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OBJECTIVE High cervical spinal cord injury (SCI) results in complete loss of upper-limb function, resulting in debilitating tetraplegia and permanent disability. Spontaneous motor recovery occurs to varying degrees in some patients, particularly in the 1st year postinjury. However, the impact of this upper-limb motor recovery on long-term functional outcomes remains unknown. The objective of this study was to characterize the impact of upper-limb motor recovery on the degree of long-term functional outcomes in order to inform priorities for research interventions that restore upper-limb function in patients with high cervical SCI. METHODS A prospective cohort of high cervical SCI (C1–4) patients with American Spinal Injury Association Impairment Scale (AIS) grade A–D injury and enrolled in the Spinal Cord Injury Model Systems Database was included. Baseline neurological examinations and functional independence measures (FIMs) in feeding, bladder management, and transfers (bed/wheelchair/chair) were evaluated. Independence was defined as score ≥ 4 in each of the FIM domains at 1-year follow-up. At 1-year follow-up, functional independence was compared among patients who gained recovery (motor grade ≥ 3) in elbow flexors (C5), wrist extensors (C6), elbow extensors (C7), and finger flexors (C8). Multivariable logistic regression evaluated the impact of motor recovery on functional independence in feeding, bladder management, and transfers. RESULTS Between 1992 and 2016, 405 high cervical SCI patients were included. At baseline, 97% of patients had impaired upper-limb function with total dependence in eating, bladder management, and transfers. At 1 year of follow-up, the largest proportion of patients who gained independence in eating, bladder management, and transfers had recovery in finger flexion (C8) and wrist extension (C6). Elbow flexion (C5) recovery had the lowest translation to functional independence. Patients who achieved elbow extension (C7) were able to transfer independently. On multivariable analysis, patients who gained elbow extension (C7) and finger flexion (C8) were 11 times more likely to gain functional independence (OR 11, 95% CI 2.8–47, p < 0.001) and patients who gained wrist extension (C6) were 7 times more likely to gain functional independence (OR 7.1, 95% CI 1.2–56, p = 0.04). Older age (≥ 60 years) and motor complete SCI (AIS grade A–B) reduced the likelihood of gaining independence. CONCLUSIONS After high cervical SCI, patients who gained elbow extension (C7) and finger flexion (C8) had significantly greater independence in feeding, bladder management, and transfers than those with recovery in elbow flexion (C5) and wrist extension (C6). Recovery of elbow extension (C7) also increased the capability for independent transfers. This information can be used to set patient expectations and prioritize interventions that restore these upper-limb functions in patients with high cervical SCI.

OBJECTIVE The objective of this study was to test whether regenerating motor axons from a donor nerve can travel in a retrograde fashion using sensory branches to successfully reinnervate a motor nerve end organ. METHODS This study has two parts. In part I, rats (n = 30) were assigned to one of five groups for obturator nerve (ON)–to–femoral nerve transfer: group 1, ON-to–saphenous nerve (SN) distal stump; group 2, ON-to-SN proximal stump without femoral nerve proper (FNP) injury; group 3, ON-to-SN proximal stump with FNP crush injury; group 4, ON-to-SN proximal stump with FNP transection injury; and group 5, gold standard transfer, ON-to–motor femoral nerve (MFN) branch. At 8 weeks, retrograde labeling was done from the distal MFN, and the spinal cords were examined to assess the degree of obturator motor axon regeneration across the five groups. In part II, only group 4 was examined (n = 8). Through use of immunostaining and optical tissue clearing methods, the nerve transfer networks were cleared and imaged using light-sheet fluorescence microscopy to visualize the regeneration pathways in 2D and 3D models at 2- and 8-week time points. RESULTS Proximal FNP transection (group 4) enabled a significantly higher number of retrogradely regenerated motor axons compared with control groups 1–3. Moreover, group 4 had modest, but nonsignificant, superiority of motor neuron counts compared with the positive control group, group 5. Optical tissue clearing demonstrated that the axons traveled in a retrograde fashion from the recipient sensory branch to the FNP mixed stump, then through complex turns, down to distal branches. Immunostaining confirmed the tissue clearing findings and suggested perineurium disruption as a means by which axons could traverse across fascicular boundaries. CONCLUSIONS Sensory branches can transmit regenerating axons from donor nerves back to main mixed recipient nerves, then distally toward target organs. The extent of retrograde regeneration is markedly influenced by the type and severity of injury sustained by the recipient nerve. Using a sensory branch as a bridge for retrogradely regenerating axons can open new potential horizons in nerve repair surgery for severely injured mixed nerves.

We have recently isolated and expanded skin-derived Schwann cells (Sk-SCs) from human skin and showed that they are largely similar to nerve-derived Schwann cells (N-SCs). Here, we extend our investigation into functional assessments of the nude rats that received human Sk-SCs and N-SCs after intraneural delivery into crushed and decellularized tibial nerve in adult nude rats.Sk-SCs, N-SCs, dermal fibroblasts, or control culture medium was injected into the crushed and decellularized tibial nerve using in situ repeated freeze-thaw cycles. Animals were then subjected to a ladder rung walking test, nociceptive von Frey testing, and walking gait analysis weekly. Animals were euthanized 6 weeks after surgery, gastrocnemius and soleus muscles were weighed, distal nerves were harvested, and whole semithin cross-sections were analyzed using segmentation software.N-SC-injected and dermal fibroblast-injected animals improved significantly at 4 to 6 weeks postinjury in nociceptive assessment compared with medium-injected controls. Sk-SCs recovered more rapidly in tibial functional index at 2 weeks postinjury compared with medium-injected controls. No significant difference was observed for the ladder rung walking test or muscle weight ratio. Histologically, the number of myelinated axons was significantly higher in all cell injection groups compared with medium-injected controls. No significant difference was observed in g ratio, axon diameter, or myelin thickness.Cell injection significantly improved axon regeneration across an in situ decellularized nerve segment. However, a more human cell-permissive animal model is required to delineate functional differences between cell types for preclinical transplantation studies.

OBJECTIVEThe aim of this study was to examine the role of intraoperative neuromonitoring (IONM) during resection of benign peripheral nerve sheath tumors in achieving gross-total resection (GTR) and in reducing postoperative neurological complications.METHODSData from consecutive adult patients who underwent resection of a benign peripheral nerve sheath tumor at 7 participating institutions were combined. Propensity score matching was used to balance covariates. The primary outcomes of interest were the association between IONM and GTR and the association of IONM and the development of a permanent postoperative neurological complication. The secondary outcomes of interest were the association between IONM and GTR and the association between IONM and the development of a permanent postoperative neurological complication in the subgroup of patients with tumors involving a motor or mixed nerve. Univariate and multivariate logistic regression were then performed on the propensity score–matched samples to assess the ability of the independent variables to predict the outcomes of interest.RESULTSA total of 337 patients who underwent resection of benign nerve sheath tumors were included. In multivariate analysis, the use of IONM (OR 0.460, 95% CI 0.199–0.978; p = 0.047) was a significant negative predictor of GTR, whereas none of the variables, including IONM, were associated with the occurrence of a permanent postoperative neurological complication. Within the subgroup of motor/mixed nerve tumors, in the multivariate analysis, IONM (OR 0.263, 95% CI 0.096–0.723; p = 0.010) was a significant negative predictor of a GTR, whereas IONM (OR 3.800, 95% CI 1.925–7.502; p < 0.001) was a significant positive predictor of a permanent postoperative motor deficit.CONCLUSIONSOverall, 12% of the cohort had a permanent neurological complication, with new or worsened paresthesias most common, followed by pain and then weakness. The authors found that formal IONM was associated with a reduced likelihood of GTR and had no association with neurological complications. The authors believe that these data argue against IONM being considered standard of care but do not believe that these data should be used to universally argue against IONM during resection of benign nerve sheath tumors.

Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility.

Cell therapy has shown potential in the field of peripheral nerve repair, and research using rodents is a critical and essential step toward clinical development of this approach. Traditionally, most experimental peripheral nerve injuries are conducted in inbred Lewis or outbred Sprague–Dawley strains. However, transplantation of xenogeneic cells such as human-derived cells typically triggers rejection in these animals. An alternative approach is to use immunodeficient animals, such as athymic nude rats. The lack of functional T cells in these animals renders them more accommodating to foreign cells from a different host. Currently, no literature exists regarding sensorimotor behavioral assessment of nude rats after peripheral nerve injury. To this end, we compared the functional recovery during a 6-wk period of behavioral testing of Lewis and nude rats after unilateral sciatic nerve crushing injury. Three sensorimotor behavioral assessments were performed weekly: a ladder rungwalking task to assess slip ratio and cross duration, von Frey nociception testing to determine the paw withdrawal threshold thus monitoring the regaining of sensory function, and sciatic functional index evaluation to monitor the recovery of integrated motor function. Both strains demonstrated significant sensory and motor deficits in the first week after injury, with a slight regain of sensory function, reduced slip ratio, and increased sciatic functional index starting at 2 wk. No significance difference existed between nude and Lewis rats in their recovery courses. We conclude that nude rats are a suitable model for behavioral training and assessment for cell transplantation studies in peripheral nerve injury and repair.

Regenerative therapies for the treatment of peripheral nerve and spinal cord injuries can require hundreds of millions of autologous cells. Current treatments involve the harvest of Schwann cells (SCs) from nerves; however, this is an invasive procedure. Therefore, a promising alternative is using skin-derived Schwann cells (Sk-SCs), in which between 3–5 million cells can be harvested from a standard skin biopsy. However, traditional static planar culture is still inefficient at expanding cells to clinically relevant numbers. As a result, bioreactors can be used to develop reproducible bioprocesses for the large-scale expansion of therapeutic cells. Here, we present a proof-of-concept SC manufacturing bioprocess using rat Sk-SCs. With this integrated process, we were able to simulate a feasible bioprocess, taking into consideration the harvest and shipment of cells to a production facility, the generation of the final cell product, and the cryopreservation and shipment of cells back to the clinic and patient. This process started with 3 million cells and inoculated and expanded them to over 200 million cells in 6 days. Following the harvest and post-harvest cryopreservation and thaw, we were able to maintain 150 million viable cells that exhibited a characteristic Schwann cell phenotype throughout each step of the process. This process led to a 50-fold expansion, producing a clinically relevant number of cells in a 500 mL bioreactor in just 1 week, which is a dramatic improvement over current methods of expansion.

Peripheral nerve and spinal cord injuries affect millions of people worldwide often leading to lifelong disability and pain. Cell therapy using autologous Schwann cells are currently being investigated for the treatment of these injuries and are showing great potential. In particular, skin‐derived Schwann cells offer a non‐invasive option for highly therapeutic cells that have been shown to regenerate tissue and restore function. However, it is estimated that for an effective therapy, hundreds of millions of Schwann cells are needed, and current static and spinner flask methods cannot generate these clinical numbers. Therefore, this study utilizes control systems facilitated by stirred suspension bioreactors to monitor and maintain the pH and dissolved oxygen at levels that promote cell proliferation and survival. By adapting the current culture practices to scalable bioreactor platforms, we were able to control the dissolved oxygen creating new expansion protocols that enabled the bioreactor process to grow to clinical scale, producing 667 × 106 cells in a single 500 mL bioreactor in 7 days. Since individual injury is different in terms of complexity and severity, and therefore cell needs, by changing the available surface area in the bioreactor, this developed process is highly adjustable to meet the cell quantity needs for each individual treatment. Following the bioprocess, cells that were harvested from the microcarriers continue to express typical Schwann cell markers suggesting that they retain phenotypic stability. By increasing the production potential of skin‐derived Schwann cells, this therapy is one step closer to the clinic with the hope for ultimately restoring function and improving quality of life to patients with neural injuries.

Objective. Focused ultrasound (FUS) use with and without microbubbles (MB) for investigation of the blood-nerve barrier (BNB) within the peripheral nervous system (PNS) has been performed in this study. We evaluate the feasibility of BNB opening in a rodent sciatic nerve model by direct vision FUS treatment and provide preliminary results of magnetic resonance guided FUS (MRgFUS). Approach. Twenty rodent bilateral sciatic nerves were investigated. Rodents were treated using a benchtop FUS system to directly visualize nerve FUS studies. Definity MB, Evans blue dye (EB) and latex micro beads were injected during studies. Selected animals underwent further compound muscle action potential (CMAP) studies. Sonication peak pressure (MPa), width, duty-cycle and duration as well as MB concentration were varied to investigate effective pressure threshold. Further preliminary MRgFUS studies were performed on selected animals. Immunohistochemistry and histological analysis under florescent microscopy were performed at termination of experiments to verify treatment outcomes. Main results. Three ultrasound pressures and three microbubble concentrations at a single sonication frequency (476.5 kHz) were performed under direct open targeting. Histological analysis demonstrated nerve internal architecture disruption at 1.2 MPa with 166.7 μl kg−1 while 0.3 MPa, with 40 μl kg−1 MB concentration was the lower threshold for consistently observed disruption of the BNB without anatomical microarchitecture disruption. EB leakage was confirmed at the target region in histological evaluation of nerve following MB injection and FUS sonication. Supra-harmonic emissions were detected during FUS exposures following MB injection but not at baseline reference, indicating effective MB response and stable cavitation. CMAP amplitudes showed delayed onset latency and lower amplitudes in sonicated nerves compared to control nerves without evidence of complete conduction block, suggesting a transient BNB disruption, while at lower limit pressure subtle conduction changes were observed. In MRgFUS, targeted nerves demonstrated further contrast agent leak as well as supra-harmonic frequency detection. Significance. Opening of the BNB in the PNS was achieved using FUS and MB in a rodent model. Ongoing work aims to refine FUS parameters for drug delivery into the nerve after experimental transient BNB disruption.

Peripheral nerve injury remains a major cause of morbidity in trauma patients. Despite advances in microsurgical techniques and improved understanding of nerve regeneration, obtaining satisfactory outcomes after peripheral nerve injury remains a difficult clinical problem. There is a growing body of evidence in preclinical animal studies demonstrating the supportive role of stem cells in peripheral nerve regeneration after injury. The characteristics of both mesoderm-derived and ectoderm-derived stem cell types and their role in peripheral nerve regeneration are discussed, specifically focusing on the presentation of both foundational laboratory studies and translational applications. The current state of clinical translation is presented, with an emphasis on both ethical considerations of using stems cells in humans and current governmental regulatory policies. Current advancements in cell-based therapies represent a promising future with regard to supporting nerve regeneration and achieving significant functional recovery after debilitating nerve injuries.

OBJECTIVEPatients with cervical spinal cord injury (SCI)/tetraplegia consistently rank restoring arm and hand function as their top functional priority to improve quality of life. Motor nerve transfers traditionally used to treat peripheral nerve injuries are increasingly used to treat patients with cervical SCIs. In this article, the authors present early results of a prospective clinical trial using nerve transfers to restore upper-extremity function in tetraplegia.METHODSParticipants with American Spinal Injury Association (ASIA) grade A–C cervical SCI/tetraplegia were prospectively enrolled at a single institution, and nerve transfer(s) was performed to improve upper-extremity function. Functional recovery and strength outcomes were independently assessed and prospectively tracked.RESULTSSeventeen participants (94.1% males) with a median age of 28.4 years (range 18.2–76.3 years) who underwent nerve transfers at a median of 18.2 months (range 5.2–130.8 months) after injury were included in the analysis. Preoperative SCI levels ranged from C2 to C7, most commonly at C4 (35.3%). The median postoperative follow-up duration was 24.9 months (range 12.0–29.1 months). Patients who underwent transfers to median nerve motor branches and completed 18- and 24-month follow-ups achieved finger flexion strength Medical Research Council (MRC) grade ≥ 3/5 in 4 of 15 (26.7%) and 3 of 12 (25.0%) treated upper limbs, respectively. Similarly, patients achieved MRC grade ≥ 3/5 wrist flexion strength in 5 of 15 (33.3%) and 3 of 12 (25.0%) upper limbs. Among patients who underwent transfers to the posterior interosseous nerve (PIN) for wrist/finger extension, MRC grade ≥ 3/5 strength was demonstrated in 5 of 9 (55.6%) and 4 of 7 (57.1%) upper limbs 18 and 24 months postoperatively, respectively. Similarly, grade ≥ 3/5 strength was demonstrated in 5 of 9 (55.6%) and 4 of 7 (57.1%) cases for thumb extension. No meaningful donor site deficits were observed. Patients reported significant postoperative improvements from baseline on upper-extremity–specific self-reported outcome measures.CONCLUSIONSMotor nerve transfers are a promising treatment option to restore upper-extremity function after SCI. In the authors’ experience, nerve transfers for the reinnervation of hand and finger flexors showed variable functional recovery; however, transfers for the reinnervation of arm, hand, and finger extensors showed a more consistent and meaningful return of strength and function.

In this review article, the authors offer their perspective on nerve surgery for nerve injury, with a focus on recent evolution of management and the current surgical management. The authors provide a brief historical perspective to lay the foundations of the modern understanding of clinical nerve injury and its evolving management, especially over the last century. The shift from evaluation of the nerve injury using macroscopic techniques of exploration and external neurolysis to microscopic interrogation, interfascicular dissection, and internal neurolysis along with the use of intraoperative electrophysiology were important advances of the past 50 years. By the late 20th century, the advent and popularization of interfascicular nerve grafting techniques heralded a major advance in nerve reconstruction and allowed good outcomes to be achieved in a large percentage of nerve injury repair cases. In the past 2 decades, there has been a paradigm shift in surgical nerve repair, wherein surgeons are not only directing the repair at the injury zone, but also are deliberately performing distal-targeted nerve transfers as a preferred alternative in an attempt to restore function. The peripheral rewiring approach allows the surgeon to convert a very proximal injury with long regeneration distances and (often) uncertain outcomes to a distal injury and repair with a greater potential of regenerative success and functional recovery. Nerve transfers, originally performed as a salvage procedure for severe brachial plexus avulsion injuries, are now routinely done for various less severe brachial plexus injuries and many other proximal nerve injuries, with reliably good to even excellent results. The outcomes from nerve transfers for select clinical nerve injury are emphasized in this review. Extension of the rewiring paradigm with nerve transfers for CNS lesions such as spinal cord injury and stroke are showing great potential and promise. Cortical reeducation is required for success, and an emerging field of rehabilitation and restorative neurosciences is evident, which couples a nerve transfer procedure to robotically controlled limbs and mind-machine interfacing. The future for peripheral nerve repair has never been more exciting.

ImportanceCervical spinal cord injury (SCI) causes devastating loss of upper extremity function and independence. Nerve transfers are a promising approach to reanimate upper limbs; however, there remains a paucity of high-quality evidence supporting a clinical benefit for patients with tetraplegia.ObjectiveTo evaluate the clinical utility of nerve transfers for reanimation of upper limb function in tetraplegia.Design, Setting, and ParticipantsIn this prospective case series, adults with cervical SCI and upper extremity paralysis whose recovery plateaued were enrolled between September 1, 2015, and January 31, 2019. Data analysis was performed from August 2021 to February 2022.InterventionsNerve transfers to reanimate upper extremity motor function with target reinnervation of elbow extension and hand grasp, pinch, and/or release.Main Outcomes and MeasuresThe primary outcome was motor strength measured by Medical Research Council (MRC) grades 0 to 5. Secondary outcomes included Sollerman Hand Function Test (SHFT); Michigan Hand Outcome Questionnaire (MHQ); Disabilities of Arm, Shoulder, and Hand (DASH); and 36-Item Short Form Health Survey (SF-36) physical component summary (PCS) and mental component summary (MCS) scores. Outcomes were assessed up to 48 months postoperatively.ResultsTwenty-two patients with tetraplegia (median age, 36 years [range, 18-76 years]; 21 male [95%]) underwent 60 nerve transfers on 35 upper limbs at a median time of 21 months (range, 6-142 months) after SCI. At final follow-up, upper limb motor strength improved significantly: median MRC grades were 3 (IQR, 2.5-4; P = .01) for triceps, with 70% of upper limbs gaining an MRC grade of 3 or higher for elbow extension; 4 (IQR, 2-4; P P Conclusions and RelevanceIn this prospective case series, nerve transfer surgery was associated with improvement of upper limb motor strength and functional independence in patients with tetraplegia. Nerve transfer is a promising intervention feasible in both subacute and chronic SCI.

OBJECTIVE Nerve transfers are increasingly being utilized in the treatment of chronic tetraplegia, with increasing literature describing significant improvements in sensorimotor function up to years after injury. However, despite technical advances, clinical outcomes remain heterogenous. Preoperative electrodiagnostic testing is the most direct measure of nerve health and may provide prognostic information that can optimize preoperative patient selection. The objective of this study in patients with spinal cord injury (SCI) was to determine various zones of injury (ZOIs) via electrodiagnostic assessment (EDX) to predict motor outcomes after nerve transfers in tetraplegia. METHODS This retrospective review of prospectively collected data included all patients with tetraplegia from cervical SCI who underwent nerve transfer at the authors’ institution between 2013 and 2020. Preoperative demographic data, results of EDX, operative details, and postoperative motor outcomes were extracted. EDX was standardized into grades that describe donor and recipient nerves. Five zones of SCI were defined. Motor outcomes were then compared based on various zones of innervation. RESULTS Nineteen tetraplegic patients were identified who underwent 52 nerve transfers targeting hand function, and 75% of these nerve transfers were performed more than 1 year postinjury, with a median interval to surgery following SCI of 24 (range 8–142) months. Normal recipient compound muscle action potential and isolated upper motor neuron injury on electromyography (EMG) were associated with greater motor recovery. When nerve transfers were stratified based on donor EMG, greater motor gains were associated with normal than with abnormal donor EMG motor unit recruitment patterns. When nerve transfers were separated based on donor and recipient nerves, normal flexor donors were more crucial than normal extensor donors in powering their respective flexor recipients. CONCLUSIONS This study elucidates the relationship of the preoperative innervation zones in SCI patients to final motor outcomes. EDX studies can be used to tailor surgical therapies for nerve transfers in patients with tetraplegia. The authors propose an algorithm for optimizing nerve transfer strategies in tetraplegia, whereby understanding the ZOI and grade of the donor/recipient nerve is critical to predicting motor outcomes.

This chapter will provide succinctly the key points to a successful surgery of benign peripheral nerve tumors. Briefly, the authors will discuss the clinical management of schwannomas and neurofibromas of the peripheral nerves, indications that range from a conservative management to a microsurgical resection. Furthermore, the authors will present important details for the successful microsurgical resection of these two types of neoplasm (illustrated by four case figures), including a step-wise technique for the safe tumor removal with preservation of all the functioning fascicles.

Schwann cells are the principal glial cells of the peripheral nervous system, and their development into myelinating glia is critically dependent on MEK/ERK signaling. Ets-domain transcription factors (Etv1, Etv4, Etv5) are common downstream effectors of MEK/ERK signalling, but so far, only Etv1 has been ascribed a role in Schwann cell development, and only in non-myelinating cells. Here, we examined the role of Etv5, which is expressed in Schwann cell precursors, including neural crest cells and satellite glia, in Schwann cell lineage development. We analysed Etv5tm1Kmm mutants (designated Etv5−/−) at embryonic days (E) 12.5, E15.5 and E18.5, focusing on dorsal root ganglia. At these embryonic stages, satellite glia (glutamine synthetase) and Schwann cell markers, including transcriptional regulators (Sox10, Sox9, Tfap2a, Pou3f1) and non-transcription factors (Ngfr, BFABP, GFAP), were expressed in the DRG of wild-type and Etv5−/− embryos. Furthermore, by E18.5, quantification of Sox10+ Schwann cells and NeuN+ neurons revealed that these cells were present in normal numbers in the Etv5−/− dorsal root ganglia. We next performed peripheral nerve injuries at postnatal day 21, revealing that Etv5−/− mice had an enhanced injury response, generating more Sox10+ Schwann cells compared to wild-type animals at five days post-injury. Thus, while Etv5 is not required for Schwann cell development, possibly due to genetic redundancy with Etv1 and/or Etv4, Etv5 is an essential negative regulator of the peripheral nerve injury repair response.SIGNIFICANCE STATEMENTOur study sought to determine whether the ets domain transcription factor, Etv5, plays a role in regulating Schwann cell development and nerve repair. By using an embryonically and postnatally viable hypomorphic Etv5 mutant allele, we demonstrated that Etv5 is not required for the development of Schwann cells or other neural crest derivatives in the dorsal root ganglia, including satellite glia and neurons. Surprisingly, loss of Etv5 had a direct impact on the Schwann cell repair response post-injury, resulting in more Schwann cells populating the distal injured nerve site compared to wild-type animals. Thus, this work describes for the first time a role for Etv5 in regulating the Schwann cell repair response after peripheral nerve injury.

OBJECTIVEThe current clinical standard of harvesting a nerve autograft for repair of long-segment peripheral nerve injuries (PNIs) is associated with many potential complications. Guidance channels offer an alternative therapy. The authors investigate whether autologous Schwann cells (SCs) implanted within a novel collagen-glycosaminoglycan conduit will improve axonal regeneration in a long-segment PNI model.METHODSNovel NeuraGen 3D collagen matrix conduits were implanted with autologous SCs to investigate axonal regeneration across a critical size defect (13 mm) in male Fischer rat sciatic nerve. Reversed sciatic nerve autografts served as positive controls, and conduits filled with serum only as negative controls. Electrophysiological assessments were made in vivo. Animals were killed at 4 or 16 weeks postinjury, muscle weights were measured, and grafts underwent immunohistochemical and morphometric analysis.RESULTSSC survival was confirmed by the presence of green fluorescent protein–labeled SCs within regenerated fibers. Regeneration and elongation of myelinated axons in all segments of the graft were significantly enhanced at 16 weeks in the SC-filled conduits compared to the conduit alone and were statistically similar to those of the autograft. Nerves repaired with SC-filled conduits exhibited onset latencies and nerve conduction amplitudes similar to those of the contralateral controls and autograft (p < 0.05). Adding SCs to the conduit also significantly reduced muscle atrophy compared to conduit alone (p < 0.0001).CONCLUSIONSRepair of long-segment PNI of rat sciatic nerve is significantly enhanced by SC-filled NeuraGen 3D conduits. Improvements in the total number of myelinated axons, axon diameter, and myelin thickness throughout SC-filled conduits allow for significant recovery in nerve conduction and a decrease in muscle atrophy.

Susceptibility to root-knot nematodes (Meloidogyne spp.) is one of the major factors limiting mungbean production in South and South-East Asia. Host-pest-environment interaction in mungbean and root-knot nematode (M. incognita) was investigated in multi-location field evaluation using 38 promising mungbean genotypes extracted from initial evaluation of 250 genotypes under sick plots considering second stage freshly hatched juvenile as inoculants. The extent of environmental and genotype-by-environment interactions (GGE) was assessed to comprehend the dynamism of resistance and identification of durable resistant mungbean genotypes. Among environmental factors, nematode activity was highly influenced by rainfall and minimum temperature. The GGE biplot and multiple comparison tests detected a higher proportion of genotype × environment (GE) interaction followed by genotype and environment on number of nematode galls, gall index and reproduction factor. The first two principal components (PCs) explained 64.33% and 66.99% of the total variation of the environment-centered gall scoring and reproduction factor data, respectively. The high GE variation indicated the presence of non-cross over interactions which justify the necessities of multi-location testing. Detection of non-redundant testing locations would expedite optimum resource utilization in future. The GGE biplot analysis identified genotypes such as PM-10-12, IPM-410-3 and NVL-641 as the outperforming and desirable genotypes with durable resistance against M. incognita which can be exploited in mungbean breeding programmes globally. On the contrary, the highest gall scoring and reproduction factor were recorded in genotype IPM-9901-8. Computation of confidence interval (CI) at 95% level through bootstrapping increased precision of GGE biplot towards genotype recommendation. Furthermore, total phenol content, ascorbic acid, phenlylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) activities were also higher in identified resistant genotypes and this information would be useful for devising mungbean breeding strategies in future for resistance against root-knot nematodes.

Background Brief electrical stimulation (ES) therapy to the nerve may improve outcome in lacerated, repaired nerves. However, most human nerve injuries leave the nerve in continuity with variable and often poor functional recovery from incomplete axon regeneration and reinnervation. Objective To evaluate the effect of brief ES in an experimental model for neuroma-in-continuity (NIC) injuries in rodents. Methods Lewis rats were randomly assigned to 1 of 4 groups: NIC injury immediately followed by brief (1 h) ES; NIC injury without ES; sham-operated controls; sciatic nerve transection without repair. Outcome measures included serial behavioral evaluation and electrophysiology together with terminal retrograde spinal cord motor neuron labeling and histomorphological analysis for axonal regeneration. Results Applying brief ES immediately after in-continuity nerve injury resulted in earlier recovery and significantly improved locomotion function at 4 and 6 wk. At 8 wk, brief ES resulted in higher compound action potential amplitude. By 12 wk there was no significant difference between the 2 groups in behavior or electrophysiology. Histomorphological analysis demonstrated a significantly higher percentage of neural tissue in the brief ES group. Spinal cord motor neuron pool cell counts revealed a preference for regeneration into a motor over a sensory nerve, for the group receiving ES. Conclusion The application of brief ES for in-continuity nerve injury promotes faster recovery, although in a rat model where regeneration distances are short the control group ultimately recovers to a similar degree. Brief EF requires further evaluation as a promising therapy for in-continuity nerve injuries in humans.

OBJECTIVEIntravital spectral imaging of the large, deeply situated nerves in the rat peripheral nervous system (PNS) has not been well described. Here, the authors have developed a highly stable platform for performing imaging of the tibial nerve in live rodents, thus allowing the capture of high-resolution, high-magnification spectral images requiring long acquisition times. By further exploiting the qualities of the topically applied myelin dye Nile red, this technique is capable of visualizing the detailed microenvironment of peripheral nerve demyelination injury and recovery, while allowing us to obtain images of exogenous Schwann cell myelination in a living animal.METHODSThe authors caused doxorubicin-induced focal demyelination in the tibial nerves of 25 Thy-1 GFP rats, of which 2 subsets (n = 10 each) received either BFP-labeled SKP-SCs or SCs to the zone of injury. Prior to acquiring images of myelin recovery in these nerves, a tibial nerve window was constructed using a silicone hemitube, a fast drying silicone polymer, and a small coverslip. This construct was then affixed to a 3D-printed nerve stage, which in turn was affixed to an external fixation/microscope stage device. Myelin visualization was facilitated by the topical application of Nile red.RESULTSThe authors reliably demonstrated intravital peripheral nerve myelin imaging with micron-level resolution and magnification, and minimal movement artifact. The detailed microenvironment of nerve remyelination can be vividly observed, while exogenously applied Schwann cells and skin-derived precursor Schwann cells can be seen myelinating axons.CONCLUSIONSTopically applied Nile red enables intravital study of myelin in the living rat PNS. Furthermore, the use of a tibial nerve window facilitates stable intravital peripheral nerve imaging, making possible high-definition spectral imaging with long acquisition times.

Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

Background To optimize survival evaluation of Schwann cells (SCs) in vivo , we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 β (NRG1β) improves in vivo survival. New method We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1β in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14 μm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed. Results Percentage cells counted in with- and without-NRG condition in both the groups were 0.83 ± 0.13% and 0.06 ± 0.04% (Group 1) & 2.83* ± 1.95% and 0.23* ± 0.29% (Group 2). Comparison to existing method We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome. Conclusions Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1β in the injectate media can improve survival.

Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

OBJECTIVEPatients with cervical spinal cord injury (SCI)/tetraplegia consistently rank restoring arm and hand function as their top functional priority to improve quality of life. Motor nerve transfers traditionally used to treat peripheral nerve injuries are increasingly being used to treat patients with cervical SCIs. In this study, the authors performed a systematic review summarizing the published literature on nerve transfers to restore upper-extremity function in tetraplegia.METHODSA systematic literature search was conducted using Ovid MEDLINE 1946–, Embase 1947–, Scopus 1960–, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and clinicaltrials.gov to identify relevant literature published through January 2019. The authors included studies that provided original patient-level data and extracted information on clinical characteristics, operative details, and strength outcomes after nerve transfer procedures. Critical review and synthesis of the articles were performed.RESULTSTwenty-two unique studies, reporting on 158 nerve transfers in 118 upper limbs of 92 patients (87 males, 94.6%) were included in the systematic review. The mean duration from SCI to nerve transfer surgery was 18.7 months (range 4 months–13 years) and mean postoperative follow-up duration was 19.5 months (range 1 month–4 years). The main goals of reinnervation were the restoration of thumb and finger flexion, elbow extension, and wrist and finger extension. Significant heterogeneity in transfer strategy and postoperative outcomes were noted among the reports. All but one case report demonstrated recovery of at least Medical Research Council grade 3/5 strength in recipient muscle groups; however, there was greater variation in the results of larger case series. The best, most consistent outcomes were demonstrated for restoration of wrist/finger extension and elbow extension.CONCLUSIONSMotor nerve transfers are a promising treatment option to restore upper-extremity function after SCI. Flexor reinnervation strategies show variable treatment effect sizes; however, extensor reinnervation may provide more consistent, meaningful recovery. Despite numerous published case reports describing good patient outcomes with nerve transfers, there remains a paucity in the literature regarding optimal timing and long-term clinical outcomes with these procedures.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

In this review article, the authors offer their perspective on nerve surgery for nerve injury, with a focus on recent evolution of management and the current surgical management. The authors provide a brief historical perspective to lay the foundations of the modern understanding of clinical nerve injury and its evolving management, especially over the last century. The shift from evaluation of the nerve injury using macroscopic techniques of exploration and external neurolysis to microscopic interrogation, interfascicular dissection, and internal neurolysis along with the use of intraoperative electrophysiology were important advances of the past 50 years. By the late 20th century, the advent and popularization of interfascicular nerve grafting techniques heralded a major advance in nerve reconstruction and allowed good outcomes to be achieved in a large percentage of nerve injury repair cases. In the past 2 decades, there has been a paradigm shift in surgical nerve repair, wherein surgeons are not only directing the repair at the injury zone, but also are deliberately performing distal-targeted nerve transfers as a preferred alternative in an attempt to restore function. The peripheral rewiring approach allows the surgeon to convert a very proximal injury with long regeneration distances and (often) uncertain outcomes to a distal injury and repair with a greater potential of regenerative success and functional recovery. Nerve transfers, originally performed as a salvage procedure for severe brachial plexus avulsion injuries, are now routinely done for various less severe brachial plexus injuries and many other proximal nerve injuries, with reliably good to even excellent results. The outcomes from nerve transfers for select clinical nerve injury are emphasized in this review. Extension of the rewiring paradigm with nerve transfers for CNS lesions such as spinal cord injury and stroke are showing great potential and promise. Cortical reeducation is required for success, and an emerging field of rehabilitation and restorative neurosciences is evident, which couples a nerve transfer procedure to robotically controlled limbs and mind-machine interfacing. The future for peripheral nerve repair has never been more exciting.

Total brachial plexus injury (BPI) typically results from high-energy vehicular accidents, affects mostly young adult males, and produces a flail, insensate limb. Because of the association of total BPI with head and cervical spine injuries, diagnosis might be delayed. Recognizing patients with total BPI and using electrodiagnostic and imaging tests in a timely fashion are critical. Advances in microsurgical techniques, primary nerve transfer, appropriate nerve graft utilization from a remaining intact (often C5) spinal nerve root, and free muscle transfers have improved outcomes. However, limited recovery even after reconstruction and severe deafferentation pain both remain challenging problems that further advancements will need to overcome.