Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans

Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regenerative capacity of neurons and the regenerative support of Schwann cells over time. Brief low-frequency electrical stimulation accelerates motor and sensory axon outgrowth across injury sites that, even after delayed surgical repair of injured nerves in animal models and patients, enhances nerve regeneration and target reinnervation. The stimulation elevates neuronal cyclic adenosine monophosphate and, in turn, the expression of neurotrophic factors and other growth-associated genes, including cytoskeletal proteins. Electrical stimulation of denervated muscles immediately after nerve transection and surgical repair also accelerates muscle reinnervation but, at this time, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical issue prior to translation to patients. Finally, the technique of inserting autologous nerve grafts that bridge between a donor nerve and an adjacent recipient denervated nerve stump significantly improves nerve regeneration after delayed nerve repair, the donor nerves sustaining the capacity of the denervated Schwann cells to support nerve regeneration. These reviewed methods to promote nerve regeneration and, in turn, to enhance functional recovery after nerve injury and surgical repair are sufficiently promising for early translation to the clinic.     

Impaired peripheral nerve regeneration in type‐2 diabetic mouse model

Peripheral neuropathy is one of the most common and serious complications of type‐2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type‐2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection‐regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type‐2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age‐matched non‐diabetic mice after axotomy. Furthermore, type‐2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type‐2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy.     

Combination of BMSCs‐laden acellular nerve xenografts transplantation and G‐CSF administration promotes sciatic nerve regeneration

Peripheral nerve gaps often lead to interrupted innervation, manifesting as severe sensory and motor dysfunctions. The repairs of the nerve injuries have not achieved satisfactory curative effects in clinic. The transplantation of bone marrow stromal cells (BMSCs)‐laden acellular nerve xenografts (ANX) has been proven more effective than the acellular nerve allografting. Besides, granulocyte colony‐stimulating factor (G‐CSF) can inhibit inflammation and apoptosis, and thus is conducive to the microenvironmental improvement of axonal regeneration. This study aims to investigate the joint effect of BMSCs‐seeded ANX grafting and G‐CSF administration, and explore the relevant mechanisms. Adult SD rats were divided into five groups randomly: ANX group, ANX combined with G‐CSF group, BMSCs‐laden ANX group, BMSCs‐laden ANX combined with G‐CSF group, and autograft group. Eight weeks after transplantation, the detection of praxiology and neuroelectrophysiology was conducted, and then the morphology of the regenerated nerves was analyzed. The inflammatory response and apoptosis in the nerve grafts as well as the expression of the growth‐promoting factors in the regenerated tissues were further assayed. G‐CSF intervention and BMSCs implanting synergistically promoted peripheral nerve regeneration and functional recovery following ANX bridging, and the restoration effect was matchable with that of the autologous nerve grafting. Moreover, local inflammation was alleviated, the apoptosis of the seeded BMSCs was decreased, and the levels of the neuromodulatory factors were elevated. In conclusion, the union application of BMSCs‐implanted ANX and G‐CSF ameliorated the niche of neurotization and advanced nerve regeneration substantially. The strategy achieved the favorable effectiveness as an alternative to the autotransplantation.     

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3 V, 20 Hz, 20 min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24 weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5 mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro‐Gold retrograde‐labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24 weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain‐derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF‐mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.     

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57 : 821–828, 2018     

Granulocyte‐macrophage colony‐stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush

Peripheral nerve injuries severely impair patients’ quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM‐CSF potentiated early axon growth, as indicated by the enhanced expression of growth‐associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM‐CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM‐CSF stimulated early brain‐derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM‐CSF treatment. The present results suggest that GM‐CSF may have beneficial effects on early axonal regeneration.     

Sensory nerves impair sympathetic reinnervation and recovery of smooth muscle function.

Neuronal populations projecting to a common target may compete for neurotrophic substances. To determine if competition impairs target reinnervation, we examined the effect of capsaicin-induced sensory denervation on sympathetic nerve ingrowth to the sympathectomized rat superior tarsal smooth muscle. In tarsal muscles with intact sympathetic innervation, capsaicin injection on Day 2 reduced numbers of perimuscular CGRP-ir sensory nerves by 68% at 3-4 months; however, it did not alter dopamine-beta-hydroxylase-ir nerve density, response to nerve stimulation, or tarsal muscle adrenoceptor-mediated contraction. Tarsal muscles denervated by ipsilateral superior cervical ganglionectomy on Postnatal Day 4 were partially reinnervated by fibers from the contralateral ganglion, as noted in previous studies. Sensory denervation by capsaicin improved sympathetic reinnervation, as evidenced by a 174% increase in numbers of DBH-ir nerves and a 62% increase in neurally mediated smooth muscle contraction evoked by electrical stimulation of the contralateral pathway relative to reinnervated muscles of vehicle-injected rats; smooth muscle function was also influenced, as indicated by a decrease toward normal in adrenoceptor sensitivity. Tarsal muscles denervated at 30 days were not reinnervated in either vehicle-injected or capsaicin-treated rats, indicating that sensory denervation does not extend the developmental window during which contralateral reinnervation can occur. Both the vehicle-injected and capsaicin-treated preparations with sustained juvenile sympathectomy showed sensory hyperinnervation as adults; thus, a chronic reduction in competition from sympathetics is a sufficiently powerful stimulus to overcome the decreased nerve density induced by neonatal capsaicin treatment. We conclude that sensory nerves limit the extent of sympathetic reinnervation and functional recovery that can occur following neonatal sympathetic denervation.     

The Neuregulin‐Rac‐MKK7 pathway regulates antagonistic c‐jun/Krox20 expression in Schwann cell dedifferentiation

Schwann cells respond to nerve injury by dedifferentiating into immature states and producing neurotrophic factors, two actions that facilitate successful regeneration of axons. Previous reports have implicated the Raf‐ERK cascade and the expression of c‐jun in these Schwann cell responses. Here we used cultured primary Schwann cells to demonstrate that active Rac1 GTPase (Rac) functions as a negative regulator of Schwann cell differentiation by upregulating c‐jun and downregulating Krox20 through the MKK7‐JNK pathway, but not through the Raf‐ERK pathway. The activation of MKK7 and induction of c‐jun in sciatic nerves after axotomy was blocked by Rac inhibition. Microarray experiments revealed that the expression of regeneration‐associated genes, such as glial cell line‐derived neurotrophic factor and p75 neurotrophin receptor, after nerve injury was dependent on Rac but not on ERK. Finally, the inhibition of ErbB2 signaling prevented MKK7 activation, c‐jun induction, and Rac‐dependent gene expression in sciatic nerve explant cultures. Taken together, our results indicate that the neuregulin‐Rac‐MKK7‐JNK/c‐jun pathway regulates Schwann cell dedifferentiation following nerve injury.     

Altered cutaneous nerve regeneration in a simian immunodeficiency virus / macaque intracutaneous axotomy model

To characterize the regenerative pattern of cutaneous nerves in simian immunodeficiency virus (SIV)‐infected and uninfected macaques, excisional axotomies were performed in nonglabrous skin at 14‐day intervals. Samples were examined after immunostaining for the pan‐axonal marker PGP 9.5 and the Schwann cell marker p75 nerve growth factor receptor. Collateral sprouting of axons from adjacent uninjured superficial dermal nerve bundles was the initial response to axotomy. Both horizontal collateral sprouts and dense vertical regeneration of axons from the deeper dermis led to complete, rapid reinnervation of the epidermis at the axotomy site. In contrast to the slower, incomplete reinnervation previously noted in humans after this technique, in both SIV‐infected and uninfected macaques epidermal reinnervation was rapid and completed by 56 days postaxotomy. p75 was densely expressed on the Schwann cells of uninjured nerve bundles along the excision line and on epidermal Schwann cell processes. In both SIV‐infected and uninfected macaques, Schwann cell process density was highest at the earliest timepoints postaxotomy and then declined at a similar rate. However, SIV‐infection delayed epidermal nerve fiber regeneration and remodeling of new sprouts at every timepoint postaxotomy, and SIV‐infected animals consistently had lower mean epidermal Schwann cell densities, suggesting that Schwann cell guidance and support of epidermal nerve fiber regeneration may account for altered nerve regeneration. The relatively rapid regeneration time and the completeness of epidermal reinnervation in this macaque model provides a useful platform for assessing the efficacy of neurotrophic or regenerative drugs for sensory neuropathies including those caused by HIV, diabetes mellitus, medications, and toxins. J. Comp. Neurol. 514:272–283, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of FK506 on nerve regeneration and reinnervation after graft or tube repair of long nerve gaps.

We compared the effects of FK506 administration on regeneration and reinnervation after sciatic nerve resection and repair with an autologous graft or with a silicone tube leaving a 6-mm gap in the mouse. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and sweating functions in the hindpaw over 4 months after operation. Morphometric analysis of the regenerated nerves was performed at the end of follow-up. The nerve graft allowed for faster and higher levels of reinnervation in the four functions tested than silicone tube repair. Treatment with FK506 (for the first 9 weeks only) resulted in a slight, although not significant, improvement of the onset of reinnervation and of the maximal degree of recovery achieved after autografting. The recovery of pain sensibility and of the compound nerve action potentials in the digital nerves, which directly depend on axonal regeneration, showed better progression with FK506 than reinnervation of muscles and sweat glands, which require reestablishment of synaptic contacts with target cells. The myelinated fibers in the regenerated nerve showed a more mature appearance in the FK506-treated rats. However, FK506 showed a marginal effect in situations in which regeneration was limited, as in a silicone tube bridging a 6-mm gap in the mouse sciatic nerve. In conclusion, treatment with FK506 improved the rate of functional recovery after nerve resection and autograft repair.     

Factors limiting motor recovery after facial nerve transection in the rat: combined structural and functional analyses

It is believed that a major reason for the poor functional recovery after peripheral nerve lesion is collateral branching and regrowth of axons to incorrect muscles. Using a facial nerve injury protocol in rats, we previously identified a novel and clinically feasible approach to combat axonal misguidance--the application of neutralizing antibodies against neurotrophic factors to the injured nerve. Here, we investigated whether reduced collateral branching at the lesion site leads to better functional recovery. Treatment of rats with antibodies against nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factor, insulin-like neurotrophic factor I, ciliary neurotrophic factor or glial cell line-derived neurotrophic factor increased the precision of reinnervation, as evaluated by multiple retrograde labelling of motoneurons, more than two-fold as compared with control animals. However, biometric analysis of vibrissae movements did not show positive effects on functional recovery, suggesting that polyneuronal reinnervation--rather than collateral branching --may be the critical limiting factor. In support of this hypothesis, we found that motor end-plates with morphological signs of multiple innervation were much more frequent in reinnervated muscles of rats that did not recover after injury (51% of all end-plates) than in animals with good functional performance (10%). Because polyneuronal innervation of muscle fibres is activity-dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies could be soon designed and tested.     

Effects of motor and sensory nerve transplants on amount and specificity of sciatic nerve regeneration

Nerve regeneration after complete transection does not allow for adequate functional recovery mainly because of lack of selectivity of target reinnervation. We assessed if transplanting a nerve segment from either motor or sensory origin may improve specifically the accuracy of sensory and motor reinnervation. For this purpose, the rat sciatic nerve was transected and repaired with a silicone guide containing a predegenerated segment of ventral root (VR) or dorsal root (DR), compared to a silicone guide filled with saline. Nerve regeneration and reinnervation was assessed during 3 months by electrophysiologic and functional tests, and by nerve morphology and immunohistochemistry against choline acetyltransferase (ChAT) for labeling motor axons. Functional tests showed that reinnervation was successful in all the rats. However, the two groups with a root allotransplant reached higher degrees of reinnervation in comparison with the control group. Group VR showed the highest reinnervation of muscle targets, whereas Group DR had higher levels of sensory reinnervation than VR and saline groups. The total number of regenerated myelinated fibers was similar in the three groups, but the number of ChAT+ fibers was slightly lower in the VR group in comparison with DR and saline groups. These results indicate that a predegenerated root nerve allotransplant enhances axonal regeneration, leading to faster and higher levels of functional recovery. Although there is not clear preferential reinnervation, regeneration of motor axons is promoted at early times by a motor graft, whereas reinnervation of sensory pathways is increased by a sensory graft.     

The impact of motor and sensory nerve architecture on nerve regeneration

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

Accuracy of reinnervation by peripheral nerve axons regenerating across a 10-mm gap within an impermeable chamber.

The axon regeneration following a peripheral nerve injury often fails to restore a complete functional recovery. One of the causes of this unsatisfactory result has been attributed to regrowth of regenerating fibers to inappropriate peripheral targets. The accuracy of reinnervation by axons regenerating across a 10-mm gap within an impermeable chamber has been studied by using a sequential retrograde double-labeling technique. Despite the long gap between the nerve stumps, at 4 weeks a mean of 30.5% of the regenerating axons can reinnervate the original muscular area. These data confirm previous studies in which a preferential reinnervation is reported not to be absolutely dependent on the axon's mechanical alignment.     

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Schwann cells seeded in acellular nerve grafts improve functional recovery

Introduction: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold‐preserved acellular nerve grafts (CP‐ANGs) would improve functional regeneration compared with nerve isografts. Methods: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP‐ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14‐mm rat sciatic injury model and compared with isografts. Results: At 14 days, motor or sensory‐derived SCs increased expression of growth factors in CP‐ANGs versus isografts. After 42 days, histomorphometric analysis found CP‐ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP‐ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. Conclusions: SCs transplanted into CP‐ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect. Muscle Nerve 49 : 267–276, 2014