Axonal injury-dependent induction of the peripheral benzodiazepine receptor in small-diameter adult rat primary sensory neurons

The peripheral benzodiazepine receptor (PBR), a benzodiazepine but not γ‐aminobutyric acid‐binding mitochondrial membrane protein, has roles in steroid production, energy metabolism, cell survival and growth. PBR expression in the nervous system has been reported in non‐neuronal glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand, [³H]PK11195, in dorsal root ganglion (DRG) neurons following injury to the sciatic nerve. In naïve animals, PBR mRNA, protein expression and ligand binding are undetectable in the DRG. Three days after sciatic nerve transection, however, PBR mRNA begins to be expressed in injured neurons, and 4 weeks after the injury, expression and ligand binding are present in 35% of L4 DRG neurons. PBR ligand binding also appears after injury in the superficial dorsal horn of the spinal cord. The PBR expression in the DRG is restricted to small and medium‐sized neurons and returns to naïve levels if the injured peripheral axons are allowed to regrow and reinnervate targets. No non‐neuronal PBR expression is detected, unlike its putative endogenous ligand the diazepam binding inhibitor (DBI), which is expressed only in non‐neuronal cells, including the satellite cells that surround DRG neurons. DBI expression does not change with sciatic nerve transection. PBR acting on small‐calibre neurons could play a role in the adaptive survival and growth responses of these cells to injury of their axons.     

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.     

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues.     

Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the injured spinal cord of adult rats – possible roles of brain-derived neurotrophic factor, TrkB and p75 neurotrophin receptor

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague–Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.     

Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.     

Sciatic nerve injury in adult rats causes distinct changes in the central projections of sensory neurons expressing different glial cell line‐derived neurotrophic factor family receptors

Most small unmyelinated neurons in adult rat dorsal root ganglia (DRG) express one or more of the coreceptors targeted by glial cell line‐derived neurotrophic factor (GDNF), neurturin, and artemin (GFRα1, GFRα2, and GFRα3, respectively). The function of these GDNF family ligands (GFLs) is not fully elucidated but recent evidence suggests GFLs could function in sensory neuron regeneration after nerve injury and peripheral nociceptor sensitization. In this study we used immunohistochemistry to determine if the DRG neurons targeted by each GFL change after sciatic nerve injury. We compared complete sciatic nerve transection and the chronic constriction model and found that the pattern of changes incurred by each injury was broadly similar. In lumbar spinal cord there was a widespread increase in neuronal GFRα1 immunoreactivity (IR) in the L1‐6 dorsal horn. GFRα3‐IR also increased but in a more restricted area. In contrast, GFRα2‐IR decreased in patches of superficial dorsal horn and this loss was more extensive after transection injury. No change in calcitonin gene‐related peptide‐IR was detected after either injury. Analysis of double‐immunolabeled L5 DRG sections suggested the main effect of injury on GFRα1‐ and GFRα3‐IR was to increase expression in both myelinated and unmyelinated neurons. In contrast, no change in basal expression of GFRα2‐IR was detected in DRG by analysis of fluorescence intensity and there was a small but significant reduction in GFRα2‐IR neurons. Our results suggest that the DRG neuronal populations targeted by GDNF, neurturin, or artemin and the effect of exogenous GFLs could change significantly after a peripheral nerve injury. J. Comp. Neurol. 518:3024–3045, 2010. © 2010 Wiley‐Liss, Inc.     

Dorsal root ganglion neurons up-regulate the expression of laminin-associated integrins after peripheral but not central axotomy

The favorable prognosis of regeneration in the peripheral nervous system after axonal lesions is generally regarded as dependent on the Schwann cell basal lamina. Laminins, a heterotrimeric group of basal lamina molecules, have been suggested to be among the factors playing this supportive role. For neurons to utilize laminin as a substrate for growth, an expression of laminin binding receptors, integrins, is necessary. In this study, we have examined the expression of laminin binding integrin subunits in dorsal root ganglion (DRG) neurons after transection to either their peripherally projecting axons, as in the sciatic nerve, followed by regeneration, or the centrally projecting axons in dorsal roots, followed by no or weak regenerative activity. In uninjured DRG, immunohistochemical staining revealed a few neurons expressing integrin subunit alpha6, whereas integrin subunits alpha7 and foremost beta1 were expressed in a majority of neurons. After an injury to the sciatic nerve, mRNAs encoding all three integrins were up-regulated in DRG neurons. By anterograde tracing, immunoreactivity for all studied integrins was also found in association with growing axons after a sciatic nerve crush lesion in vivo. In contrast, mRNA levels remained constant in DRG neurons after a dorsal root injury. Together with previous findings, this suggests that integrin subunits alpha6, alpha7, and beta1 have an important role in the regenerative response following nerve injury and that the lack of regenerative capacity following dorsal root injury could in part be explained by the absence of response in integrin regulation.     

The distribution of C-Fos protein immunolabeled cells in the spinal cord of the rat after electrical and noxious thermal stimulation following sciatic nerve crush, or transection and repair

The distribution of stimulus evoked Fos protein-like immunoreactivity in spinal cord neurons was studied in adult rats at different survival times after sciatic nerve crush or transection and epineural repair. Fos protein-like immunoreactivity was induced either by electrical stimulation of the sciatic nerve central to the injury, at C-fiber strength, at 21, 39, and 92 days post-lesion, or by noxious heat applied to the skin of the hind paw 92 days post-lesion. The contralateral uninjured side served as control. The results with electrical stimulation showed, with some exceptions, that the distribution of c-fos expressing cells in the spinal cord on the normal and on the previously injured side were similar after both crush and transection with repair. The main finding was an up-regulation of the number of Fos protein immunoreactive neurons in the inner portion of Rexed's lamina II. The results following heat stimulation 92 days post-lesion showed a decrease in the number of labeled neurons in most laminae after both types of injury. This was more pronounced in cases with sciatic nerve transection with repair compared to cases with crush. The results indicate time-dependent alterations in the distribution of stimulus evoked c-fos expression in spinal cord neurons during regeneration after nerve injury. Furthermore, the results from heat stimulation may indicate a slower and perhaps more incomplete restoration process after transection with repair than after crush.     

Selective expression of Jun proteins following axotomy and axonal transport block in peripheral nerves in the rat: evidence for a role in the regeneration process

Expression of the protein products of the immediate-early genes (IEGs), members of the fos, jun and krox families (Jun, Fos, and Krox, resp.) was investigated in the spinal cord and sensory ganglia (DRG) of normal rats; and following transection of, block of axonal transport in, or electrical stimulation of their peripheral axons. The nuclei of many moto- and DRG neurons showed a faint basal immunoreactivity (IR) for Jun proteins, but not for Fos or Krox proteins. There was a strong and selective induction of Jun-IR in moto- and DRG neurons after peripheral nerve transection or crush, or colchicine- or vinblastine-induced block of axonal transport. The Jun-IR induced by nerve transection disappeared after nerve regeneration. In contrast, Jun, Fos and Krox proteins were all induced transynaptically in spinal dorsal horn neurons following electrical stimulation of the C-fibers in the afferent nerves. Thus in differentiated neurons in vivo these IEG proteins can be expressed either independently or concomitantly depending on the type of stimulus.     

Effects of injury discharge on the persistent expression of spinal cord fos-like immunoreactivity produced by sciatic nerve transection in the rat

We recently reported that peripheral nerve injury produced by sciatic nerve transection induces a persistent increase in the expression of the immunoreactive Fos protein product of the c-fos proto-oncogene, an indicator of neuronal activity, in the lumbar spinal cord of the rat and that local anesthetic blockade of the peripheral neuroma attenuates this long-term expression of Fos^6,7. In addition to the sustained activity of the injured afferents, the nerve transection itself results, acutely, in a massive injury-induced neural discharge. In this study we evaluated the effect of blocking this massive injury discharge on the persistence of Fos expression. Just prior to nerve transection we applied the short-acting local anesthetic, lidocaine, to the sciatic nerve. Control injections were made subcutaneously on the dorsum of the neck. We report that injection of the local anesthetic, by either route, significantly reduced the number of fos-like immunoreactive neurons at 2 days after nerve transection. The effect was only observed on neurons in the superficial dorsal horn. These results indicate that along with sustained activity of injured afferents and of reorganization of central circuits after injury, the initial brief discharge at the time of nerve injury contributes to a prolonged increase in the activity of spinal cord neurons.     

Primary motor neurons fail to up-regulate voltage-gated sodium channel Na(v)1.3/brain type III following axotomy resulting from spinal cord injury

Epilepsy occurs in a small proportion of patients with spinal cord injury (SCI), but whether it is due to concomitant traumatic head injury or to changes in cortical motor neurons secondary to axotomy within the spinal cord is not known. Na(v)1.3/brain type III sodium channel expression is up-regulated following peripheral axotomy of dorsal root ganglion (DRG) and facial motor neurons, but, to date, Na(v)1.3 expression has not been examined in upper (cortical) motor neurons following axotomy associated with SCI. In the present study, we examine Na(v)1.3 expression in upper motor neurons within rat primary motor cortex following midthoracic (T9) dorsal column transection, which severs the axons of those cells. Axotomized pyramidal cells were identified by retrograde transport of fluorogold. Immunolabeled cells were confined to layer V of the primary motor cortex and exhibited low levels of Na(v)1.3 staining. After axotomy, no significant changes were detected in Na(v)1.3 density or distribution in injured or uninjured cells, compared with control brains, in contrast to up-regulation of Na(v)1.3 in ipsilateral DRG neurons after sciatic nerve transection. These results do not preclude a role for voltage-gated sodium channels in post-SCI epilepsy but suggest that up-regulated expression of Na(v)1.3 channel is not involved.     

Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin-1 and plexin-A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A-positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A-positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin-C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin-C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.     

A-fiber sprouting in spinal cord dorsal horn is attenuated by proximal nerve stump encapsulation

Peripheral nerve transection in the rat alters the spinal cord dorsal horn central projections from both small and large DRG neurons. Injured neurons with C-fibers exhibit transganglionic degeneration of their terminations within lamina II of the spinal cord dorsal horn, while peripheral nerve injury of medium to large neurons induces collateral sprouting of myelinated A-fibers from lamina I and III/IV into lamina II in rats, cats, and primates. To date, it is not known what sequelae are responsible for the collateral sprouting of A-fibers after peripheral nerve injury, although target-derived factors are thought to play an important role. To determine whether target-derived factors are necessary for changes in A-fiber laminar terminations in rat spinal cord dorsal horn, we unilaterally transected the sciatic nerve and ensheathed the proximal nerve stump in a silicone cap. Three days before sacrifice of rat, the injured sciatic nerve was injected with cholera toxin beta-subunit conjugated to horseradish peroxidase (betaHRP) that effectively labels both peripheral and central A-fiber axons. The effect of the ligature, axotomy, and silicone cap treatment was evaluated by analyzing the extent of betaHRP-, Substance P-(SP-), and isolectin B4- (IB4-) immunoreactive (ir) fibers in the somatotopically appropriate spinal cord dorsal horn regions. In all animals, 2-5 weeks after nerve transection (treated or otherwise), IB4- and SP-ir is absent from lamina II. Animals without nerve cap treatment exhibited robust fiber sprouting into lamina II at 2 weeks. In sharp contrast, animals treated with silicone caps did not exhibit betaHRP-ir fibers in lamina II at 2 weeks. This observation was extended up to 5 weeks postinjury. These results suggest that axotomy-induced expansion of betaHRP-ir primary afferent central terminations in the spinal cord dorsal horn is dependent on factors produced in the injury site milieu. While our understanding of local repair mechanisms of injured peripheral nerves is incomplete, it is clear that the time-dependent production of growth factors in the nerve injury microenvironment favor nerve fiber outgrowth, both peripherally and centrally.     

Expression of c-Jun as a Response to Dorsal Root and Peripheral Nerve Section in Damaged and Adjacent Intact Primary Sensory Neurons in the Rat

It was previously shown that the immediate early gene, c-jun , was highly expressed over long periods, in both peripheral sensory and motor neurons following axon damage or block of axoplasmic transport. Here we have examined the question of whether the expression of c-Jun protein is related to axon injury per se or to the process of axon growth. We have examined dorsal root ganglion (DRG) cells subjected to different manipulations which are associated with varying degrees of regrowth, as follows: (i) after peripheral nerve section, where it appears that all damaged neurons make some regenerative effort. 1 – 24 days after sciatic nerve section and ligation most cells in L4/L5 DRG were c-Jun-positive; (ii) after section of the central processes of the DRG cells, which then showed a slow and limited regrowth of their axons towards, but not into, the spinal cord. This resulted in a variable, but significant, expression of c-Jun in a small number of DRG cells; (iii) in intact sensory neurons that were offered the opportunity to sprout into adjacent denervated peripheral tissue. The sciatic nerve was ligated and the response of cells in the L3 ganglia (many of which project to the saphenous nerve) was measured. A small but significant number of cells were c-Jun-positive; (iv) in intact sensory neurons that were offered the opportunity to sprout centrally into partialy denervated neuropil of the spinal cord. We examined neurons in the L3 DRG after rhizotomy of the adjacent L4/L5 dorsal roots. Previous work suggests that sensory neurons show at best a very limited growth under these conditions. No significant increase was seen in c-Jun expression in these cases. These results suggest that c-Jun expression is closely correlated with growth and regeneration, and not simply a consequence of neuronal injury.     

Immediate anti‐tumor necrosis factor‐α (etanercept) therapy enhances axonal regeneration after sciatic nerve crush

Peripheral nerve regeneration begins immediately after injury. Understanding the mechanisms by which early modulators of axonal degeneration regulate neurite outgrowth may affect the development of new strategies to promote nerve repair. Tumor necrosis factor‐α (TNF‐α) plays a crucial role in the initiation of degenerative cascades after peripheral nerve injury. Here we demonstrate using real‐time Taqman quantitative RT‐PCR that, during the time course (days 1–60) of sciatic nerve crush, TNF‐α mRNA expression is induced at 1 day and returned to baseline at 5 days after injury in nerve and the corresponding dorsal root ganglia (DRG). Immediate therapy with the TNF‐α antagonist etanercept (fusion protein of TNFRII and human IgG), administered systemically (i.p.) and locally (epineurially) after nerve crush injury, enhanced the rate of axonal regeneration, as determined by nerve pinch test and increased number of characteristic clusters of regenerating nerve fibers distal to nerve crush segments. These fibers were immunoreactive for growth associated protein‐43 (GAP‐43) and etanercept, detected by anti‐human IgG immunofluorescence. Increased GAP‐43 expression was found in the injured nerve and in the corresponding DRG and ventral spinal cord after systemic etanercept compared with vehicle treatments. This study established that immediate therapy with TNF‐α antagonist supports axonal regeneration after peripheral nerve injury. © 2009 Wiley‐Liss, Inc.