Inflammation near the nerve cell body enhances axonal regeneration

Although crushed axons in a dorsal spinal root normally regenerate more slowly than peripheral axons, their regeneration can be accelerated by a conditioning lesion to the corresponding peripheral nerve. These and other observations indicate that injury to peripheral sensory axons triggers changes in their nerve cell bodies that contribute to axonal regeneration. To investigate mechanisms of activating nerve cell bodies, an inflammatory reaction was provoked in rat dorsal root ganglia (DRG) through injection of Corynebacterium parvum. This inflammation enhanced regeneration in the associated dorsal root, increasing 4-fold the number of regenerating fibers 17 d after crushing; peripheral nerve regeneration was not accelerated. A milder stimulation of dorsal root regeneration was detected after direct injection of isogenous macrophages into the ganglion. It is concluded that changes favorable to axonal regeneration can be induced by products of inflammatory cells acting in the vicinity of the nerve cell body. Satellite glial cells and other unidentified cells in lumbar DRG were shown by thymidine radioautography to proliferate after sciatic nerve transection or injection of C. parvum into the ganglia. Intrathecal infusion of mitomycin C suppressed axotomy-induced mitosis of satellite glial cells but did not impede axonal regeneration in the dorsal root or the peripheral nerve. Nevertheless, the similarity in reactions of satellite glial cells during 2 processes that activate neurons adds indirect support to the idea that non-neuronal cells in the DRG might influence regenerative responses of primary sensory neurons.     

Rho-kinase inhibition enhances axonal regeneration after peripheral nerve injury

In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho-kinase, retards axon regeneration of injured motoneurons, we employed a Rho-kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho-kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho-kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.     

Sciatic nerve regeneration across gaps within silicone chambers: long-term effects of NGF and consideration of axonal branching

We examined whether the short-term beneficial effects of nerve growth factor (NGF) upon regeneration are sustained over a prolonged period of time across 8-mm gaps within silicone chambers. Rat sciatic nerve regeneration both with and without NGF was examined after 10 weeks. Myelinated counts from the regenerated sciatic and distal tributary nerves were correlated to the numbers of motor and sensory neurons retrogradely labeled with horseradish peroxidase (HRP) applied distal to the regenerated segment. Regenerated sciatic and sural nerves were examined ultrastructurally for morphological analysis. Both regenerated groups by 10 weeks achieved essentially complete counts of myelinated axons in the distal tributary nerves and the regenerated segment of the sciatic nerve compared to the uninjured controls. There were similar numbers of retrogradely labeled sensory and motor neurons in the dorsal root ganglia (DRG) and lumbar spinal cord of both groups and, surprisingly, of the uninjured normal control group. Ultrastructural analysis demonstrated no difference in the distribution of axonal diameters or myelin thickness between the regenerated groups. In evaluating regeneration in experimental silicone chamber models, it is important to determine such parameters as the percentage of neurons that grow across the gap and the incidence of axonal sprouting. One can then make accurate assessments of experimental perturbations and predict whether they improve the naturally occurring regeneration through chambers. These results must ultimately be compared with equivalent determinations in the uninjured nerve. At 10 weeks there was essentially complete regeneration of both the NGF and control regenerative groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.     

Prior collateral sprouting enhances axonal regeneration

Regeneration is accelerated in axons which have undergone collateral sprouting prior to crushing.     

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3‐treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine α‐bungarotoxin and neurofilament antibody. Four weeks after surgery, most end‐plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS‐treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission. © 2010 Wiley‐Liss, Inc.     

Basic fibroblast growth factor released from synthetic guidance channels facilitates peripheral nerve regeneration across long nerve gaps

Basic fibroblast growth factor (b-FGF) has been shown to enhance the in vitro survival and neurite extension of various types of neurons including dorsal root ganglia (DRG) cells. Alpha-1 glycoprotein (alpha 1-GP), an acute phase reactant, has been reported to enhance the in vitro neuritic extensions of chick DRG cells. In the present study, we investigated the ability of synthetic nerve guidance channels, which release sustained controlled amounts of b-FGF and/or alpha 1-GP, to support the regeneration of a transected peripheral nerve over a 15 mm long gap, a distance that does not permit regeneration in conventional polymeric tubes. Tubes releasing bovine serum albumin (BSA), cytochrome C, BSA and b-FGF, BSA and denatured b-FGF, BSA and alpha 1-GP, or BSA, b-FGF, and alpha 1-GP were fabricated by a dip-molding technique. In vitro kinetic studies of protein release from these channels showed an initial burst during the 1st day, followed by a linear release for at least 2 weeks thereafter. In vitro studies indicated that the b-FGF released from the polymer was biologically active as assessed by the ability of channels releasing b-FGF to induce neurite extensions in PC12 cells. For in vivo studies, the various types of tubes were used as nerve guidance channels for the repair of a 15 mm nerve gap in the sciatic nerve of rats. Four weeks postimplantation, only the tubes releasing b-FGF or b-FGF and alpha 1-GP displayed regenerated cables bridging both nerve stumps, which contained nerve fascicles with myelinated and unmyelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.     

Intraspinal microinjection of chondroitinase ABC following injury promotes axonal regeneration out of a peripheral nerve graft bridge

Chondroitin sulfate proteoglycans (CSPG) within the glial scar formed after central nervous system (CNS) injury are thought to play a crucial role in regenerative failure. We previously showed that delivery of the CSPG-digesting enzyme chondroitinase ABC (ChABC) via an osmotic minipump allowed axonal regeneration and functional recovery in a peripheral nerve graft (PNG)-bridging model. In this study, we sought to overcome the technical limitations associated with minipumps by microinjecting ChABC directly into the distal lesion site in the PN bridging model. Microinjection of ChABC immediately rostral and caudal to an injury site resulted in extensive CSPG digestion. We also demonstrate that this delivery technique is relatively atraumatic and does not result in a noticeable inflammatory response. Importantly, microinjections of ChABC into the lesion site permitted more regenerating axons to exit a PNG and reenter spinal cord tissue than saline injections. These results are similar to our previous findings when ChABC was delivered via a minipump and suggest that microinjecting ChABC is an effective method of delivering the potentially therapeutic enzyme directly to an injury site.     

Exercise training and axonal regeneration after sciatic nerve injury.

In the present study, we aimed to investigate the relationship between exercise training and peripheral nerve regeneration after crush injury. For this purpose, HRP neurohistochemistry and modified Pal-Weigert methods were used to assess the axonal regeneration. In the 2nd and 3rd regeneration week groups, myelin debris was observed, and there was no significant difference between exercise trained and sedentary groups. In the 4th regeneration week group, it was seen that myelin debris was removed, and some myelinated fibers were observed in the exercise trained group. On the other hand, there was no myelinated fiber in the sedentary group, and there was a significant difference between exercise trained and sedentary groups. Consequently, we think that exercise is effective in the 4th regeneration week.     

Calcium channel inhibition-mediated axonal stabilization improves axonal regeneration after optic nerve crush

正Axonal projections are specialized neuronal compartments and the longest parts of neurons.Axonal degeneration is a common pathological feature in many neurodegenerative disorders,such as Parkinson’s disease,amyotrophic lateral sclerosis,glaucoma,as well as in traumatic lesions of the central nervous system(CNS),such as spinal cord injury.In many neurological disorders,the axon is     

Valproic acid enhances axonal regeneration and recovery of motor function after sciatic nerve axotomy in adult rats

It has recently been demonstrated that valproic acid (VPA) robustly promotes neurite outgrowth, activates the extracellular signal regulated kinase pathway, and increases growth cone-associated protein 43 and bcl-2 levels in cultured human neuroblastoma SH-SY5Y cells. We hypothesized that VPA could also enhance peripheral nerve regeneration in adult animals. To test this hypothesis, we examined the effects of VPA (300 mg/kg daily for 16 weeks) on sciatic axonal regeneration following single or conditional axotomies in rats. The results showed that in VPA-treated rats there was a significant increase in the total numbers of regenerated myelinated nerve fibers and reinnervated muscle fibers in comparison with those rats not treated with VPA. As measured by sciatic function index and toe spread index, the motor function of the reinnervated hind limbs of rats receiving single axotomy without VPA treatment significantly improved at week 8 and reached plateau levels at about week 11, whereas the motor function of the reinnervated hind limbs of rats receiving single axotomy plus VPA and rats receiving conditional axotomy with or without VPA treatment significantly improved at week 4 and reached plateau levels at about week 8; there was no significant difference of the motor function among the three later groups. The results demonstrated that VPA is able to enhance sciatic nerve regeneration and recovery of motor function in adult rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.     

Inhibition of p38 MAP kinase activity enhances axonal regeneration

Tumor necrosis factor alpha (TNF)-induced cellular signaling through the p38 mitogen-activated protein kinase (p38 MAPK) pathway plays a critical role in Wallerian degeneration and subsequent regeneration, processes that depend on Schwann cell (SC) activity. TNF dose-dependently induces Schwann cell and macrophage activation in vivo and apoptosis in primary SC cultures in vitro, while inhibition of p38 MAPK is thought to block these cellular processes. We show with Western blots that after sciatic nerve crush injury, phosphorylated p38 (p-p38) MAPK is significantly increased (P < 0.01) in distal nerve segments. In tissue sections, p38 co-localized immunohistochemically with activated Schwann cells (GFAP) and to a lesser degree with macrophages (ED-1). In other experiments, animals were gavaged with Scios SD-169 (10 or 30 mg/kg) or excipient (PEG300) 1 day before and daily after crush injury to the sciatic nerve. SD-169 is a proprietary oral inhibitor of p38 MAPK activity. The rate of axonal regeneration was determined by the functional pinch test and was significantly increased in treated animals 8 days after crush injury (P < 0.05; 30 mg/kg dose). In SD-169-treated animals with nerve transection, nerve fibers regenerating through a silicone chamber were morphologically more mature than untreated nerves when observed 28 days after transection. TNF immunofluorescence of distal nerve segments after crush injury suggested that SD-169 reduced SC TNF protein. In support of these findings, SD-169 significantly reduced (P < 0.05) TNF-mediated primary SC death in culture experiments. We conclude that inhibition of p38 activity promotes axonal regeneration through interactions with SC signaling and TNF activity.     

Chondroitinase ABC promotes axonal regeneration of Clarke's neurons after spinal cord injury

We examined whether enzymatic digestion of chondroitin sulfate (CS) promoted the axonal regeneration of neurons in Clarke's nucleus (CN) into a peripheral nerve (PN) graft following injury of the spinal cord. After hemisection at T11, a segment of PN graft was implanted at the lesion site. Either vehicle, brain-derived neurotrophic factor (BDNF) or chondroitinase ABC was applied at the implantation site. The postoperative survival period was 4 weeks. Treatment with vehicle or BDNF did not promote the axonal regeneration of CN neurons into the PN graft. Application of 2.5 unit/ml chondroitinase ABC resulted in a significant increase (12.8%) in the number of regenerated CN neurons. Double labeling with Fluoro-Gold and NADPH-diaphorase histochemistry showed that the regenerated CN neurons did not express nitric oxide synthase (NOS). Our results suggest that CS is inhibitory to the regeneration of CN neurons following injury of the spinal cord.     

Neuregulin is associated with nerve regeneration in axonal neuropathies

Neuregulins (NRG), the only trophic factors known to support Schwann cell survival, have a potential for use in treatment of patients with chronic neuropathies. Expression of one NRG isoform, NRG-beta1, was increased in axons in the early phase of axonal degeneration in nerve biopsy specimens from patients with neuropathy. Regenerating nerve fibers contained abundant NRG-beta1 in axons, but nerves from patients with chronic axonal neuropathy showed less. NRG-beta1 may act as an axonally derived signal having a trophic effect on denervated Schwann cells, facilitating their supportive role in axonal regeneration.     

In vitro pre-degenerated nerve autografts support CNS axonal regeneration

In the present study we compared, in adult rats, the axonal regeneration of central respiratory neurons within autologous fresh (f-; grafted immediately after removal) and pre-degenerated (pd-; grafted after being stored during 3 days in saline at + 8°C) peripheral nerve grafts (PNGs) implanted within the C2 cervical spinal cord. The proximal end of the left peroneal nerve was implanted in the site of projection of medullary respiratory neurons (ventro-lateral quadrant) and the distal part of each nerve graft was left unconnected (blind-ended graft). PNGs were examined 2 to 4 months after grafting. Central neurons regenerating axons within the PNGs were studied by recording spontaneous unit activity from small strands teased from the grafts. In control f-PNGs (n = 9), 248 filaments had spontaneous activities, 58 of these were respiratory-related, i.e. had discharge patterns identical to those of normal respiratory (inspiratory and expiratory) neurons. The presence of regenerated nerve fibers with spontaneous unitary impulse traffic (n = 216) was found in all pd-PNGs (n = 5). Thirty-four had respiratory patterns identical to those found within f-PNGs and corresponded to efferent activity. No statistically significant differences in axonal regrowth were found between f- and pd-PNGs. In conclusion, f- and pd-PNGs were equally capable of promoting axonal regeneration of central neurons. The neural components (Schwann cells and others) required for axonal regeneration of adult central neurons are still effective following 3 days of in vitro peripheral nerve degeneration without special storage conditions (oxygenation, medium inducing ATP synthesis). These results have clinical implications for nerve graft surgery when time is required for typing the tissues of both donor and recipient (post-mortem allografts) or transportation of graft material.     

Controlled release of nerve growth factor enhances sciatic nerve regeneration

Based on previous studies demonstrating the potential of growth factors to enhance peripheral nerve regeneration, we developed a novel growth factor delivery system to provide sustained delivery of nerve growth factor (NGF). This delivery system uses heparin to immobilize NGF and slow its diffusion from a fibrin matrix. This system has been previously shown to enhance neurite outgrowth in vitro, and in this study, we evaluated the ability of this delivery system to enhance nerve regeneration through conduits. We tested the effect of controlled NGF delivery on peripheral nerve regeneration in a 13-mm rat sciatic nerve defect. The heparin-containing delivery system was studied in combination with three doses of NGF (5, 20, or 50 ng/mL) and the results were compared with positive controls (isografts) and negative controls (fibrin alone, NGF alone, and empty conduits). Nerves were harvested at 6 weeks postoperatively for histomorphometric analysis. Axonal regeneration in the delivery system groups revealed a marked dose-dependent effect. The total number of nerve fibers at both the mid-conduit level and in the distal nerve showed no statistical difference for NGF doses at 20 and 50 ng/mL from the isograft (positive control). The results of this study demonstrate that the incorporation of a novel delivery system providing controlled release of growth factors enhances peripheral nerve regeneration and represents a significant contribution toward enhancing nerve regeneration across short nerve gaps.     

Chondroitinase applied to peripheral nerve repair averts retrograde axonal regeneration

Antegrade, target-directed axonal regeneration is the explicit goal of nerve repair. However, aberrant and dysfunctional regrowth is commonly observed as well. At the site of surgical nerve coaptation, axonal sprouts encounter fibrotic connective tissue rich in growth-inhibiting chondroitin sulfate proteoglycan that may contribute to misdirection of axonal regrowth. In the present study, we tested the hypothesis that degradation of chondroitin sulfate proteoglycan by application of chondroitinase at the site of nerve repair can decrease aberrant axonal growth. Adult rats received bilateral sciatic nerve transection and end-to-end repair. One nerve was injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone. After 28 weeks, retrograde axonal regeneration was assessed proximal to the repair by scoring neurofilament-immunopositive axons within the nerve (intrafascicular) and outside the nerve proper (extrafascicular). Intrafascicular retrograde axonal growth was equivalent in both control and chondroitinase treatment conditions. In contrast, chondroitinase treatment caused a pronounced (93%) reduction in extrafascicular retrograde axonal growth. The decrease in axon egress from the nerve was coincident with an increase in antegrade regeneration and improved recovery of motor function. Based on these findings, we conclude that chondroitinase applied at the site of nerve transection repair averts dysfunctional extrafascicular retrograde axonal growth.     

Spontaneous axonal regeneration after optic nerve injury in adult rat

Optic nerves of adult rats were crushed 2 mm behind the eye to examine the ability of retinal ganglion cells (RGCs) to regenerate their axons. Some animals were treated with the immunophilin ligands FK 506 or GPI 1046 for up to 4 weeks. After 10 days to 16 months, regenerating RGC axons were visualized using anterograde tracing and/or electron microscopy. A small proportion of RGC axons regenerated across the lesion site and grew very slowly along the entire optic nerve. Immunophilin ligands had no obvious effect. The regenerating axons were about 0.2 microm in diameter, and usually in clusters surrounded by astrocyte processes. Thus, some CNS axons can spontaneously regenerate long distances within degenerate white matter and this slow regeneration is not accelerated by immunophilin ligands.     

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