Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.

Background Neuropathic pain is a major pathology of the central nervous system associated with neuroinflammation. Ryk (receptor-like tyrosine kinase) receptors act as repulsive axon-guidance molecules during development of central nervous system and neural injury. Increasing evidence suggests the potential involvement of Wnt/Ryk (wingless and Int) signaling in the pathogenesis of neuropathic pain. However, its underlying mechanism remains unknown. Results The expression and location of Ryk receptor as well as its ligand Wnt1 were detected by qPCR, Western blot, and immunohistochemistry. We found that Ryk, a specific Wnt receptor, was expressed in IB4+ (Isolectin B4) and CGRP+ (calcitonin gene-related peptide) dorsal root ganglia neurons and their ascending unmyelinated fibers in the dorsal horn of the spinal cord. Ryk was upregulated after spinal nerve ligation surgery. Wnt1 was also increased in activated astrocytes in the dorsal horn after spinal nerve ligation. The presynaptic mechanism of Ryk in regulation of neuropathic pain was determined by electrophysiology in spinal slice. Spinal nerve ligation model was established, and the therapeutic potential of inhibiting Ryk receptor was determined. Spine-specific blocking of the Wnt/Ryk receptor signaling attenuated the spinal nerve ligation-induced mechanical allodynia but not thermal hyperalgesia. Further, it also blocked Ca2+-dependent signals including CaMKII and PKCγ, subsequent release of CCL2 (CCR-like protein) in the dorsal horn. An in vitro study showed that inactivating Ryk receptors with anti-Ryk antibodies or lentiviral Ryk shRNA led to the inactivation of Wnt1 for excitatory synaptic transmission in spinal slices and subsequent decrease in CCL2 expression in the dorsal root ganglia neurons. Conclusion These studies demonstrate the existence of critical crosstalk between astrocytes and unmyelinated fibers, which indicate the presynaptic mechanism of Ryk in cytokine transmission of neuropathic pain and the therapeutic potential for Wnt/Ryk signaling pathway in the treatment of neuropathic pain.

Histomorphometric studies of unmyelinated fibers of the rat fibular nerves are uncommon, and side-to-end neurorrhaphy studies using the fibular nerve investigate primarily motor fibers. We investigated side-to-end tubulization (SET) technique, in which occurs collateral sprouting from the intact donor nerve fibers to the distal stump of receptor nerve, with muscle reinnervation and functional rehabilitation, to assess whether there is a successful growth of unmyelinated fibers in this model.

Pain during mechanical stimulation of the joint and spontaneous pain are major symptoms of arthritis. An important neuronal process of mechanical hypersensitivity of the joint is the sensitization of thin myelinated Adelta fibers and unmyelinated C fibers innervating the joint. Because interleukin-6 (IL-6) is a major inflammatory mediator, we investigated whether this cytokine has the potential to sensitize joint afferents to mechanical stimuli.

Postherpetic neuralgia (PHN) is a common and exceptionally drug-resistant neuropathic pain condition. In this cross-sectional skin biopsy study, seeking information on the responsible pathophysiological mechanisms we assessed how ophthalmic PHN affects sensory and autonomic skin innervation. We took 2-mm supraorbital punch skin biopsies from the affected and unaffected sides in 10 patients with ophthalmic PHN. Using indirect immunofluorescence and a large panel of antibodies including protein gene product (PGP) 9.5 we quantified epidermal unmyelinated, dermal myelinated and autonomic nerve fibers. Although skin biopsy showed reduced epidermal and dermal myelinated fiber density in specimens from the affected side, the epidermal/dermal myelinated nerve fiber ratio was lower in the affected than in the unaffected side (p < 0.001), thus suggesting a predominant epidermal unmyelinated nerve fiber loss. Conversely, autonomic skin innervation was spared. Our study showing that ophthalmic PHN predominantly affects unmyelinated nerve fiber and spares autonomic nerve fiber might help to understand the pathophysiological mechanisms underlying this difficult-to-treat condition.

In mammalian peripheral nerves, unmyelinated C-fibers usually outnumber myelinated A-fibers. By using transmission electron microscopy, we recently showed that the saphenous nerve of the naked mole-rat (Heterocephalus glaber) has a C-fiber deficit manifested as a substantially lower C:A-fiber ratio compared with other mammals. Here we determined the uniqueness of this C-fiber deficit by performing a quantitative anatomical analysis of several peripheral nerves in five further members of the Bathyergidae mole-rat family: silvery (Heliophobius argenteocinereus), giant (Fukomys mechowii), Damaraland (Fukomys damarensis), Mashona (Fukomys darlingi), and Natal (Cryptomys hottentotus natalensis) mole-rats. In the largely cutaneous saphenous and sural nerves, the naked mole-rat had the lowest C:A-fiber ratio (∼1.5:1 compared with ∼3:1), whereas, in nerves innervating both skin and muscle (common peroneal and tibial) or just muscle (lateral/medial gastrocnemius), this pattern was mostly absent. We asked whether lack of hair follicles alone accounts for the C-fiber paucity by using as a model a mouse that loses virtually all its hair as a consequence of conditional deletion of the β-catenin gene in the skin. These β-catenin loss-of function mice (β-cat LOF mice) displayed only a mild decrease in C:A-fiber ratio compared with wild-type mice (4.42 compared with 3.81). We suggest that the selective cutaneous C-fiber deficit in the cutaneous nerves of naked mole-rats is unlikely to be due primarily to lack of skin hair follicles. Possible mechanisms contributing to this unique peripheral nerve anatomy are discussed.

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level [Formula: see text] score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems.

SSeCKS (Src SupprEssed C Kinase Substrate) is a proposed protein kinase C substrate/A kinase anchoring protein (AKAP) that has recently been characterized in the rat peripheral nervous system. It has been shown that approximately 40% of small primary sensory neurons contain SSeCKS-immunoreactivity in a population largely separate from substance P (95.2%), calcitonin gene related peptide (95.3%), or fluoride resistant acid phosphatase (55.0%) labeled cells. In the spinal cord, it was found that SSeCKS-immunoreactive axon collaterals terminate in the dorsal third of lamina II outer in a region similar to that of unmyelinated C-, or small diameter myelinated Adelta-, fibers. However, the precise characterization of the anatomical profile of the primary sensory neurons containing SSeCKS remains to be determined. Here, immunohistochemical labeling at the light and ultrastructural level is used to clarify the myelination status of SSeCKS-containing sensory neuron axons and to further clarify the morphometric, and provide insight into the functional, classification of SSeCKS-IR sensory neurons.

Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Existing vagus nerve stimulation (VNS) therapies activate vagal fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. The transverse and longitudinal arrangement of fibers inside the vagal trunk with respect to the functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using micro-computed tomography imaging, we tracked fascicular trajectories and found that, in swine, sensory and motor fascicles are spatially separated cephalad, close to the nodose ganglion, and merge caudad, towards the lower cervical and upper thoracic region; larynx-, heart- and lung-specific fascicles are separated caudad and progressively merge cephalad. Using quantified immunohistochemistry at single fiber level, we identified and characterized all vagal fibers and found that fibers of different morphological types are differentially distributed in fascicles: myelinated afferents and efferents occupy separate fascicles, myelinated and unmyelinated efferents also occupy separate fascicles, and small unmyelinated afferents are widely distributed within most fascicles. We developed a multi-contact cuff electrode to accommodate the fascicular structure of the vagal trunk and used it to deliver fascicle-selective cervical VNS in anesthetized and awake swine. Compound action potentials from distinct fiber types, and physiological responses from different organs, including laryngeal muscle, cough, breathing, and heart rate responses are elicited in a radially asymmetric manner, with consistent angular separations that agree with the documented fascicular organization. These results indicate that fibers in the trunk of the vagus nerve are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.

The distribution of enkephalin-like immunoreactivity in the cochlea of the guinea pig and cat was studied. Indirect immunofluorescence immunohistochemistry using antisera generated against a methionine enkephalin-bovine thyroglobulin conjugate was applied to surface preparations of the organ of Corti and cryostat sections of the whole of the cochlea. In the cochlear osseous spiral lamina, immunofluorescence was localized to unmyelinated fibers of the intraganglionic spiral bundle. In the organ of Corti, immunofluorescence was localized to a small number of fibers at inner hair cells, the inner spiral bundle, and tunnel spiral bundle, to tunnel crossing fibers at the level of the tunnel floor, to an occasional spiral outer fiber, and to the synaptic region of outer hair cells in the three rows of the basal turn of the cochlea. Less immunofluorescence was found in this region as one progressed towards the apex, with none seen at the apex. At the most apical region the inner spiral bundle became patchy and the tunnel spiral bundle developed arcades. There was no immunofluorescence found in spiral ganglion cells, in auditory nerve fibers, or in the hair cells of the organ of Corti. The findings were the same in cat as in guinea pig, the latter being studied in more detail. It was concluded that efferent, olivocochlear neurons of the cochlea, synapsing predominantly with primary auditory nerve fibers from the inner sensory cells or with the sensory cells, contain enkephalin-like immunoreactivity. Also, the findings indicate that endings of olivocochlear neurons that synapse predominantly with outer hair cells contain enkephalin-like immunoreactivity. It has previously been shown that olivocochlear neurons are likely to be cholinergic.

Biophysically based computational models of nerve fibers are important tools for designing electrical stimulation therapies, investigating drugs that affect ion channels, and studying diseases that affect neurons. Although peripheral nerves are primarily composed of unmyelinated axons (i.e., C-fibers), most modeling efforts focused on myelinated axons. We implemented the single-compartment model of vagal afferents from Schild et al. (1994) (Schild JH, Clark JW, Hay M, Mendelowitz D, Andresen MC, Kunze DL. J Neurophysiol 71: 2338-2358, 1994) and extended the model into a multicompartment axon, presenting the first cable model of a C-fiber vagal afferent. We also implemented the updated parameters from the Schild and Kunze (1997) model (Schild JH, Kunze DL. J Neurophysiol 78: 3198-3209, 1997). We compared the responses of these novel models with those of three published models of unmyelinated axons (Rattay F, Aberham M. IEEE Trans Biomed Eng 40: 1201-1209, 1993; Sundt D, Gamper N, Jaffe DB. J Neurophysiol 114: 3140-3153, 2015; Tigerholm J, Petersson ME, Obreja O, Lampert A, Carr R, Schmelz M, Fransén E. J Neurophysiol 111: 1721-1735, 2014) and with experimental data from single-fiber recordings. Comparing the two models by Schild et al. (1994, 1997) revealed that differences in rest potential and action potential shape were driven by changes in maximum conductances rather than changes in sodium channel dynamics. Comparing the five model axons, the conduction speeds and strength-duration responses were largely within expected ranges, but none of the models captured the experimental threshold recovery cycle-including a complete absence of late subnormality in the models-and their action potential shapes varied dramatically. The Tigerholm et al. (2014) model best reproduced the experimental data, but these modeling efforts make clear that additional data are needed to parameterize and validate future models of autonomic C-fibers.NEW & NOTEWORTHY Peripheral nerves are primarily composed of unmyelinated axons, and there is growing interest in electrical stimulation of the autonomic nervous system to treat various diseases. We present the first cable model of an unmyelinated vagal nerve fiber and compare its ion channel isoforms and conduction responses with other published models of unmyelinated axons, establishing important tools for advancing modeling of autonomic nerves.

Vagal reflexes regulate homeostasis in visceral organs and systems through afferent and efferent neurons and nerve fibers. Small, unmyelinated, C-type afferents comprise over 80% of fibers in the vagus and form the sensory arc of autonomic reflexes of the gut, lungs, heart and vessels and the immune system. Selective bioelectronic activation of C-afferents could be used to mechanistically study and treat diseases of peripheral organs in which vagal reflexes are involved, but it has not been achieved.

The injection of safe doses of botulinum neurotoxin A (BoNT/A) have been reported to be useful for the treatment of neuropathic pain, but it is still unknown how functional recovery is induced after peripheral nerve injury. We evaluated the effects of intranerve application of BoNT/A, on regeneration and sensorimotor functional recovery in partial and complete peripheral nerve injuries in the mouse. After sciatic nerve crush (SNC) and intranerve delivery of BoNT/A (15pg), axonal regeneration was measured by nerve pinch test at different days. Regeneration of myelinated and unmyelinated fibers was assessed by immunohistochemical double labeling for NF200/GAP43 and CGRP/GAP43. S100 was used as Schwann cells marker. Medial footpad skin reinnervation was assessed by PGP staining. Motor functions were assessed by means of nerve conduction tests. In other mice groups, nerve conduction tests were performed also after chronic constriction injury (CCI) of the sciatic nerve and intraplantar injection of BoNT/A (15pg). In SNC mice, BoNT/A increased the rate of axonal regeneration. The advantage of regrowing myelinated axons after BoNT/A injection was evidenced by longer NF200+ nerve profiles and confirmed by nerve histology. We observed also a higher expression of S100 in the distal portion of BoNT/A-injected regenerated nerves. In CCI mice, BoNT/A induced an increase in reinnervation of gastrocnemius and plantar muscles. These results show that a low dose of BoNT/A, insufficient to produce muscular dysfunction, conversely speeds up sensorimotor recovery by stimulating myelinated axonal regeneration, and points out its application as a multipotent treatment for peripheral neuropathies.

Kv3 family K+ channels, by ensuring speedy repolarization of action potential, enable rapid and high frequency neuronal firing and high precision temporal coding of auditory information in various auditory synapses in the brain. Expression of different Kv3 subtypes within the auditory end organ has been reported. Yet, their precise role at the hair cell synaptic transmission has not been fully elucidated. Using immunolabeling and confocal microscopy we examined the expression pattern of different Kv3 family K+ channel subunits in the nerve fibers innervating the cochlear hair cells. Kv3.1b was found in NKA-positive type 1 afferent fibers, exhibiting high signal intensity at the cell body, the unmyelinated dendritic segment, first heminode and nodes of Ranvier. Kv3.3 signal was detected in the cell body and the unmyelinated dendritic segment of NKA-positive type 1 afferent fibers but not in peripherin-positive type 2 afferent. Kv3.4 was found in ChAT-positive LOC and MOC efferent fibers as well as peripherin-positive type 2 afferent fibers. Such segregated expression pattern implies that each Kv3 subunits participate in different auditory tasks, for example, Kv3.1b and Kv3.3 in ascending signaling while Kv3.4 in feedback upon loud noise exposure.

The pars distalis of the adenohypophysis has recently been proved to be innervated by substance P-like immunoreactive nerve fibers in the monkey and the dog. The present study investigated immuno-electron microscopically the morphology of these nerve fibers and their relationship to anterior pituitary gland cells in the dog. The nerve fibers were unmyelinated and varicose. Direct contacts between them and every cell type of the gland could be ascertained, including folliculostellate cells. Typical synapses were identified on somatotropes and corticotropes. Most of them were of asymmetrical type with round to oval small clear vesicles and scattered large dense-cored vesicles. Occasional symmetrical type synapses were also seen. The results imply that substance P-like immunoreactive nerve fibers have an effector role in the pars distalis of the dog.

The vagus nerve is dominated by afferent fibers that convey sensory information from the viscera to the brain. Most vagal afferents are unmyelinated, slow-conducting C-fibers, while a smaller portion are myelinated, fast-conducting A-fibers. Vagal afferents terminate in the nucleus tractus solitarius (NTS) in the dorsal brainstem and regulate autonomic and respiratory reflexes, as well as ascending pathways throughout the brain. Vagal afferents form glutamatergic excitatory synapses with postsynaptic NTS neurons that are modulated by a variety of channels. The organization of vagal afferents with regard to fiber type and channels is not well understood. In the present study, we used tract tracing methods to identify distinct populations of vagal afferents to determine if key channels are selectively localized to specific groups of afferent fibers. Vagal afferents were labeled with isolectin B4 (IB4) or cholera toxin B (CTb) to detect unmyelinated and myelinated afferents, respectively. We find that TRPV1 channels are preferentially found in unmyelinated vagal afferents identified with IB4, with almost half of all IB4 fibers showing co-localization with TRPV1. These results agree with prior electrophysiological findings. In contrast, we found that the ATP-sensitive channel P2X3 is found in a subset of both myelinated and unmyelinated vagal afferent fibers. Specifically, 18% of IB4 and 23% of CTb afferents contained P2X3. The majority of CTb-ir vagal afferents contained neither channel. Since neither channel was found in all vagal afferents, there are likely further degrees of heterogeneity in the modulation of vagal afferent sensory input to the NTS beyond fiber type.

Diabetic peripheral neuropathy (DPN) is a major complication of diabetes mellitus, and hypertension is considered to be a risk factor for DPN in patients with type 1 diabetes (T1DM). However, the morphological effects of hypertension on DPN are unclear. In this study, we investigated the effect of hypertension on DPN by investigating the changes in unmyelinated and myelinated nerve fibers in hypertensive rats with alloxan (AL)-induced T1DM. Thirteen-week-old WBN/Kob rats with AL-induced diabetes were allocated to receive tap water only (AL group), tap water containing 0.5% saline (0.5AN group), or tap water containing 0.75% saline (0.75AN group) for 15 weeks. Hyperglycemia was maintained for 15 weeks, and the animals were euthanized at 28 weeks. By 23 weeks of age, the systolic blood pressure was significantly higher in the 0.75AN and 0.5AN groups than in the AL group and was unchanged in all groups at 28 weeks. The number of intraepidermal sensory unmyelinated nerve fibers was significantly smaller in the 0.75AN and 0.5AN groups than in the AL group. The axonal size in the myelinated tibial and sural nerve fibers was significantly smaller in the 0.75AN group than in the AL group. Furthermore, luminal narrowing and endothelial hypertrophy were observed in the endoneurial tibial nerve vessels in the 0.75AN group. These findings suggest that superimposing hypertension on hyperglycemia may accelerate a reduction in the number of small unmyelinated sensory nerve fibers in the skin and induce mild axonal atrophy in myelinated tibial and sural nerve fibers in rats with AL-induced T1DM.

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.

Recently, we have shown that green tea (GT) consumption improves both reflexes and sensation in unilateral chronic constriction injury to the sciatic nerve. Considering the substantial neuroprotective properties of GT polyphenols, we sought to investigate whether (-)-epigallocatechin-3-gallate (EGCG) could protect the sciatic nerve and improve functional impairments induced by a crushing injury. We also examined whether neuronal cell apoptosis induced by the crushing injury is affected by EGCG treatment. Histological examination of sciatic nerves from EGCG-treated (50mg/kg; i.p.) showed that axonotmized rats had a remarkable axonal and myelin regeneration with significant decrease in the number of myelinated axonal fibers compared to vehicle-treated crush group. Similarly, ultrastructural evaluation of EGCG-treated nerves displayed normal unmyelinated and myelinated axons with regular myelin sheath thickness and normalized appearance of Schmidt-Lantermann clefts. Extracellular matrix displayed normal collagen fibers appearance with distinctively organized distribution similar to sham animals. Analysis of foot position and extensor postural thrust test showed a progressive and faster recovery in the EGCG-treated group compared to vehicle-treated animals. EGCG-treated rats showed significant increase in paw withdrawal thresholds to mechanical stimulation compared to vehicle-treated crush group. EGCG treatment also restored the mRNA expression of Bax, Bcl-2 and survivin but not that of p53 to sham levels on days 3 and 7 post-injury. Our results demonstrate that EGCG treatment enhanced functional recovery, advanced morphological nerve rescue and accelerated nerve regeneration following crush injury partly due to the down regulation of apoptosis related genes.