Pattern Of Reinnervation Research Articles

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1’s role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Morphological Changes in Neural Progenitors Derived from Human Induced Pluripotent Stem Cells and Transplanted into the Striatum of a Parkinson's Disease Rat Model

Introduction. Development of cell therapy for Parkinson's disease (PD) requires protocols based on transplantation of neurons derived from human induced pluripotent stem cells (hiPSCs) into the damaged area of the brain.
 Objective: to characterize neurons transplanted into a rat brain and evaluate neural transplantation efficacy using a PD animal model.
 Materials and methods. Neurons derived from hiPSCs (IPSRG4S line) were transplanted into the striatum of rats after intranigral injection of 6-hydroxydopamine (6-OHDA). Immunostaining was performed to identify expression of glial and neuronal markers in the transplanted cells within 224 weeks posttransplant.
 Results. 4 weeks posttransplant we observed increased expression of mature neuron markers, decreased expression of neural progenitor markers, and primary pro-inflammatory response of glial cells in the graft. Differentiation and maturation of neuronal cells in the graft lasted over 3 months. At 3 and 6 months we detected 2 graft zones: one mainly contained the transplanted neurons and the other human astrocytes. We detected human neurites in the corpus callosum and surrounding striatal tissue and large human tyrosine hydroxylase-expressing neurons in the graft.
 Conclusion. With graft's morphological characteristics identified at different periods we can better understand pathophysiology and temporal patterns of new dopaminergic neurons integration and striatal reinnervation in a rat PD model in the long-term postoperative period.

Slow motor neurons resist pathological TDP-43 and mediate motor recovery in the rNLS8 model of amyotrophic lateral sclerosis

In the intermediate stages of amyotrophic lateral sclerosis (ALS), surviving motor neurons (MNs) that show intrinsic resistance to TDP-43 proteinopathy can partially compensate for the loss of their more disease-susceptible counterparts. Elucidating the mechanisms of this compensation may reveal approaches for attenuating motor impairment in ALS patients. In the rNLS8 mouse model of ALS-like pathology driven by doxycycline-regulated neuronal expression of human TDP-43 lacking a nuclear localization signal (hTDP-43ΔNLS), slow MNs are more resistant to disease than fast-fatigable (FF) MNs and can mediate recovery following transgene suppression. In the present study, we used a viral tracing strategy to show that these disease-resistant slow MNs sprout to reinnervate motor endplates of adjacent muscle fibers vacated by degenerated FF MNs. Moreover, we found that neuromuscular junctions within fast-twitch skeletal muscle (tibialis anterior, TA) reinnervated by SK3-positive slow MNs acquire resistance to axonal dieback when challenged with a second course of hTDP-43ΔNLS pathology. The selective resistance of reinnervated neuromuscular junctions was specifically induced by the unique pattern of reinnervation following TDP-43-induced neurodegeneration, as recovery from unilateral sciatic nerve crush did not produce motor units resistant to subsequent hTDP-43ΔNLS. Using cross-reinnervation and self-reinnervation surgery in which motor axons are disconnected from their target muscle and reconnected to a new muscle, we show that FF MNs remain hTDP-43ΔNLS-susceptible and slow MNs remain resistant, regardless of which muscle fibers they control. Collectively, these findings demonstrate that MN identity dictates the susceptibility of neuromuscular junctions to TDP-43 pathology and slow MNs can drive recovery of motor systems due to their remarkable resilience to TDP-43-driven degeneration. This study highlights a potential pathway for regaining motor function with ALS pathology in the advent of therapies that halt the underlying neurodegenerative process.

Evaluation of Laryngeal Motor Neuropathy Using Transcranial Magnetic Stimulation-Mediated Evoked Potentials.

Contemporary evaluation of vocal fold motion impairment largely relies on clinical laryngoscopy, with the diagnoses of vocal fold paresis (VFPa) and paralysis (VFP) being based on identification of partial and complete restriction of gross vocal fold motion, respectively. No consensus exists on the diagnostic criteria of VFPa. Laryngeal electromyography does not offer any insight into nerve conduction velocity without the adjunction of nerve conduction studies, which are impractical to perform on laryngeal nerves due to their anatomic location. The present study aims to assess the feasibility of laryngeal nerve conduction studies using transcranial magnetic stimulation (TMS)-mediated myogenic evoked potentials in the evaluation of laryngeal motor nerve function. Prospective controlled cohort study. Enrollment of three groups of subjects defined as healthy volunteers, subjects with clinically diagnosed unilateral VFP, and subjects with clinically diagnosed unilateral VFPa of peripheral etiology. Electrodiagnostic studies consisting of bilateral stimulation of the laryngeal motor cortex, proximal cisternal, and peripheral portions of the vagus nerves were performed using figure-of-eight magnetic stimulation coils, and myogenic evoked potentials recorded from bilateral thyroarytenoid, cricothyroid, and posterior cricoarytenoid muscles using indwelling hook wire electrodes. Conduction latencies were plotted against demographic and anthropometric variables. Values obtained in healthy volunteers were used as normative references and compared to aggregated latencies of VFP and VFPa groups. Enrolled subjects included 19 healthy volunteers, 5 subjects with VFP, and 4 subjects with VFPa. Normative laryngeal nerve conduction latency ranges measured in healthy subjects were comparable to prior published values, and recorded latencies increased in positive correlation with age. VFPa subjects exhibited increased latencies in affected nerve sites, while VFP subjects presented more variability in electrophysiologic manifestations, mostly dependent on their degree of compensatory reinnervation. Aberrant and synkinetic reinnervation patterns were more predominant in the VFP group than the VFPa group. Laryngeal nerve conduction studies using TMS-mediated myogenic evoked potentials are safely feasible. They may serve as a useful complement to laryngeal electromyography in the evaluation of motor laryngeal neuropathy and represent a promising diagnostic modality in the evaluation of VFPa. Based on the present study's findings, the commonly accepted notion of VFPa as a manifestation of a less severe form of neuropathy than VFP may be unsubstantiated. Aging may contribute to progressive motor nerve dysfunction. Future investigations are needed to ascertain the role of nerve conduction studies in clinical laryngology practice. 3 Laryngoscope, 132:S1-S12, 2022.

Reinnervation as measured by the motor unit size index is associated with preservation of muscle strength in amyotrophic lateral sclerosis, but not all muscles reinnervate.

The motor unit size index (MUSIX) may provide insight into reinnervation patterns in diseases such as amyotrophic lateral sclerosis (ALS). However, it is not known whether MUSIX detects clinically relevant changes in reinnervation, or if all muscles manifest changes in MUSIX in response to reinnervation after motor unit loss. Fifty-seven patients with ALS were assessed at 3-month intervals for 12 months in four centers. Muscles examined were abductor pollicis brevis, abductor digiti minimi, biceps brachii, and tibialis anterior. Results were split into two groups: muscles with increases in MUSIX and those without increases. Longitudinal changes in MUSIX, motor unit number index (MUNIX), compound muscle action potential amplitude, and Medical Research Council strength score were investigated. One hundred thirty-three muscles were examined. Fifty-nine percent of the muscles exhibited an increase in MUSIX during the study. Muscles with MUSIX increases lost more motor units (58% decline in MUNIX at 12 months, P< .001) than muscles that did not increase MUSIX (34.6% decline in MUNIX at 12 months, P< .001). However, longitudinal changes in muscle strength were similar. When motor unit loss was similar, the absence of a MUSIX increase was associated with a significantly greater loss of muscle strength (P= .002). MUSIX increases are associated with greater motor unit loss but relative preservation of muscle strength. Thus, MUSIX appears to be measuring a clinically relevant response that can provide a quantitative outcome measure of reinnervation in clinical trials. Furthermore, MUSIX suggests that reinnervation may play a major role in determining the progression of weakness.

Assessment of axonal sprouting and motor performance after hypoglossal-facial end-to-side nerve repair: experimental study in rats.

Hypoglossal-facial nerve anastomosis (HFA) aims to reanimate denervated mimic muscles with hypoglossal axons when the transected facial nerve is not accessible. The aim of this study was to evaluate the recovery of HFA using a "Y" tube in two variants: (1) the proximal stump of the hypoglossal nerve was entubulated to the "Y" tube (classic "Y" tube HFA) and (2) the "Y" tube was sutured to an epineurial window of a slightly damaged hypoglossal nerve (end-to-side "Y" tube HFA). A total of 48 adult female rats were divided into four groups: intact controls (group 1), sham operated (group 2), classic "Y" tube HFA (group 3) and end-to-side "Y" tube HFA (group 4). The abdominal aorta with both common iliac arteries of isogeneic male rats served as the Y-tube conduit. Animals from group 4 recovered better than those from group 3: the degree of collateral axonal branching (3 ± 1%) was significantly lower than that determined in group 3 (13 ± 1%). The mean deviation of the tongue from the midline was significantly smaller in group 4 (6 ± 4°) than that measured in animals from group 3 (41 ± 6°). In the determination of vibrissal motor function in group 3 and group 4, a decrease in amplitude was found to be - 66% and - 92%, respectively. No differences in the reinnervation pattern of the target muscles were detected. As a result, these surgical models were not determined to be able to improve vibrissal movements. It was concluded that performance of end-to-side "Y" tube HFA diminishes collateral axonal branching at the lesion site, which in turn, promotes better recovery of tongue- and vibrissal-motor performance.

Evaluation of Intrinsic Hand Musculature Reinnervation following Supercharge End-to-Side Anterior Interosseous-to-Ulnar Motor Nerve Transfer.

Supercharge end-to-side anterior interosseous-to-ulnar motor nerve transfer is commonly performed in the authors' institution to augment intrinsic hand function. Following observations of recovery patterns, the authors hypothesized that despite its more distal innervation, the first dorsal interosseous muscle recovers to a greater extent than the abductor digiti minimi muscle. The objective of this work was to evaluate the clinical and electrodiagnostic pattern of reinnervation of intrinsic hand musculature following supercharge end-to-side anterior interosseous-to-ulnar motor nerve transfer. A retrospective cohort of prospectively collected data included all patients who underwent a supercharge end-to-side anterior interosseous-to-ulnar motor nerve transfer. Two independent reviewers performed data collection. Reinnervation was assessed with two primary outcome measures: (1) clinically, with serial Medical Research Council strength assessments; and (2) electrodiagnostically, with serial motor amplitude measurements. Statistical analysis was performed using nonparametric statistics. Seventeen patients (65 percent male; mean age, 56.9 ± 13.3 years) were included with a mean follow-up of 16.7 ± 8.5 months. Preoperatively, all patients demonstrated clinically significant weakness and electrodiagnostic evidence of denervation. Postoperatively, strength and motor amplitude increased significantly for both the first dorsal interosseous muscle (p = 0.002 and p = 0.016) and the abductor digiti minimi muscle (p = 0.044 and p = 0.015). Despite comparable preoperative strength (p = 0.098), postoperatively, the first dorsal interosseous muscle achieved significantly greater strength when compared to the abductor digiti minimi muscle (p = 0.023). Following supercharge end-to-side anterior interosseous-to-ulnar motor nerve transfer, recovery of intrinsic muscle function differs between the abductor digiti minimi and the first dorsal interosseous muscles, with better recovery observed in the more distally innervated first dorsal interosseous muscle. Further work to elucidate the underlying physiologic and anatomical basis for this discrepancy is indicated. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, IV.

Exploring intrinsic triggers for functional facial electrostimulation based on intramuscular electromyography recordings.

Based on univariate intramuscular electromyography (EMG) recordings of facial muscles of patients suffering from chronic idiopathic facial palsy we propose a data-driven feature selection process for the discrimination of different mimic maneuvers. Following fundamental ideas of automatic EMG decompositions based on templates defined by motor unit action potentials, the proposed approach relies on a multiple template matching. Yet, the novel methodology utilizes templates derived from the intramuscular EMG signal itself without any supervisor interaction or a priori information by identifying abundant short signal sections (motifs). Focusing on motifs as individual, characteristical graphoelements of an EMG recording implies a high level of flexibility. In connection with facial palsy such a flexibility is necessary, since unique individual, also pathological, EMG patterns can be expected due to the high spatial variability of intramuscular recordings combined with random patterns of aberrant reinnervation. The proposed methodology is applied to EMG data of frontalis, zygomaticus, and orbicularis oculi muscle without patient- or muscle-specific adaptations.

An unusual complication of sacral nerve root injury following bone marrow harvesting: a case report

BackgroundHematopoietic stem cell transplantation (HSCT) remains an important therapeutic option for many hematologic malignancies. Bone marrow harvesting from an appropriate donor must be conducted for hematopoietic stem cell transplantation (HSCT). Many previous studies show complications of the recipient after hematopoietic stem cell transplantation (HSCT). However, complications of the donor after bone marrow harvesting are rare. We here report a unique case of a patient who developed sacral nerve root injury after bone marrow harvesting.Case presentationA 26-year-old man was admitted to our medical center complaining of acute onset painful burning and tingling sensation at the left posterior thigh and calf. He was a bone marrow donor for his brother’s bone marrow transplantation. He had underwent a bone marrow harvesting procedure two days before admission as a bone marrow donor, using both posterior superior iliac spine (PSIS) as the puncture site.Pelvic magnetic resonance image (MRI) showed enhancement around the left S2 nerve root in T1 and T2-weighted images. Nerve conduction studies (NCS) revealed normal conduction velocity and amplitude on both lower extremities. Electromyography (EMG) presented abnormal spontaneous activity and neurogenic motor unit potentials on the S2-innervated intrinsic foot muscle and gastrocnemius, soleus muscle on the left.The patient was treated with pregabalin for pain control. The patient was followed up after 3, 6, and 12 months. Neuropathic pain improved to Visual Analogue Scale (VAS) 1, and recovery state was confirmed by re-innervation patterns of motor unit potentials in electromyography.ConclusionBone marrow harvesting is a relatively safe procedure. However, variable complications may occur. Accurate anatomical knowledge and carefulness are required to avoid sacral nerve root injury when performing the bone marrow harvesting procedure.

Corneal re-innervation following refractive surgery treatments.

Laser refractive surgery is one of the most performed surgical procedures in the world. Although regarded safe and efficient, it has side effects. All of the laser based refractive surgical procedures invoke corneal nerve injury to some degree. The impact of this denervation can range from mild discomfort to neurotrophic corneas. Currently, three techniques are widely used for laser vision correction: small incision lenticule extraction, laser-assisted keratomileusis in situ and photorefractive keratotomy. Each of these techniques affects corneal innervation differently and has a different pattern of nerve regeneration. The purpose of this review is to summarize the different underlying mechanisms for corneal nerve injury and compare the different patterns of corneal reinnervation.

Influence of Netrin-1 on reinnervation of laryngeal muscles following recurrent laryngeal nerve injury

Following recurrent laryngeal nerve (RLN) injury, recovery results in poor functional restitution of the paralyzed vocal fold. Netrin-1 has been found to be upregulated in the rat posterior cricoarytenoid muscle (PCA) during nerve regeneration. We evaluated the effect of ectopic Netrin-1 in the PCA during RLN reinnervation. The right RLN was transected and Netrin-1 was injected into the PCA (2.5, 5, 10, 15, 20μg/ml). At 7 days post injury fluorescent retrograde tracer was injected into the PCA and Thyroarytenoid (TA) muscles. At 9 days tissues were harvested. Immunostaining showed reinnervation patterns in the laryngeal muscles and labelled motoneurons in the nucleus ambiguus. Lower concentrations of Netrin-1 (2.5 and 5μg/ml) showed no significant changes in laryngeal muscles reinnervation. Higher concentrations of Netrin-1 significantly reduced motor end plate innervation. The most effective dose was 10μg/ml showing reduced number of innervated motor endplates in the PCA. The somatotopic organization of the nucleus ambiguus was altered in all concentrations of Netrin-1 injection. These findings indicate that injection of Netrin-1 into the PCA changes the reinnervation pattern of the RLN.

Changes in neurotrophic factors of adult rat laryngeal muscles during nerve regeneration

Injury to the recurrent laryngeal nerve (RLN) leads to the loss of ipsilateral laryngeal fold movement, with dysphonia, and occasionally dysphagia. Functional movement of the vocal folds is never restored due to misrouting of regenerating axons to agonist and antagonist laryngeal muscles. Changes of neurotrophic factor expression within denervated muscles occur after nerve injury and may influence nerve regeneration, axon guidance and muscle reinnervation. This study investigates the expression of certain neurotrophic factors in the laryngeal muscles during the course of axonal regeneration using RT-PCR. The timing of neurotrophic factor expression was correlated to the reinnervation of the laryngeal muscles by motor axons. Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Netrin-1 (NTN-1) increased their expression levels in laryngeal muscles after nerve section and during regeneration of RLN. The upregulation of trophic factors returned to control levels following regeneration of RLN. The expression levels of the neurotrophic factors were correlated with the innervation of regenerating axons into the denervated muscles. The results suggest that certain neurotrophic factor expression is strongly correlated to the reinnervation pattern of the regenerating RLN. These factors may be involved in guidance and neuromuscular junction formation during nerve regeneration. In the future, their manipulation may enhance the selective reinnervation of the larynx.

Blockade of glial-derived neurotrophic factor in laryngeal muscles promotes appropriate reinnervation.

Synkinetic reinnervation of the laryngeal muscles is one of the causes of the poor functional recovery after a recurrent laryngeal nerve (RLN) injury. Glial-derived neurotrophic factor (GDNF) is elevated in rat laryngeal muscles during RLN reinnervation. The specific aim of this investigation was to evaluate the effect of anti-GDNF on RLN reinnervation. Anti-GDNF antibody was injected into the posterior cricoarytenoid (PCA) 3 days following RLN transection and anastomosis. Larynges were harvested at 7, 14, 28, 56, and 112 days post injury (DPI). Prior to sacrifice, the vocal fold mobility was assessed. Immunostaining to identify neuromuscular junctions was used to evaluate the extent of axonal reinnervation of the PCA, lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA). After anti-GDNF injection into PCA, RLN reinnervation in all muscles was altered when compared to the controls. PCA innervation was delayed. At 7 DPI, only a few axons made synapses in the PCA. In contrast, axons prematurely innervated the LTA and MTA when compared to controls. Innervation was similar to controls at 56 and 112 DPI. Vocal fold motion was enhanced in 10 of 24 animals studied. After injection of anti-GDNF into the PCA, early arriving axons bypass the PCA and enter the LTA. Later arriving axons innervate the PCA and MTA. Vocal fold function is improved as compared to controls. Anti-GDNF injection into the PCA influences the pattern of reinnervation and may result in less synkinetic, more functional innervation. NA Laryngoscope, 126:E337-E342, 2016.

Complex reinnervation pattern after unilateral renal denervation in rats.

Renal denervation (DNX) is a treatment for resistant arterial hypertension. Efferent sympathetic nerves regrow, but reinnervation by renal afferent nerves has only recently been shown in the renal pelvis of rats after unilateral DNX. We examined intrarenal perivascular afferent and sympathetic efferent nerves after unilateral surgical DNX. Tyrosine hydroxylase (TH), CGRP, and smooth muscle actin were identified in kidney sections from 12 Sprague-Dawley rats, to distinguish afferents, efferents, and vasculature. DNX kidneys and nondenervated kidneys were examined 1, 4, and 12 wk after DNX. Tissue levels of CGRP and norepinephrine (NE) were measured with ELISA and mass spectrometry, respectively. DNX decreased TH and CGRP labeling by 90% and 95%, respectively (P < 0.05) within 1 wk. After 12 wk TH and CGRP labeling returned to baseline with a shift toward afferent innervation (P < 0.05). Nondenervated kidneys showed a doubling of both labels within 12 wk (P < 0.05). CGRP content decreased by 72% [3.2 ± 0.3 vs. 0.9 ± 0.2 ng/gkidney; P < 0.05] and NA by 78% [1.1 ± 0.1 vs. 0.2 ± 0.1 pmol/mgkidney; P < 0.05] 1 wk after DNX. After 12 wk, CGRP, but not NE, content in DNX kidneys was fully recovered, with no changes in the nondenervated kidneys. The use of phenol in the DNX procedure did not influence this result. We found morphological reinnervation and transmitter recovery of afferents within 12 wk after DNX. Despite morphological evidence of sympathetic regrowth, NE content did not fully recover. These results suggest a long-term net surplus of afferent influence on the DNX kidney may be contributing to the blood pressure lowering effect of DNX.

Microarray Analysis Gene Expression Profiles in Laryngeal Muscle After Recurrent Laryngeal Nerve Injury.

The pathophysiology of recurrent laryngeal nerve (RLN) transection injury is rare in that it is characteristically followed by a high degree of spontaneous reinnervation, with reinnervation of the laryngeal adductor complex (AC) preceding that of the abducting posterior cricoarytenoid (PCA) muscle. Here, we aim to elucidate the differentially expressed myogenic factors following RLN injury that may be at least partially responsible for the spontaneous reinnervation. F344 male rats underwent RLN injury (n = 12) or sham surgery (n = 12). One week after RLN injury, larynges were harvested following euthanasia. The mRNA was extracted from PCA and AC muscles bilaterally, and microarray analysis was performed using a full rat genome array. Microarray analysis of denervated AC and PCA muscles demonstrated dramatic differences in gene expression profiles, with 205 individual probes that were differentially expressed between the denervated AC and PCA muscles and only 14 genes with similar expression patterns. The differential expression patterns of the AC and PCA suggest different mechanisms of reinnervation. The PCA showed the gene patterns of Wallerian degeneration, while the AC expressed the gene patterns of reinnervation by adjacent axonal sprouting. This finding may reveal important therapeutic targets applicable to RLN and other peripheral nerve injuries.

Endplate denervation correlates with Nogo-A muscle expression in amyotrophic lateral sclerosis patients.

ObjectiveData from mouse models of amyotrophic lateral sclerosis (ALS) suggest early morphological changes in neuromuscular junctions (NMJs), with loss of nerve–muscle contact. Overexpression of the neurite outgrowth inhibitor Nogo-A in muscle may play a role in this loss of endplate innervation.MethodsWe used confocal and electron microscopy to study the structure of the NMJs in muscle samples collected from nine ALS patients (five early-stage patients and four long-term survivors). We correlated the morphological results with clinical and electrophysiological data, and with Nogo-A muscle expression level.ResultsSurface electromyography assessment of neuromuscular transmission was abnormal in 3/9 ALS patients. The postsynaptic apparatus was morphologically altered for almost all NMJs (n = 430) analyzed using confocal microscopy. 19.7% of the NMJs were completely denervated (fragmented synaptic gutters and absence of nerve terminal profile). The terminal axonal arborization was usually sparsely branched and 56.8% of innervated NMJs showed a typical reinnervation pattern. Terminal Schwann cell (TSC) morphology was altered with extensive cytoplasmic processes. A marked intrusion of TSCs in the synaptic cleft was seen in some cases, strikingly reducing the synaptic surface available for neuromuscular transmission. Finally, high-level expression of Nogo-A in muscle was significantly associated with higher extent of NMJ denervation and negative functional outcome.InterpretationOur results support the hypothesis that morphological alterations of NMJs are present from early-stage disease and may significantly contribute to functional motor impairment in ALS patients. Muscle expression of Nogo-A is associated with NMJ denervation and thus constitutes a therapeutic target to slow disease progression.

Daily Electrical Muscle Stimulation Enhances Functional Recovery Following Nerve Transection and Repair in Rats

Background. Incomplete recovery following surgical reconstruction of damaged peripheral nerves is common. Electrical muscle stimulation (EMS) to improve functional outcomes has not been effective in previous studies. Objective. To evaluate the efficacy of a new, clinically translatable EMS paradigm over a 3-month period following nerve transection and immediate repair. Methods. Rats were divided into 6 groups based on treatment (EMS or no treatment) and duration (1, 2, or 3 months). A tibial nerve transection injury was immediately repaired with 2 epineurial sutures. The right gastrocnemius muscle in all rats was implanted with intramuscular electrodes. In the EMS group, the muscle was electrically stimulated with 600 contractions per day, 5 days a week. Terminal measurements were made after 1, 2, or 3 months. Rats in the 3-month group were assessed weekly using skilled and overground locomotion tests. Neuromuscular junction reinnervation patterns were also examined. Results. Muscles that received daily EMS had significantly greater numbers of reinnervated motor units with smaller average motor unit sizes. The majority of muscle endplates were reinnervated by a single axon arising from a nerve trunk with significantly fewer numbers of terminal sprouts in the EMS group, the numbers being small. Muscle mass and force were unchanged but EMS improved behavioral outcomes. Conclusions. Our results demonstrated that EMS using a moderate stimulation paradigm immediately following nerve transection and repair enhances electrophysiological and behavioral recovery.

Innervation mapping of the hind paw of the rat using Evans Blue extravasation, Optical Surface Mapping and CASAM.

Although numerous studies investigate sensory regeneration and reinnervation of the hind paw of the rat after nerve damage, no comprehensive overview of its normal innervation is present in literature. The Evans Blue extravasation technique is a well-known technique to study patterns of skin innervation. This technique has been performed differently by various groups but was never used to study the entire skin innervation in rats' hind paw including all three branches of the sciatic nerve and the saphenous nerve in detail. In this paper, we have used the Evans Blue extravasation technique to chart the skin areas innervated by the sural, peroneal, tibial and/or saphenous nerves, which together innervate the entire hind paw of the rat, and use a new technique to analyze the distribution, overlap and variability of the results. The technique is based on analysis of whole hind paws using Optical Surface Mapping (OSM) in combination with the Computer Assisted Surgical Anatomy Mapping (CASAM) technology. While the plantar hind paw is mainly innervated by the tibial nerve, the dorsal hind paw is supplied by the sural, peroneal and the saphenous nerve. Although our results basically concur with the general nerve-specific innervation of the rat hind paw, they show considerable detail in their areas of overlap as well as in the amount of variability between animals. These results will be invaluable to study and evaluate patterns of innervation and reinnervation of intact and damaged nerve fibers.

Assessment of sensory thresholds and nociceptive fiber growth after sciatic nerve injury reveals the differential contribution of collateral reinnervation and nerve regeneration to neuropathic pain

Following traumatic peripheral nerve injury reinnervation of denervated targets may be achieved by regeneration of injured axons and by collateral sprouting of neighbor undamaged axons. Experimental models commonly use sciatic nerve injuries to assess nerve regeneration and neuropathic pain, but behavioral tests for evaluating sensory recovery often disregard the pattern of hindpaw innervation. This may lead to confounding attribution of recovery of sensory responses to improvement in sciatic nerve regeneration instead of collateral reinnervation by the undamaged saphenous nerve. We used a standardized methodology to assess the separate contribution of collateral and regenerative skin reinnervation on sensory responses. Section and suture of the sciatic nerve induced loss of sensibility in the lateral and central areas of the injured paw, but nociceptive responses rapidly recovered by expansion of the intact saphenous innervation territory. We used electronic Von Frey and Plantar test devices to measure mechanical and thermal withdrawal thresholds in specific sites of the injured paw: lateral site innervated by the sciatic nerve, medial site that remained innervated by the saphenous nerve, and central site originally innervated by the sciatic nerve but affected by saphenous sprouting. After sciatic section, signs of early hyperalgesia developed in medial and central paw areas due to saphenous sprouting and expansion. The regenerating sciatic nerve fibers reached the paw at 3–4weeks and a late mechanical hyperalgesia was observed at the lateral site. Immunohistochemical staining of sensory fibers innervating the medial and lateral areas revealed a different pattern of skin reinnervation. Hypersensitivity in the intact saphenous nerve area was paralleled by early fiber sprout growth in the subepidermal plexus, but not entering the epidermis. On the other side, late sciatic hyperalgesia was accompanied by gradual skin reinnervation after 4weeks.The standardization of algesimetry testing in sciatic nerve injury models, as proposed in this study, provides a suitable model for studying in parallel neuropathic pain and sensory nerve regeneration processes. Our results also indicate that collateral sprouting and axonal regeneration contribute differently in the initiation and maintenance of neuropathic pain.

Differentiated Adipose-derived Stem Cells Promote Reinnervation of Rat Skin Flaps.

Background:Sensory recovery of skin flaps is generally poor unless they are elevated as an innervated flap. The aim of this study was to elucidate if Schwann cell (SC)-like cells differentiated from adipose-derived stem cells (ASCs) could improve the cutaneous nerve regeneration in skin flaps.Methods:Microvascular island groin flaps were elevated bilaterally in 32 Lewis rats. On the right flap, the epigastric nerve was resected and ligated (noninnervated flap), and on the left flap, the nerve was crushed (innervated flap). ASCs, SC-like cells differentiated from ASCs (dASCs), SCs, or vehicle were simultaneously injected to the dermal and hypodermal layers of flap. After 20 weeks, the reinnervation pattern of flap was assessed immunohistochemically using a neuronal marker, PGP9.5.Results:dASC cultures produced significantly higher amount of nerve growth factor and brain-derived neurotrophic factor compared with ASC cultures (P < 0.01), and the level was comparable to that of SC cultures. Although a long-term survival of the transplanted cells was not found, dASCs and SCs significantly increased reinnervation density in the periphery of both types of flaps (P < 0.05), and this effect was more pronounced in noninnervated flaps. On the other hand, ASC transplantation showed no statistically significant effect on the peripheral reinnervation (P > 0.05). In the center of flap, there was no statistically significant increase in reinnervation density in all groups irrespective of flap innervation (P > 0.05).Conclusions:dASCs could improve flap reinnervation by 2 mechanisms: First, neurotrophic factors produced by dASCs facilitated regrowth of cutaneous axons from the surroundings of flap. Second, nerve growth factor released by dASCs induced the collateral sprouting of undamaged axons in adjacent tissues. In addition to the use of innervated flaps, dASC transplantation therapy could be a new approach to improve the sensory recovery of skin flaps.