefulness of a novel nanostructured fibrin-agarose bio-artificial nerve substitute (Nano) used alone or in combination with NeuraGen® collagen type I conduits (Coll-Nano) in laboratory rats with a 10-mm sciatic nerve defect. Control animals were subjected to the gold-standard autograft technique (Auto). Results first demonstrated that the percentage of self-amputations was lower in Nano and Coll-Nano groups as compared to the Auto group. Neurotrophic ulcers were more abundant in the Auto group (60%, with 66.6% of them being >2-mm) than Nano and Coll-Nano groups (0%) at 4 weeks, although Nano showed more ulcers after 12 weeks. Foot length was significantly altered in Auto animals due to neurogenic retraction, but not in Nano and Coll-Nano groups after 12 weeks. At the functional level, all animals showed a partial sensory recovery as determined by the pinch test, especially in Nano and Auto groups, but did not reach the levels of native animals. Toe-spread test revealed a partial motor function recovery only in Nano animals at 4 weeks and Auto and Nano at 12 weeks. Electromyography showed clear denervation signs in all experimental groups, with few differences between Auto and Nano animals. After 12 weeks, an important denervation decrease and an increase of the reinnervation process was found in Auto and Nano groups, with no differences between these groups. Histological analyses demonstrated an active peripheral nerve regeneration process with newly formed peripheral nerve fascicles showing S-100, GAP-43 and myelin in all experimental groups. The peripheral nerve regeneration process was more abundant in Auto group, followed by Nano group, and both were better than Coll-Nano group. Muscle histology confirmed the electromyography results and showed some atrophy and fibrosis signs and an important weight and volume loss in all groups, especially in the Coll-Nano group (56.8% weight and 60.4% volume loss). All these results suggest that the novel Nano substitutes used in in vivo were able to contribute to bridge a 10-mm peripheral nerve defect in rats.

Rehabilitation of Upper Extremity Nerve Injuries Using Surface EMG Biofeedback: Protocols for Clinical Application.

Motor recovery following nerve transfer surgery depends on the successful re-innervation of the new target muscle by regenerating axons. Cortical plasticity and motor relearning also play a major role during functional recovery. Successful neuromuscular rehabilitation requires detailed afferent feedback. Surface electromyographic (sEMG) biofeedback has been widely used in the rehabilitation of stroke, however, has not been described for the rehabilitation of peripheral nerve injuries. The aim of this paper was to present structured rehabilitation protocols in two different patient groups with upper extremity nerve injuries using sEMG biofeedback. The principles of sEMG biofeedback were explained and its application in a rehabilitation setting was described. Patient group 1 included nerve injury patients who received nerve transfers to restore biological upper limb function (n = 5) while group 2 comprised patients where biological reconstruction was deemed impossible and hand function was restored by prosthetic hand replacement, a concept today known as bionic reconstruction (n = 6). The rehabilitation protocol for group 1 included guided sEMG training to facilitate initial movements, to increase awareness of the new target muscle, and later, to facilitate separation of muscular activities. In patient group 2 sEMG biofeedback helped identify EMG activity in biologically “functionless” limbs and improved separation of EMG signals upon training. Later, these sEMG signals translated into prosthetic function. Feasibility of the rehabilitation protocols for the two different patient populations was illustrated. Functional outcome measures were assessed with standardized upper extremity outcome measures [British Medical Research Council (BMRC) scale for group 1 and Action Research Arm Test (ARAT) for group 2] showing significant improvements in motor function after sEMG training. Before actual movements were possible, sEMG biofeedback could be used. Patients reported that this visualization of muscle activity helped them to stay motivated during rehabilitation and facilitated their understanding of the re-innervation process. sEMG biofeedback may help in the cognitively demanding process of establishing new motor patterns. After standard nerve transfers individually tailored sEMG biofeedback can facilitate early sensorimotor re-education by providing visual cues at a stage when muscle activation cannot be detected otherwise.

A Systematic Review of Zosteriform Rash in Breast Cancer Patients: An Objective Proof of Flap Reinnervation and a Management Algorithm.

Zosteriform rash in cancer patients provides objective clues to the process of reinnervation of the reconstructed breast. This rash should also raise suspicion for metastasis, which can be confused with herpes zoster. The aims of this study were to explain the reconstruction flap sensory reinnervation mechanism based on the clinical findings and provide a diagnostic and management algorithm of zosteriform rash in breast cancer patients. On November 15, 2017, we conducted a search of published articles in MEDLINE and Cochrane databases. All the articles describing a zosteriform rash in a patient with a history of breast cancer were included in this review. Eleven articles from the literature and 1 case from our practice were selected for inclusion in this systematic review. Five patients had a breast reconstruction with a flap. The flap skin was affected by the rash in 4 of these patients, providing an objective proof of the reinnervation of the reconstructed breast. In 6 patients, the presentation was typical, and the diagnosis of herpes zoster was made without additional diagnostic testing. In 4 cases, the eruption was atypical, and a biopsy was done to confirm the diagnosis of a cancer metastasis. In 2 patients, the rash was multidermatomal, and a polymerase chain reaction was done to confirm the diagnosis of disseminated herpes zoster. Zoster reactivation in breast reconstructed patients is an objective proof of the reinnervation of the skin flap. Moreover, zosteriform rash in cancer patients should raise suspicion for metastasis, which can be confused with herpes zoster.

Striatal Reinnervation Process after Acute Methamphetamine-Induced Dopaminergic Degeneration in Mice.

Methamphetamine (METH), an amphetamine derivate, may increase the risk of developing Parkinson's disease (PD). Human and animal studies have shown that METH produces persistent dopaminergic neurotoxicity in the nigrostriatal pathway, despite initial partial recovery. To determine the processes leading to early compensation, we studied the detailed morphology and distribution of tyrosine hydroxylase immunoreactive fibers (TH-ir) classified by their thickness (types I-IV) before and after METH. Applying three established neurotoxic regimens of METH: single high dose (1 × 30mg/kg), multiple lower doses (3 × 5mg/kg) or (3 × 10mg/kg), we show that METH primarily damages type I fibers (the thinner ones), and to a much lesser extend types II-IV fibers including sterile axons. The striatal TH terminal partial recovery process, consisting of a progressive regrowth increases in types II, III, and IV fibers, demonstrated by co-localization of GAP-43, a sprouting marker, was observed 3days post-METH treatment. In addition, we demonstrate the presence of growth-cone-like TH-ir structures, indicative of new terminal generation as well as improvement in motor functions after 3days. A temporal relationship was observed between decreases in TH-expression and increases in silver staining, a marker of degeneration. Striatal regeneration was associated with an increase in astroglia and decrease in microglia expression, suggesting a possible role for the neuroimmune system in regenerative processes. Identification of regenerative compensatory mechanisms in response to neurotoxic agents could point to novel mechanisms in countering the neurotoxicity and/or enhancing the regenerative processes.

TLR4 Gene Expression and Pro-Inflammatory Cytokines in Alzheimer’s Disease and in Response to Hippocampal Deafferentation in Rodents

One important aspect in Alzheimer's disease pathology is the presence of chronic inflammation. Considering its role as a key receptor in the microglial innate immune system, TLR4 was shown to regulate the binding and phagocytosis of amyloid plaques by microglia in several mouse models of amyloidosis, as well as the production of pro-inflammatory cytokines. To our knowledge, TLR4 and its association with cytokines have not been thoroughly examined in the brains of subjects affected with Alzheimer's disease. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in postmortem human brains, we observed increased expression for the TLR4 and TNF genes (p = 0.001 and p = 0.025, respectively), as well as a trend for higher IL6 gene expression in the frontal cortex of AD subjects when compared to age-matched controls. Similarly, using a mouse model of hippocampal deafferentation without amyloidosis, (i.e., the entorhinal cortex lesioned mouse), we observed significant increases in the expression of both the Tlr4 (p = 0.0367 and p = 0.0193 compared to sham-lesioned mice or to the contralateral side, respectively) and Il1b (p = 0.0055 and p = 0.0066 compared to sham-lesioned mice or to the contralateral side, respectively) genes in the deafferentation phase, but not during the ensuing reinnervation process. In conclusion, we suggest that the modulation of cytokines by TLR4 is differentially regulated whether by the presence of amyloid plaques or by the ongoing deafferentation process.

Platform Session – Electromyography: Two novel electrophysiological methods: In the evaluation of ALS patients – MScan MUNE and MVRC

Introduction Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive loss of motor units, but electromyography (EMG) with quantitative motor unit potential (MUP) analysis does not directly quantify the denervation process. A more extensive clinical use of Motor Unit Estimation Methods: (MUNE), like MScan MUNE, could hence be of potentially great value. Muscle Velocity Recovery Cycle (MVRC) recordings can provide in vivo information about changes in muscle fiber membrane properties, and may enlighten the effect of the denervation process on the muscle fibers. The aim of this study was to examine the clinical utility of MScan MUNE and MVRC in ALS patients; which also contribute to a better understanding of disease pathophysiology. Methods 27 ALS patients and 23 age and sex-matched healthy control subjects were included. All participants were evaluated electrophysiologically by a standard quantitative EMG and by the two novel methods: MScan MUNE and MVRC, all in the anterior tibial muscle (TA). MScan MUNE, MVRC and EMG parameters were compared between ALS patients and healthy controls using parametric unpaired t-tests. ROC curves were used to test how these methods distinguish patients from healthy controls. MVRC parameters were correlated to EMG results and MScan parameters using linear regression analysis. Results The MScan MUNE value for ALS patients (55 (95% CI: 42–68)) was significantly lower than for the healthy controls (108 (95% CI: 98–117)) (p 5 sites) and controls. And there was no significant correlation between MVRC and MUP parameters or incidence of spontaneous activity. Conclusion The novel MScan MUNE method is a sensitive quantitative measure of loss of motor units in ALS patients in TA. The finding of no difference in MVRC for ALS patients compared to healthy controls indicate, that the muscle fiber membranes are not prominently affected by the denervation process. This may be explained by the compensatory process of collateral reinnervation, which initially is strong and ensures that the vast majority of muscle fibers are innervated and behave normally.

T158. Sympathetic reinnervation in patient with transplanted upper limbs

Introduction Autonomic peripheral nerve reinnervation for skin vasculature and eccrine sweat glands has not been reported or analyzed before. As upper limb transplantation (ULT) remains an uncommon procedure sympathetic physiologic reinnervation process still remains a challenge and an important question to be answered in patients who undergo anastomosis of a peripheral nerve after a complete section. Methods A 52 year old male, underwent forearm transplantation 16 months after a high current electrical burn. Autonomic evaluation yearly during 4 years after transplant included: (1) evaluation of skin sympathetic response (SSR); (2) measurement of temperature with an infrared thermometer of high sensitivity (Nubee© range 50–350 °C, with one decimal point of discrimination); (3) evaluation of skin blood flow (SBF) with an infrared photopletysmograph; (4) capillary filling at the nails; (5) observation of the presence or absence of atrophic changes of the skin; (6) observation for the presence or absence of edema; (7) presence or absence of allodynia; (8) systolic blood pressure changes at the heart level and lowering the arm 60° (Portapres©); (9) presence or absence of the veno-arteriolar reflex Results SSR on both palmar surfaces was evident and reliable on the third year postransplantation. The patient clinically also referred sweating in both hands. No powder sweating was done Temperature of both hands had normal values, compared to the arms above transplant. No trophic changes were observed. The humidity of the skin appeared normal. There was no edema. No allodynia was present. The SBF demonstrated that the arteriolar vessels responded normally to stimuli such as breathing, baroreceptor stimulation, gravity pressor activation, and had normal blood pressure changes to breathing and lowering the arm. Capillary filling of the nail was normal. The veno-arteriolar reflex was present. Conclusion The eccrine sweat glands of the glabrous skin appear to be reinnervated as observed by the normal humidity, trophism of the hand, and presence of sympathetic skin response. The skin was not dry or scamous but soft and humid. The exocrine sweat glands of the non-glabrous skin of the forearm also had normal humidity and trophism which strongly suggests normal function of these glands. Piloerection could not be evaluated. The normal temperature, the absence of edema, the absence of trophic changes of the skin and more importantly the normal response of the SBF and SBP to deep breathing, postural changes and baroreceptor stimulation suggests that the arterial and venous system are reinnervated. The diffuse distributed sympathetic system seem to reinnervate skin and probably muscular vasculature after several years of a limb transplant.

La paralysie faciale périphérique a frigore

Idiopathic peripheral facial palsy, also named Bell's palsy, is the most common cause of peripheral facial palsy in adults. Although it is considered as a benign condition, its social and psychological impact can be dramatic, especially in the case of incomplete recovery. The main pathophysiological hypothesis is the reactivation of HSV 1 virus in the geniculate ganglia, leading to nerve edema and its compression through the petrosal bone. Patients experience an acute (less than 24 hours) motor deficit involving ipsilateral muscles of the upper and lower face and reaching its peak within the first three days. Frequently, symptoms are preceded or accompanied by retro-auricular pain and/or ipsilateral face numbness. Diagnosis is usually clinical but one should look for negative signs to eliminate central facial palsy or peripheral facial palsy secondary to infectious, neoplastic or autoimmune diseases. About 75% of the patients will experience spontaneous full recovery, this rate can be improved with oral corticotherapy when introduced within the first 72 hours. To date, no benefit has been demonstrated by adding an antiviral treatment. Hemifacial spasms (involuntary muscles contractions of the hemiface) or syncinesia (involuntary muscles contractions elicited by voluntary ones, due to aberrant reinnervation) may complicate the disease's course. Electroneuromyography can be useful at different stages: it can first reveal the early conduction bloc, then estimate the axonal loss, then bring evidence of the reinnervation process and, lastly, help for the diagnosis of complications.

S14-3. F-wave and spinal excitability in neurological disease

Depending on the neurological condition, the excitability of lower motoneuron (LMN) changes. Shortest latency, conduction velocity, frequency and amplitude are F-wave parameters we are able to measure. Because F-wave travels the entire length of peripheral nerve, parameters of latency and conduction velocity are used to evaluate motor neuropathies. However, frequency and amplitude are considered to reflect the excitability of LMN. Normally, LMN receives presynaptic inhibition from upper motoneuron (UMN) or Renshaw inhibitory cell. In case of central nervous system disorders like stroke and myelopathy, LMN excitability becomes higher because of disinhibition from those inhibitory neuron system, and is leading cause of F-wave frequency and amplitude increase. In diseases that lower LMN excitability such as motor neuron disease and neuropathy, F-wave frequency diminishes. Evaluation of amplitude would be somewhat complicated. Even though F-wave frequency diminishes, F-wave amplitude increases in relation to motor unit size, which is affected by reinnervation process. In amyotrophic lateral sclerosis, where both UMN and LMN progressively disappear, the balance of both neurons decides the excitability of LMN. Therefore, the frequency and amplitude of F-wave varies. Repeater F-wave might be the key to analyze this complicated condition.

End-to-side neurorrhaphy: expectations and reality

An increasing number of injuries with damage of peripheral nerves, including a substantial number of injuries with extensive defects of large nerve trunks, requires improvement in approach to surgical treatment of patients. High-energy impact injuries in peacetime and wartime often lead to persistent violations of musculoskeletal apparatus, a significant part of which is due to injuries of peripheral nerves. During peacetime, various types of peripheral nerve injuries occur in 2-6% of trauma patients. In today’s armed conflicts, various types of peripheral nerve injuries occur in nearly 80% of neurosurgical patients and are mostly of combined injury. Improvements in surgical methods of recovery of lost innervation due to an injury are directly associated with the development of fundamental understanding of the nature of occurring degenerative and regenerative processes in damaged nerve fiber. Nerve suture is only the creation of appropriate conditions that contribute to regeneration of nerve fibers and recovery of lost functions. When extensive defects of nerve trunks exist, connection of distal part damaged nerve with side surface of the intact nerve is the alternative to traditional replacement of extensive defects with nerve grafts. Positive outcomes of such interventions that were seen during tests on laboratory animals led to trials of this method on patients and resulted in a number of positive outcomes as well. Despite the fact that the question of morphological substantiation of reinnervation processes of the distal portion of a damaged nerve is the cause of heated disputes among scientists, some surgeons make attempts to define a set of specific conditions needed to justify the use of end-to-side neurorrhaphy.

Co-Administered Vitamin E Isoforms d-α-tocopherol and d-δ-tocotrienol Rich Fraction Promote Regeneration of Skeletal Muscle in Diabetics

In diabetes the structural and functional recovery of skeletal muscle is impaired due to persistent hyperglycemia-induced oxidative stress. Vitamin E is known to be essential antioxidant for maintains the skeletal muscle homeostasis thus preventing oxidative damages. This study is designed to explore the effect of d-α-tocopherol and d-δ-tocotrienol rich fraction (d-δ-TRF) on crushed muscle regeneration in both healthy and diabetic rats. Diabetes was induced through single subcutaneous injection of aqueous alloxan at the dose of 100 mg/kg. Twenty four albino rats were divided into four groups; healthy control, diabetic control, healthy treated and diabetic treated. Treated groups received 100 mg/kg of d-α-tocopherol and d-δ-TRF each, orally, daily for three weeks. Through a horizontal mid-thigh skin incision and splitting of the fascia gluteus maximus was approached and crushed with Kocher’s forceps. Skin wound was closed with an absorbable suture. The crush-induced degenerative and regenerative changes in the muscle were studied by assessing the histological features, histomorphological measurements and biochemical analyses at the end of 3^rd weeks. One- way ‘ANOVA’ and Student’s t-test were used for statistical analysis. All results revealed that the vitamin E isoforms have potency to maintain glycemic level, improve the antioxidant capacity and hasten the process of regeneration, revascularization, reinnervation and connective tissue remodeling in skeletal muscle after crush injury. It is therefore, concluded that the vitamin E isoforms are useful nutritional supplements for skeletal muscle functional and structural recovery in both healthy and diabetics.

S69 From outside: MU characteristics by stimulation methods during acute and chronic denervation

Introduction We can observe different characteristics of the motor units and their compound activity by stimulating the nerve directed to the relevant muscle and looking through different magnifications of our electrophysiological microscope. Methods Low magnification: CMAP, medium magnification: estimating the number of motor units innervating the muscle (MUNE), higher magnification: CMAPscan and F waves. Results CMAP: The most simple, easy and inclusive of all. Gives an overall impression about the condition of the muscle. However it is not specific, has wide normal ranges and tends to veil the abnormality by reorganizational changes of the constituent MUs. MUNE methods: To unveil the amount of MU loss caused by the lesion, but more time consuming. Each method has its own drawbacks. Incorrect results can be caused by the reinnervation process itself. May behave differently in chronic and acute denervation. CMAP scan: To observe the general appearance of the constituent MUs, especially those enlarged due to the denervation-reinnervation process. Can be used for qualitative observation or for follow-up with quantitative parameters. F-wave characteristics: To observe the components of CMAP through a narrow window. We can deduce information about the MUs and the physiological conditions acting on them, especially by taking their less frequently used parameters into consideration, e.g. persistence, amplitude and repeater Fs. Conclusion We can extract more information about the pathophysiology of MUs and their compound function by looking concurrently through the different magnifications provided by our routine neurophysiological device.

Delayed Reinnervation During Muscle Regeneration After NO‐Donor Pre‐Treatment

Activation of satellite cells (SCs) toward myogenesis leads to structural regeneration of damaged myofibers and reinnervation is essential to muscle function. SC activation is mediated by NO (a signaling and neurotransmitter molecule) and manipulation of the NO signal is shown to enhance muscle repair and promote growth in older mice in combination with exercise. SCs up‐regulate mRNA expression of a neural chemorepellent, semaphorin 3A (Sema3A) that peaks 6–8 days after muscle injury. Since Sema3A expression is implicated in regulating the timing of axon growth toward regenerating fibers, we hypothesized that premature activation of SC before an injury would advance Sema3A synthesis and disrupt the process of reinnervation. The time‐ course of reinnervation in regenerating tibialis anterior muscle (TA) of 6–10‐week‐old C57BL6 mice was examined after injury by crush (a large traumatic injury) or injection of cardiotoxin (CTX, which damages only fibers). We tested whether early activation of SC before injury using a 2‐day daily pre‐treatment with isosorbide dinitrate (ISDN, an NO‐donor drug) affects the process of new‐fiber innervation post‐injury. Muscle was collected 0, 4, 6, 8, 10, and 14 days after unilateral TA injury (n=4–5 per time point in crush, CTX, and respective control groups). The position of the nuclei and the length of the newly‐formed myotubes were used to track muscle regeneration on coded slides. CTX‐injured muscles had noticeably more advanced formation of neuromuscular junctions (NMJs) and faster fiber regeneration compared to crush‐ injured muscles at the same time point post‐injury. Combined staining of NMJs for acetylcholinesterase (AchE, silver staining) and acetylcholine receptors (AchRs, α‐bungarotoxin epifluorescence) on coded slides, revealed immature or mature NMJs (positive for AchE and AchRs) by day 14 in untreated crush‐ or CTX‐injured muscles, respectively. ISDN pre‐treatment in both crush‐ and CTX‐injured muscles delayed NMJ development on myotubes up to day 14 post‐injury. Results are consistent with the action of Sema3A as a neural chemorepellent, since SC activation before injury resulted in delayed myotube reinnervation. Therapeutic manipulation of Sema3A during regeneration, in time and/or concentration, may have potential to improve reinnervation and muscle function in conditions where nerve‐muscle interactions are disrupted by injury or disease.Support or Funding InformationSupported by a NSERC Discovery grant (to JEA) and a University of Manitoba Graduate Fellowship (to ND)

Electrical stimulation delays reinnervation in denervated rat muscle.

It is not clear if electrical stimulation (ES) can affect muscle reinnervation. This study aimed to verify if ES affects neuromuscular recovery after nerve crush injury in rats. Denervated muscles were electrically stimulated daily for 6 or 14 days. Neuromuscular performance and excitability, and muscle morphology were determined. Muscle trophism markers (atrogin-1, MuRF-1, and myoD), as well as neuromuscular junction (NMJ) organization (muscle-specific receptor tyrosine kinase [MuSK], cytoplasmic protein downstream of kinase-7 [Dok-7], nicotinic ACh receptor [nAChR], and neural cell adhesion molecule [N-CAM]) were assessed. ES impaired neuromuscular recovery at day 14 postdenervation. Muscle hypoexcitability was accentuated by ES at 6 and 14 days postdenervation. Although ES reduced the accumulation of atrogin-1, MuRF1, and myoD mRNAs, it increased muscle atrophy. Gene expression of MuSK, Dok-7, nAChR, and the content of N-CAM protein were altered by ES. ES can delay the reinnervation process by modulating factors related to NMJ stability and organization, and inducing dysfunction, hypoexcitability, and muscle atrophy. Muscle Nerve 56: E108-E118, 2017.

Reinnervation post-Heart transplantation

Heart transplantation results in complete denervation of the donor heart with loss of afferent and efferent nerve connections. The majority of patients remain completely denervated during the first 6-12 months following transplantation. Evidence of reinnervation is usually found during the second year after transplantation and involve the myocardial muscle, sinoatrial node, and coronary vessels, but remains incomplete and regionally limited many years post-transplant. Restoration of cardiac innervation can improve exercise capacity as well as blood flow regulation in the coronary arteries, and hence improve quality of life. As yet, there is no evidence that the reinnervation process is associated with the occurrence of allograft-related events or survival.

Peripheral Retrograde Nerve Regeneration After Sciatic Nerve Injury in a Rat Model: A New Concept in Peripheral Nerve Surgery

Background: Neuroregeneration after nerve injury occurs via anterograde (proximal in the distal) regrowth. The purpose of this study was to demonstrate that nerve regeneration occurs not only proximally to distally as classically described, but also distally to proximally using a rat model with a left sciatic nerve injury, with or without repair, compared to a healthy uninjured control group. Methods: Fifteen rats were divided into 3 groups; a healthy control (Group 1, n=5), left sciatic nerve injury (Group 2, n=5), and left sciatic nerve injury and surgical retrograde nerve repair (Group 3, n=5). In Groups 2 and 3, complete surgical resection of the left sciatic nerve was performed and retrograde reconstruction of the nerve was done in Group 3 using an original, unique technique. Neuroconductive and histological variables were analyzed and compared between groups. Results: After 15 weeks, Groups 1 (control) and 3 (retrograde reinnervation) behaved similarly, with no significant differences (p<0.05) in the proportion of Schwann cells and axons, amplitude waves, nerve conduction velocity, and latency response time (p<0.05), whereas Group 2 (injured unrepaired nerve) was significantly different from Groups 1 and 3 (p<0.05). The reconstruction of injured sciatic nerves showed histopathological and neuroconductive characteristics similar to that of the healthy control group 15 weeks after the reinnervation process. Conclusion: This preclinical study demonstrates that retrograde nerve regeneration after sciatic nerve injury is feasible in a rat model 15 weeks after reconstructive nerve surgery. These results indicate that neuroregeneration is possible in a caudo-cephalic direction.

54. Early neurophysiological signs of disease in SOD1G93A mouse model: A stimulated single-fiber electromyography study

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive degeneration of the corticospinal tract motor neurons. Growing evidence, especially derived from studies in murine models, could suggest that ALS is a distal axonopathy in which axonal degeneration occurs early during the disease, preceding the degeneration of motoneuron soma in the ventral horn of the spinal cord and the onset of symptoms (Fischer et al., 2004; Dadon-Nachum et al., 2011). A combination of molecular and subcellular events, such as protein misfolding, oxidative damage, mitochondrial alterations, impairment of glutamate transmission leading to excitotoxicity, and axonal transport impairment has been implicated in motoneuron vulnerability in ALS (Ferraiuolo et al., 2011). These observations led to consider the neuromuscular junction (NMJ) as a major player in the initiation and progression of ALS. Aim of this study was to investigate the functionality of the NMJ through all the course of the disease in the hSOD1G93A ALS mouse model, by means of single-fiber electromyography (SFEMG) technique. A comprehensive neurophysiological investigation (including sciatic nerve conduction, MEPs by transcranial electrical stimulation and sSFEMG) was carried out on 5 WT and 5 hSOD1G93A mice, at different time-points (50-70-90-110 ± 3 PND). In the early pre-symptomatic phase (50 PND), hSOD1G93A mice presented a significant jitter elongation [MCD (mean ± SE): 18.36 ± 3.21 in hSOD1G93A, 8.23 ± 0.84 in WT; p 0.021] together with increased fiber density [FD (mean ± SE): 1.62 ± 0.17 in hSOD1G93A, 1.27 ± 0.10 in WT; p 0.117] when compared with controls, suggestive of recent reinnervation process. No difference were found in peripheral conduction and MEPs parameters at the same time-point. Our results show that the neuromuscular transmission of hSOD1G93A mice is altered since the early presymptomatic phase of the disease. This probably derive from an early denervation-reinnervation process resulting in immature collateral nerve terminals and instability of neuromuscular transmission. SFEMG seems to be more sensitive in detecting the early signs of the disease compared to the other neurophysiological techniques, and could be a valuable tool for monitoring the effectiveness of new experimental therapeutic approach.

Muscle specific nucleus ambiguus neurons isolation and culturing

BackgroundPeripheral nerve injury leads to a regenerative state. However, the reinnervation process is highly non-selective. Growing axons are often misrouted and establish aberrant synapsis to abductor or adductor muscles. Determining the complex properties of abductor and adductor motoneurons in a neuron culture, may lay the groundwork for future studies on axon guidance, leading to a clinical treatment for a selective reinnervation. New methodIn the present study we develop a neuron culture protocol to isolate recurrent laryngeal nerve abductor and adductor motoneurons in order to study their unique properties.Comparison with existing methods the best period to perform the present protocol for postnatal rat cranial motoneurons isolation was determined. In addition, the method allows identification of specific motoneurons from other primary motoneurons and interneurons within brainstem. ConclusionThe present protocol will allow investigators to perform targeted and novel studies of the mechanisms of peripheral nerve regeneration.