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Intrathecal delivery of glial cell line-derived neurotrophic factor (GDNF) reverses mechanical allodynia after 5th lumbar (L5) spinal nerve ligation (SNL). However, the molecular mechanism behind this process is not fully understood. Following sciatic nerve injury, primary afferent neurons in the injured dorsal root ganglion (DRG) begin to express neuropeptide Y (NPY) that is absent in normal DRG. The aim of the current study was to determine the relationship of this de novo expression of NPY and the anti-allodynic effect of GDNF. Following L5 SNL, 73% of neurons began to express NPY mRNA in the ipsilateral L5 DRG and robust NPY-immunoreactive fibers appeared in the ipsilateral GN where the touch-sense mediating A-fiber primary afferents from the hindpaw terminate. Seven-daylong intrathecal infusion of GDNF at the L5 DRG level, starting on day three when mechanical allodynia had fully developed, reversed once-established these changes. The GN neurons normally expressed NPY Y1 receptor, but not Y2, Y4, or Y5 receptors, and L5 SNL did not change the expression pattern. Bolus intracisternal injection of BIBP3226, a Y1 receptor antagonist, dose-dependently reversed mechanical allodynia. We demonstrated that GDNF reversed once-established mechanical allodynia as well as NPY induction in the touch-sense processing pathway. NPY could facilitate touch-sense processing by Y1 receptor in the gracile nucleus after peripheral nerve injury. GDNF may exert anti-allodynic effects through mitigation of this NPY up-regulation. The effectiveness of delayed treatment further indicates the therapeutic potential of GDNF on neuropathic pain., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prialt, a synthetic version of Ca(v)2.2 antagonist ω-conotoxin MVIIA derived from Conus magus, is the first clinically approved voltage-gated calcium channel blocker for refractory chronic pain. However, due to the narrow therapeutic window and considerable side effects associated with systemic dosing, Prialt is only administered intrathecally. N-triazole oxindole (TROX-1) is a novel use-dependent and activation state-selective small-molecule inhibitor of Ca(v)2.1, 2.2 and 2.3 calcium channels designed to overcome the limitations of Prialt. We have examined the neurophysiological and behavioral effects of blocking calcium channels with TROX-1. In vitro, TROX-1, in contrast to state-independent antagonist Prialt, preferentially inhibits Ca(v)2.2 currents in rat dorsal root ganglia (DRG) neurons under depolarized conditions. In vivo electrophysiology was performed to record from deep dorsal horn lamina V/VI wide dynamic range neurons in non-sentient spinal nerve-ligated (SNL) and sham-operated rats. In SNL rats, spinal neurons exhibited reduced responses to innocuous and noxious punctate mechanical stimulation of the receptive field following subcutaneous administration of TROX-1, an effect that was absent in sham-operated animals. No effect was observed on neuronal responses evoked by dynamic brushing, heat or cold stimulation in SNL or sham rats. The wind-up response of spinal neurons following repeated electrical stimulation of the receptive field was also unaffected. Spinally applied TROX-1 dose dependently inhibited mechanically evoked neuronal responses in SNL but not sham-operated rats, consistent with behavioral observations. This study confirms the pathological state-dependent actions of TROX-1 through a likely spinal mechanism and reveals a modality selective change in calcium channel function following nerve injury., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Evidence is emerging for a role of rough endoplasmic reticulum (RER) in the form of stress granules, the unfolded protein response and protein bodies in the response of neurons to injury and in neurodegenerative diseases. Here, we have studied the role of the peripheral target in regulating the RER and polyribosomes of Nissl bodies in axotomised adult cat dorsal root ganglion (DRG) neurons where axonal regeneration and peripheral target reinnervation was either allowed or denied. Retrograde labelling with horseradish peroxidise was used as an independent marker to enable selection of only those DRG neuronal cell bodies with axons in the injured intercostal nerves. Indications of polyribosomal dispersal were seen by 6h following axotomy, and by 24h the normal orderly arrangement of lamellae of RER in Nissl bodies had become disorganised. These ultrastructural changes preceded light microscopical chromatolysis by 1-3d. The retrograde response was maximal 8-32 d after axotomy. Clusters of debris-laden satellite cells/macrophages were present at this time but no ultrastructural evidence of neuronal apoptosis or necrosis was seen and there were no differences in the initial retrograde response according to the type of injury. By 64 d following axotomy with reinnervation, approximately half the labelled DRG neurons showed restoration of the orderly arrangement of RER and polyribosomes in their Nissl bodies. This was not seen after axotomy with reinnervation denied. We propose that the target-dependent changes in Nissl body ultrastructure described here are part of a continuum that can modify neuronal protein synthesis directed towards growth, maintenance or death of the neuron. This represents a possible structural basis for mediating the varied effects of neurotrophic interactions., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Regeneration of sensory neurons is limited in response to lesion of their central axons when compared to lesion of their peripheral axons. To identify transcriptional changes underlying this differential regenerative response between dorsal root and spinal nerve axons, the L5 dorsal root ganglion (DRG) of adult rats was investigated three days after crushing the respective nerve branches by performing high density genome oligonucleotide microarrays. RT-PCR, in situ hybridization and immunohistochemistry confirmed the up-regulation of the vasodilator peptide apelin in non-neuronal cells of the DRG after dorsal root but not after spinal nerve lesion. Induction of apelin mRNA and peptide is accompanied by increased vascular permeability around neuronal cell bodies as demonstrated by Evans-blue albumin (EBA) leakage. Enhanced vasodilation and increased vascular permeability cause intraganglionic edema, which may play a key role in the reduced axonal regeneration rate after dorsal root injury., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

While the voltage-gated sodium channels (VGSCs) are the key molecules for neuronal activities, the precise distribution of them in spinal cord is not clear in previous studies. We examined the expression of mRNAs for alpha-subunits of VGSC (Navs) in adult rat spinal cord before and 7 days after L5 spinal nerve ligation (SPNL) or complete Freund's adjuvant (CFA)-induced paw inflammation by in situ hybridization histochemistry, reverse transcription-polymerase chain reaction, and immunohistochemistry. Nav1.1 and Nav1.6 mRNAs were present in all laminae, except for lamina II, including the spinothalamic tract neurons in lamina I identified by retrograde tracing of Fluoro-gold. Nav1.2 mRNA was predominantly observed in the superficial layers (laminae I, II), and Nav1.3 mRNA was more restricted to these layers. All these transcripts were expressed by the neurons characterized by immunostaining for neuron-specific nuclear protein. Nav1.7 mRNA was selectively expressed by a half of motoneurons in lamina IX. No signals for Nav1.8 or Nav1.9 mRNAs were detected. Immunohistochemistry for Nav1.1, Nav1.2, Nav1.6, and Nav1.7 proteins verified some of these neuronal distributions. L5 SPNL decreased Nav1.1 and Nav1.6 mRNAs, and increased Nav1.3 and Nav1.7 mRNAs in the axotomized spinal motoneurons, without any changes in other laminae of L4-6 spinal segments. Intradermal injection of CFA did not cause any transcriptional change. Our findings demonstrate that spinal neurons have different compositions of VGSCs according to their location in laminae. Pathophysiological changes of spinal neuronal activity may due to post-transcriptional changes of VGSCs. Comparison with our previous data concerning the subpopulation-specific distribution of Nav transcripts in primary afferent neurons provides potentially specific targets for local analgesics at the peripheral nerve and spinal levels., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Chronic neuropathic pain remains an unmet clinical problem because it is often resistant to conventional analgesics. Metabotropic glutamate receptors (mGluRs) are involved in nociceptive processing at the spinal level, but their functions in neuropathic pain are not fully known. In this study, we investigated the role of group III mGluRs in the control of spinal excitatory and inhibitory synaptic transmission in a rat model of neuropathic pain induced by L5/L6 spinal nerve ligation. Whole-cell recording of lamina II neurons was performed in spinal cord slices from control and nerve-ligated rats. The baseline amplitude of glutamatergic EPSCs evoked from primary afferents was significantly larger in nerve-injured rats than in control rats. However, the baseline frequency of GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) was much lower in nerve-injured rats than in control rats. The group III mGluR agonist l(+)-2-amino-4-phosphonbutyric acid (l-AP4) produced a greater inhibition of the amplitude of monosynaptic and polysynaptic evoked EPSCs in nerve-injured rats than in control rats. l-AP4 inhibited the frequency of miniature EPSCs in 66.7% of neurons in control rats but its inhibitory effect was observed in all neurons tested in nerve-injured rats. Furthermore, l-AP4 similarly inhibited the frequency of GABAergic and glycinergic IPSCs in control and nerve-injured rats. Our study suggests that spinal nerve injury augments glutamatergic input from primary afferents but decreases GABAergic and glycinergic input to spinal dorsal horn neurons. Activation of group III mGluRs attenuates glutamatergic input from primary afferents in nerve-injured rats, which could explain the antinociceptive effect of group III mGluR agonists on neuropathic pain.

We have previously demonstrated that CNS toll-like receptor 4 (TLR4) plays a key role in the development of behavioral hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). TLR4 is a well-known receptor for lipopolysaccharide (LPS) in innate immune responses. In the current study, we further investigated the role of CD14, an accessory molecule in the LPS-TLR4 signaling pathway, in the development of L5Tx-induced neuropathic pain. CD14 knockout (KO) mice displayed significantly decreased behavioral sensitivity (mechanical allodynia and thermal hyperalgesia) as early as day 1 post-L5Tx, indicating a nociceptive role of CD14. By flow cytometric analyses, we observed significantly elevated microglial surface CD14 expression in the ipsilateral lumbar spinal cord 3 days post-L5Tx, as well as remarkable increases in microglial size (via forward scatter (FSC)) and granularity (via side scatter (SSC)). Further, intrathecal injection of soluble CD14 induced significantly greater mechanical hypersensitivity in wild type (C3H/HeN) mice compared with TLR4-deficient (C3H/HeJ) mice. Together, these data demonstrate that CD14 plays a contributing role in TLR4-dependent nerve injury-induced neuropathic pain.

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.

We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage-either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo. This clear demonstration of CB(2) receptors on sensory neurons suggests an additional cellular target for CB(2) agonist induced analgesia, at least in neuropathic models.

The distribution of major histocompatibility complex class II (MHC II)-positive non-neuronal cells and T-lymphocytes was examined immunohistochemically in dorsal root ganglia (DRGs) up to 12 weeks following transection of one sciatic or lumbar spinal nerve in adult rats. Unlike within the brain, MHC II immunopositive (+) and T-cells are normally present within DRGs. After nerve transection, MHC II+ cell density increased (by about four times after each lesion) in DRGs projecting into lesioned nerves. Subsequently the number declined after sciatic but not spinal nerve transection. MHC II+ cells did not contain glial markers, even when these were up-regulated after the lesions. Initially, MHC II+ cells lay outside the satellite glia but, by 11 weeks, they had moved through them to lie against the somata. T-cells invaded the lesioned DRGs earlier than the MHC II+ cells. They achieved greater numbers after spinal (30 x control) than after sciatic (12 x control) nerve transection. They also increased in undamaged ganglia adjacent to the spinal nerve injury. T-cell density progressively declined after spinal but not sciatic nerve transection. Both cell types appeared to invade the DRGs initially through blood vessels and the meninges, particularly near the subarachnoid angle. At later stages, occasional neurones had dense aggregations of T-cell receptor+ and MHC II+ cells associated with them. We conclude that the magnitude and time course of changes in MHC II expression and T-cell numbers in lesioned DRGs differ from the responses within motor nuclei after axotomy. The influx of inflammatory cells may contribute to neurone survival in the short term. Their long-term presence has implications for patients. These cells have the potential to release excitatory cytokines that may generate ectopic impulse activity in sensory neurones after nerve injury and so may play a role in the generation of chronic neuropathic pain.

Neuropathic pain is induced by injury or disease of the nervous system. Studies aimed at understanding the molecular pathophysiology of neuropathic pain have so far focused on a few known molecules and signaling pathways in neurons. However, the pathophysiology of neuropathic pain appears to be very complex and remains poorly understood. A global understanding of the molecular mechanisms involved in neuropathic pain is needed for a better understanding of the pathophysiology and treatment of neuropathic pain. Towards this end, we examined global gene expression changes as well as the pathobiology at the cellular level in a spinal nerve ligation neuropathic pain model using DNA microarray, quantitative real-time PCR and immunohistochemistry. We found that the behavioral hypersensitivity that is manifested in the persistent pain state is accompanied by previously undescribed changes in gene expression. In the DRG, we found regulation of: (1) immediate early genes; (2) genes such as ion channels and signaling molecules that contribute to the excitability of neurons; and (3) genes that are indicative of secondary events such as neuroinflammation. In addition, we studied gene regulation in both injured and uninjured DRG by quantitative PCR, and observed differential gene regulation in these two populations of DRGs. Furthermore, we demonstrated unexpected co-regulation of many genes, especially the activation of neuroinflammation markers in both the PNS and CNS. The results of our study provide a new picture of the molecular mechanisms that underlie the complexity of neuropathic pain and suggest that chronic pain shares common pathobiology with progressive neurodegenerative disease., (Copyright 2002 IBRO)

Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.