Role of the peripheral benzodiazepine receptor in sensory neuron regeneration

Peripheral benzodiazepine receptor (PBR) expression increases in small dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury. To determine the functional significance of this induction, we evaluated the effects of PBR ligands on rodent sensory axon outgrowth. In vitro, Ro5-4864, a PBR agonist, enhanced outgrowth only of small peripherin-positive DRG neurons. When DRG cells were preconditioned into an active growth state by a prior peripheral nerve injury Ro5-4864 augmented and PK 11195, a PBR antagonist, blocked the injury-induced increased outgrowth. In vivo, Ro5-4864 increased the initiation of regeneration after a sciatic nerve crush injury and the number of GAP-43-positive axons in the distal nerve while PK 11195 inhibited the enhanced growth produced by a preconditioning lesion. These results show that PBR has a role in the early regenerative response of small caliber sensory axons, the preconditioning effect, and that PBR agonists enhance sensory axon regeneration.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

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.     

Comparison of c-jun,junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using ³²P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 stays and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRAG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA protein complex was reduced byc-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that cjun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection. © 1995 Wiley-Liss, Inc.     

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.     

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.     

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons

We compared the distribution of the α‐subunit mRNAs of voltage‐gated sodium channels Nav1.1–1.3 and Nav1.6–1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A‐fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, ‐1.8, and ‐1.9 mRNAs were more abundant in C‐fiber neurons compared with A‐fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and ‐1.9 were preferentially expressed by TrkA neurons, other α‐subunits were expressed independently of TrkA expression. Actually, all IB4⁺ neurons expressed both Nav1.8 and ‐1.9, and relatively limited subpopulations of IB4⁺ neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up‐regulated mainly in A‐fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell‐derived neurotrophic factor (GDNF) reversed this up‐regulation, the Nav1.3 induction was independent of either TrkA or GFRα1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. J. Comp. Neurol. 510:188–206, 2008. © 2008 Wiley‐Liss, Inc.     

Ro5-4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats

The translocator protein (18 kDa; TSPO), formerly known as the peripheral benzodiazepine receptor, is an outer mitochondrial membrane protein that associates with the mitochondrial permeability transition pore to regulate both steroidogenesis and apoptosis. TSPO expression is induced in adult dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury and a TSPO receptor ligand, Ro5-4864, enhances DRG neurite growth in vitro and axonal regeneration in vivo . We have now found that TSPO is induced in neonatal motor neurons after peripheral nerve injury and have evaluated its involvement in neonatal and adult sensory and motor neuron survival, and in adult motor neuron regeneration. The TSPO ligand Ro5-4864 rescued cultured neonatal DRG neurons from nerve growth factor withdrawal-induced apoptosis and protected neonatal spinal cord motor neurons from death due to sciatic nerve axotomy. However, Ro5-4864 had only a small neuroprotective effect on adult facial motor neurons after axotomy, did not delay onset or prolong survival in SOD1 mutant mice, and failed to protect adult DRG neurons from sciatic nerve injury-induced death. In contrast, Ro5-4864 substantially enhanced adult facial motor neuron nerve regeneration and restoration of function after facial nerve axotomy. These data indicate a selective sensitivity of neonatal sensory and motor neurons to survival in response to Ro5-4864, which highlights that survival in injured immature neurons cannot necessarily predict success in adults. Furthermore, although Ro5-4864 is only a very weak promoter of survival in adult neurons, it significantly enhances regeneration and functional recovery in adults.     

Upregulation of Ryk expression in rat dorsal root ganglia after peripheral nerve injury

To study changes of Ryk expression in dorsal root ganglia (DRG) after peripheral nerve injury, we set up an animal model of unilateral sciatic nerve lesioned rats. Changes of Ryk protein expression in DRG neurons after unilateral sciatic nerve injury were investigated by immunostaining. Changes of Ryk mRNA were also tested by semi-quantitative PCR concurrently. We found, both at the level of protein and mRNA, that Ryk could be induced in cells of ipsilateral DRG after unilateral sciatic nerve lesion. Further investigation by co-immunostaining confirmed that the Ryk-immunoreactive (Ryk-IR) cells were NeuN-immunoreactive (NeuN-IR) neurons of DRG. We also showed the pattern of Ryk induction in DRG neurons after sciatic nerve injury: the number of Ryk IR neurons peaked at 2 weeks post-lesion and decreased gradually by 3 weeks post-lesion. The proportions of different sized Ryk IR neurons were also observed and counted at various stages after nerve lesion. Analysis of Ryk mRNA by RT-PCR showed the same induction pattern as by immunostaining. Ryk mRNA was not expressed in normal or contralateral DRG, but was expressed 1, 2 and 3 weeks post-lesion in the ipsilateral DRG. Ryk mRNA levels increased slightly from 1 to 2 weeks, decreased then by 3 weeks post-lesion. These results indicate that Ryk might be involved in peripheral nerve plasticity after injury. This is a novel function apart from its well-known fundamental activity as a receptor mediating axon guidance and outgrowth.     

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.     

Gene expression profile in rat dorsal root ganglion following sciatic nerve injury and systemic neurotrophin-3 administration

Following sciatic nerve transection in adult rats, a proportion of injured dorsal root ganglion (DRG) neurons die, through apoptosis, over the following 6 months. Previous studies showed that axotomy and neurotrophin-3 administration may have effects on expression of neurotrophins and their receptors in DRG. In the current study, the fourth and fifth lumbar DRGs of rats were examined 2 weeks after right sciatic nerve transection and ligation. The effects of axotomy and systemic NT-3 treatment on neuronal genes were investigated by microarray. The results demonstrated that bone morphogenetic protein (BMP) and Janus protein tyrosine kinase signaling pathways are induced in axotomized DRG, and PI-3 kinase and BMP pathways and genes controlling various cellular functions were induced after axotomy and NT-3 administration.     

Sciatic nerve transection triggers release and intercellular transfer of a genetically expressed macromolecular tracer in dorsal root ganglia

We recently developed a genetic transneuronal tracing approach that allows for the study of circuits that are altered by nerve injury. We generated transgenic (ZW-X) mice in which expression of a transneuronal tracer, wheat germ agglutinin (WGA), is induced in primary sensory neurons, but only after transection of their peripheral axon. By following the transneuronal transport of the tracer into the central nervous system (CNS) we can label the circuits that are engaged by the WGA-expressing damaged neurons. Here we used the ZW-X mouse line to analyze dorsal root ganglia (DRG) for intraganglionic connections between injured sensory neurons and their neighboring "intact" neurons. Because neuropeptide Y (NPY) expression is strongly induced in DRG neurons after peripheral axotomy, we crossed the ZW-X mouse line with a mouse that expresses Cre recombinase under the influence of the NPY promoter. As expected, sciatic nerve transection triggered WGA expression in NPY-positive DRG neurons, most of which are of large diameter. As expected, double labeling for ATF-3, a marker of cell bodies with damaged axons, showed that the tracer predominated in injured (i.e., axotomized) neurons. However, we also found the WGA tracer in DRG cell bodies of uninjured sensory neurons. Importantly, in the absence of nerve injury there was no intraganglionic transfer of WGA. Our results demonstrate that intraganglionic, cell-to-cell communication, via transfer of large molecules, occurs between the cell bodies of injured and neighboring noninjured primary afferent neurons.     

Prostaglandin E2 contributes to the synthesis of brain-derived neurotrophic factor in primary sensory neuron in ganglion explant cultures and in a neuropathic pain model

Brain-derived neurotrophic factor (BDNF) exists in small to medium size neurons in adult rat dorsal root ganglion (DRG) and serves as a modulator at the first synapse of the pain transmission pathway in the spinal dorsal horn. Peripheral nerve injury increases BDNF expression in DRG neurons, an event involved in the genesis of neuropathic pain. In the present study, we tested the hypothesis that prostaglandin E2 (PGE2) over-produced in injured nerves contributes to the up-regulation of BDNF in DRG neurons. Two weeks after partial sciatic nerve ligation (PSNL), BDNF levels in the ipsilateral L4–L6 DRG of injured rats were significantly increased compared to the contralateral side. Perineural injection of a selective cyclooxygenase (COX2) inhibitor or a PGE2 EP4 receptor antagonist not only dose-dependently relieved PSNL elicited mechanical hypersensitivity, but also suppressed the increased BDNF levels in DRG neurons. PSNL shifted BDNF expression in the ipsilateral DRG from small to medium and larger size injured neurons. BDNF is mainly co-expressed with the EP1 and EP4 while moderately with the EP2 and EP3 receptor subtypes in naïve and PSNL rats. PSNL also shifted the expression of EP1–4 receptors to a larger size population of DRG neurons. In DRG explant cultures, a stabilized PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) or the agonists of EP1 and EP4 receptors significantly increased BDNF levels and the phosphorylated protein kinase A (PKA), extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) and cAMP response element binding protein (CREB). The EP1 and EP4 antagonists, a sequester of nerve growth factor (NGF), the inhibitors of PKA and MEK as well as CREB small interfering RNA suppressed dmPGE2-induced BDNF. Taken together, EP1 and EP4 receptor subtypes, PKA, ERK/MAPK and CREB signaling pathways as well as NGF are involved in PGE2-induced BDNF synthesis in DRG neurons. Injured nerve derived-PGE2 contributes to BDNF up-regulation in DRG neurons following nerve injury. Facilitating the synthesis of BDNF in primary sensory neurons is a novel mechanism underlying the role of PGE2 in the genesis of neuropathic pain.     

Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy

Using in situ hybridization histochemistry, we examined the expression of prepro-vasoactive intestinal polypeptide (VIP) mRNAs and preprotachykinin (PPT) mRNAs which coded for substance P (SP) in the rat dorsal root ganglion (DRG) following spinal nerve transection. VIP mRNAs increased dramatically in the DRG neurons after transection of the peripheral branch of the spinal nerve (sciatic nerve), whereas PPT mRNAs showed a gradual decrease for a few weeks. Dorsal rhizotomy or axotomy of the central branch of DRG cells had little influence on VIP-mRNAs and no effect on PPT mRNA expression. These results demonstrated an activation of VIP biosynthesis in the DRG neurons due to axotomy of the peripheral branch, which was opposite to the reaction of PPT mRNA to the same treatment.     

VR1 protein expression increases in undamaged DRG neurons after partial nerve injury

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.     

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.