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We compared the distribution of the alpha-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 alpha-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 GFRalpha1 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.

Background & Aims: Changes in the properties of visceral sensory neurons contribute to the development of gastrointestinal pain. However, little is known about the molecules involved in mechanosensation from the gastrointestinal tract. We investigated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase cascade, in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons by noxious gastric distention (GD) and its involvement in acute visceral pain in rats. Methods: Electromyographic responses to gastric balloon distention through gastrostomy were recorded from the acromiotrapezius muscle in rats after splanchnic nerve resection or vagotomy and in control rats. We then examined the phosphorylated-ERK1/2 (p-ERK1/2) labeling in the DRG and NG after GD using immunohistochemistry. Results: Gastric distention induced p-ERK1/2 in DRG and NG neurons with a peak at 2 minutes after stimulation. We found a stimulus intensity-dependent increase in the number of activated neurons, and this activation corresponded well with the incidence of the visceromotor response. Most of these p-ERK1/2–labeled neurons were small- and medium-sized neurons that coexpressed transient receptor potential vanilloid 1 ion channel and acid-sensing ion channel 3. Splanchnic nerve resection, but not vagotomy, affected the visceromotor response, and attenuated the ERK1/2 activation in DRG neurons produced by GD. Furthermore, intrathecal administration of the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126, altered the response to noxious GD. Conclusions: The activation of ERK1/2 pathways in DRG neurons by noxious GD may be correlated with functional activity, and may be involved in acute visceral pain.

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.

The cell adhesion molecule L1 (L1-CAM) plays important functional roles in the developing and adult nervous systems. Here we show that peripheral nerve injury induced dynamic post-transcriptional alteration of L1-CAM in the rat dorsal root ganglia (DRGs) and spinal cord. Sciatic nerve transection (SCNT) changed the expression of L1-CAM protein but not L1-CAM mRNA. In DRGs, SCNT induced accumulation of the L1-CAM into the surface of somata, which resulted in the formation of immunoreactive ring structures in a number of unmyelinated C-fiber neurons. These neurons with L1-CAM-immunoreactive ring structures were heavily colocalized with phosphorylated p38 MAPK. Western blot analysis revealed the increase of full-length L1-CAM and decrease of fragments of L1-CAM after SCNT in DRGs. Following SCNT, L1-CAM-immunoreactive profiles in the dorsal horn showed an increase mainly in pre-synaptic areas of laminae I–II with a delayed onset and colocalized with growth-associated protein 43. In contrast to DRGs, SCNT increased the proteolytic 80-kDa fragment of L1-CAM and decreased full-length L1-CAM in the spinal cord. The intrathecal injection of L1-CAM antibody for the extracellular domain of L1-CAM inhibited activation of p38 MAPK and emergence of ring structures of L1-CAM immunoreactivity in injured DRG neurons. Moreover, inhibition of extracellular L1-CAM binding by intrathecal administration of antibody suppressed the mechanical allodynia and thermal hyperalgesia induced by partial SCNT. Collectively, these data suggest that the modification of L1-CAM in nociceptive pathways might be an important pathomechanism of neuropathic pain.

Alterations in the intracellular signal transduction pathway in primary afferents may contribute to pain hypersensitivity. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) occurs in primary afferent neurons in response to noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of ERK1/2 and p38 MAPK in dorsal root ganglion (DRG) neurons. In the present study, we investigated the phosphorylation of ERK5, also known as big MAPK1, in the DRG by noxious stimulation using immunohistochemistry. Capsaicin injection induced phosphorylated ERK5 (p-ERK5) in small-to-medium diameter sensory neurons with a peak at 2 min after capsaicin injection. Furthermore, we examined the p-ERK5 labeling in the DRG after noxious heat and cold stimuli and found a stimulus intensity-dependent increase in the number of activated neurons. Most of these p-ERK5-immunoreactive neurons were small- and medium-sized neurons, which coexpressed transient receptor potential (TRP) ion channel TRPV1 and TRPA1 after noxious heat and cold stimuli, respectively. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. The i.t. administration of ERK5 antisense oligodeoxynucleotide reversed heat hyperalgesia, but not mechanical allodynia, produced by capsaicin injection. Taken together, these findings suggest that the in vivo activation of the ERK5 signaling pathway in sensory neurons by noxious stimulation may be, at least in part, correlated with functional activity and, further, involved in the development of pain hypersensitivity.

The p75 neurotrophin receptor (p75NTR) has been implicated in diverse neuronal responses, including survival, cell death, myelination, and inhibition of regeneration. However, the role of p75NTR in neuropathic pain, for which there is currently no effective therapy, has not been explored. Here, we report that the pharmacological blockade of p75NTR in primary sensory neurons reversed neuropathic pain after nerve injury. Nerve injury increased the expression and axonal transport of p75NTR and phosphorylation of TrkA in the uninjured primary afferents. Functional inhibition of p75NTR suppressed injury-induced neuropathic pain and decreased the phosphorylation of TrkA and p38 mitogen-activated protein kinase, and the induction of transient receptor potential channels in dorsal root ganglion (DRG) neurons. Our results show that p75NTR induced in undamaged DRG neurons facilitates TrkA signaling and contributes to heat and cold hyperalgesia.

Pain during inflammatory joint diseases is enhanced by the generation of hypersensitivity in nociceptive neurons in the peripheral nervous system. To explore the signaling mechanisms of mechanical hypersensitivity during joint inflammation, experimental arthritis was induced by injection of complete Freund's adjuvant (CFA) into the synovial cavity of rat knee joints. As a pain index, the struggle threshold of the knee extension angle was measured. In rats with arthritis, the phosphorylation of extracellular signal-regulated kinase (ERK), induced by passive joint movement, increased significantly in dorsal root ganglion (DRG) neurons innervating the knee joint compared to the naïve rats that received the same movement. The intrathecal injection of a MEK inhibitor, U0126, reduced the phosphorylation of ERK in DRG neurons and alleviated the struggle behavior elicited by the passive movement of the joint. In addition, the injection of U0126 into the joint also reduced the struggle behavior. These findings indicate that the ERK signaling is activated in both cell bodies in DRG neurons and peripheral nerve fibers and may be involved in the mechanical sensitivity of the inflamed joint. Furthermore, the phosphorylated ERK-positive neurons co-expressed the P2X3 receptor, and the injection of TNP-ATP, which antagonizes P2X receptors, into the inflamed joint reduced the phosphorylated ERK and the struggle behavior. Thus, it is suggested that the activation of the P2X3 receptor is involved in the phosphorylation of ERK in DRG neurons and the mechanical hypersensitivity of the inflamed knee joint.

Patients with neuropathic pain frequently experience hypersensitivity to cold stimulation. However, the underlying mechanisms of this enhanced sensitivity to cold are not well understood. After partial nerve injury, the transient receptor potential ion channel TRPV1 increases in the intact small dorsal root ganglion (DRG) neurons in several neuropathic pain models. In the present study, we precisely examined the incidence of cold hyperalgesia and the changes of TRPA1 and TRPM8 expression in the L4 and L5 DRG following L5 spinal nerve ligation (SNL), because it is likely that the activation of two distinct populations of TRPA1- and TRPM8-expressing small neurons underlie the sensation of cold. We first confirmed that L5 SNL rats developed cold hyperalgesia for more than 14 days after surgery. In the nearby uninjured L4 DRG, TRPA1 mRNA expression increased in trkA-expressing small-to-medium diameter neurons from the 1st to 14th day after the L5 SNL. This upregulation corresponded well with the development and maintenance of nerve injury-induced cold hyperalgesia of the hind paw. In contrast, there was no change in the expression of the TRPM8 mRNA/protein in the L4 DRG throughout the 2-week time course of the experiment. In the injured L5 DRG, on the other hand, both TRPA1 and TRPM8 expression decreased over 2 weeks after ligation. Furthermore, intrathecal administration of TRPA1, but not TRPM8, antisense oligodeoxynucleotide suppressed the L5 SNL-induced cold hyperalgesia. Our data suggest that increased TRPA1 in uninjured primary afferent neurons may contribute to the exaggerated response to cold observed in the neuropathic pain model.

We examined the precise distribution of mRNAs for six cloned rat P2Y receptor subtypes, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y14, in the dorsal root ganglion (DRG) and spinal cord by in situ hybridization histochemistry (ISHH) with 35S-labeled riboprobes. In the DRG, P2Y1 and P2Y2 mRNAs were expressed by 15% and 24% of all neurons, respectively. Although each receptor was evenly distributed between neurofilament-positive and -negative neurons, P2Y2 was rather selectively expressed by TrkA-positive neurons. Schwann cells expressed P2Y2 mRNA, and the nonneuronal cells around the DRG neurons, perhaps the satellite cells, expressed P2Y12 and P2Y14 mRNAs. No ISHH signals for P2Y4 or P2Y6 were seen in any cellular components of the DRG. In the spinal cord, P2Y1 and P2Y4 mRNAs were expressed by some of the dorsal horn neurons, whereas the motor neurons in the ventral horn had P2Y4 and P2Y6 mRNAs. In addition, astrocytes in the gray matter had P2Y1 mRNA, and the microglia throughout the spinal cord expressed P2Y12 mRNA. P2Y14 mRNA was weakly expressed by putative microglia. These findings should provide useful information in interpreting pharmacological and electrophysiological studies in this field given the lack of highly selective antagonists for each P2Y receptor subtype.

Cold hyperalgesia is a well-documented symptom of inflammatory and neuropathic pain; however, the underlying mechanisms of this enhanced sensitivity to cold are poorly understood. A subset of transient receptor potential (TRP) channels mediates thermosensation and is expressed in sensory tissues, such as nociceptors and skin. Here we report that the pharmacological blockade of TRPA1 in primary sensory neurons reversed cold hyperalgesia caused by inflammation and nerve injury. Inflammation and nerve injury increased TRPA1, but not TRPM8, expression in tyrosine kinase A-expressing dorsal root ganglion (DRG) neurons. Intrathecal administration of anti-nerve growth factor (anti-NGF), p38 MAPK inhibitor, or TRPA1 antisense oligodeoxynucleotide decreased the induction of TRPA1 and suppressed inflammation- and nerve injury-induced cold hyperalgesia. Conversely, intrathecal injection of NGF, but not glial cell line-derived neurotrophic factor, increased TRPA1 in DRG neurons through the p38 MAPK pathway. Together, these results demonstrate that an NGF-induced TRPA1 increase in sensory neurons via p38 activation is necessary for cold hyperalgesia. Thus, blocking TRPA1 in sensory neurons might provide a fruitful strategy for treating cold hyperalgesia caused by inflammation and nerve damage.

To investigate the intracellular signal transduction pathways involved in the pathophysiological mechanisms of neuropathic pain after partial nerve injury, we examined the activation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) in the chronic constriction injury (CCI) model. The CCI induced an increase in the phosphorylation of ERK in predominantly injured medium-sized and large-sized DRG neurons and in satellite glial cells. Treatment with the MAPK kinase 1/2 inhibitor, U0126, suppressed CCI-induced mechanical allodynia and partially reversed the increase in neuropeptide Y (NPY) expression in damaged DRG neurons. In contrast, the CCI induced the activation of p38, mainly in uninjured small-to-medium-diameter DRG neurons and in satellite glial cells. The p38 inhibitor, SB203580, reversed the CCI-induced heat hyperalgesia and also the increase in brain-derived neurotrophic factor (BDNF) expression in intact DRG neurons. On the other hand, the nerve growth factor (NGF)-induced increase in BDNF expression in small-to-medium-diameter neurons was reversed by SB203580, whereas the anti-NGF-induced increase in NPY in medium-sized and large-sized neurons was partially blocked by U0126. Taken together, our results demonstrate that the activation of ERK and p38 and also the changes in NPY and BDNF expression may occur in different populations of DRG neurons after CCI, partially through alterations in the target-derived NGF. These changes in injured and intact primary afferents are likely to have a substantial role in pathological states, and MAPK pathways in nociceptors may be potential targets for the development of novel analgesics.

To elucidate the role of the degeneration of motor and sensory fibers in neuropathic pain, we examined the pain-related behaviors and the changes of brain-derived neurotrophic factor (BDNF) in the L4/5 dorsal root ganglion (DRG) and the spinal cord after L5 ventral rhizotomy. L5 ventral rhizotomy, producing a selective lesion of motor fibers, produced thermal hyperalgesia and increased BDNF expression in tyrosine kinase A-containing small- and medium-sized neurons in the L5 DRG and their central terminations within the spinal cord, but not in the L4 DRG. Furthermore, L5 ventral rhizotomy up-regulated nerve growth factor (NGF) protein in small to medium diameter neurons in the L5 DRG and also in ED-1-positive cells in the L5 spinal nerve, suggesting that NGF synthesized in the degenerative fibers is transported to the L5 DRG and increases BDNF synthesis. On the other hand, L5 ganglionectomy, producing a selective lesion of sensory fibers, produced heat hypersensitivity and an increase in BDNF and NGF in the L4 DRG. These data indicate that degeneration of L5 sensory fibers distal to the DRG, but not motor fibers, might influence the neighboring L4 nerve fibers and induce neurotrophin changes in the L4 DRG. We suggest that these changes of neurotrophins in the intact primary afferents of neighboring nerves may be one of many complex mechanisms, which can explain the abnormal pain behaviors after nerve injury. The ventral rhizotomy and ganglionectomy models may be useful to investigate the pathophysiological mechanisms of neuropathic pain after Wallerian degeneration in motor or sensory or mixed nerve.

Inflammation of the primary afferent proximal to the dorsal root ganglion (DRG) and the DRG itself is known to produce radicular pain. Here, we examined pain-related behaviors and the activation of extracellular signal-regulated protein kinase (ERK) in the DRG after inflammation near the DRG somata. Inflammation of the L4/5 nerve roots and DRG induced by complete Freund's adjuvant (CFA) produced mechanical allodynia on the ipsilateral hindpaw and induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase (trk) A-expressing small- and medium-size neurons. This CFA-induced increase in ERK phosphorylation was mediated through trk receptors, because intrathecal treatment with the tyrosine kinase inhibitor, K252a, reduced the activation of ERK. On the other hand, an increase in brain-derived neurotrophic factor (BDNF) mRNA/protein in the DRG concomitant with the ERK activation was also observed. Furthermore, we found that nerve growth factor (NGF) injection directly into the L4/5 nerve roots and DRG produced mechanical allodynia, and an increase in the phosphorylation of ERK and BDNF expression in the DRG, but the mitogen-activated protein kinase (MAPK) kinase1/2 inhibitor, U0126, inhibited the effects induced by NGF. Therefore, we suggest that after inflammation near the cell body, NGF synthesized within the nerve root and DRG induces BDNF expression through trkA receptors and intracellular ERK-MAPK. The activation of MAPK in the primary afferents may be involved in the pathophysiological mechanisms of inflammation-induced radiculopathy and MAPK pathways in the primary afferents may be potential targets for pharmacological intervention for neuropathic pain produced by inflammation near the DRG somata.

We examined the expression of hepatocyte growth factor (HGF) mRNA and its receptor, c-met mRNA, in the dorsal root ganglia (DRG) and spinal cord of naive adult rats using in situ hybridization histochemistry (ISHH) and the reverse transcription-polymerase chain reaction (RT-PCR) technique. HGF mRNA was expressed in 25.0% of DRG neurons and 80.5% of HGF mRNA-positive neurons expressed trkA mRNA. In the lumbar spinal cord, c-met mRNA signals were observed in the superficial layer of dorsal horn. These results suggest that the HGF/cMet system may be involved in sensory transmission.

Afferent fibers innervating the gastrointestinal tract have major roles in consciously evoked sensations including pain. However, little is known about the molecules involved in mechanonociception from the upper gastrointestinal tract. We recently reported that activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase cascade in primary afferent neurons, was induced by noxious gastric distention in the rat, and that the activation of ERK1/2 in dorsal root ganglion (DRG) neurons can be implicated in acute visceral pain. Transient receptor potential (TRP) A1, a member of the TRP family of cation channels, was expressed in both DRG and nodose ganglion (NG) neurons innervating the stomach and in nerve fibers in the gastric wall. TRPA1 was coexpressed with ERK1/2 in gastric primary afferent neurons, and attenuation of TRPA1 activation using antisense peptides and a specific blocker led to suppression of both ERK1/2 activation and visceromotor responses. TRPA1 also significantly colocalized with substance P (SP) and calcitonin gene-related peptide (CGRP) in the thoracolumbar DRG, NG and stomach. These data indicate that SP and CGRP may also be released by TRPA1 activation in primary afferent neurons to elicit neurogenic inflammation and promote visceral hyperalgesia.

Background: Transient receptor potential (TRP)A1, a member of the TRP family of ion channels, has been proposed to function in diverse sensory processes, including thermosensation and pain. However, TRPA1 has not been directly implicated in stomach mechanosensation, and its contribution to acute visceral pain from this organ is unknown. Here, we investigated the expression of TRPA1 in primary sensory afferents and its involvement in visceral hypersensitivity in rats. Methods: We examined TRPA1 expression in the dorsal root ganglion (DRG), nodose ganglion (NG), and stomach of rats by using immunohistochemistry. Electromyographic responses to gastric distention (GD) were recorded from the acromiotrapezius muscle in TRPA1 knockdown rats and in control rats. Results: TRPA1 was predominantly expressed with sensory neuropeptides in DRG and NG neurons, and in nerve fibres in the rat stomach. Gastric distention induced the activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in DRG and NG neurons 2 min after stimulation, and most of the phosphorylated-ERK1/2-labelled DRG neurons were TRPA1-positive neurons. Intrathecal injection of TRPA1 antisense attenuated the visceromotor response, and suppressed ERK1/2 activation in the DRG, but not NG, neurons produced by GD. Furthermore, intrathecal and intraperitoneal injections of the TRPA1 inhibitor HC-03003 suppressed the response to noxious GD. Conclusions: The activation of TRPA1 in DRG neurons by noxious GD may be involved in acute visceral pain. Our findings point to the potential blockade of TRPA1 in primary afferents as a new therapeutic target for the reduction of visceral hypersensitivity.

An intracellular signalling pathway in the dorsal root ganglion (DRG) and spinal neurons is a popular target in pain research that is relevant to the neuroplastic changes that occur during chronic pain conditions. First, we examined the phosphorylation of ERK in DRG neurons after peripheral inflammation and sciatic nerve transection without any stimulation to the receptive field. We found an activation of ERK in different populations of DRG neurons after peripheral inflammation and axotomy, which developed from alterations in target-derived nerve growth factor (NGF). We observed that the ERK signalling regulates the brain-derived neurotrophic factor (BDNF) expression in DRG neurons in both conditions. We also demonstrated that very rapid phosphorylation of ERK occurred in DRG neurons that were involved in the transmission of various noxious signals under normal conditions. Further, we examined the pERK labelling after the mechanical stimuli into the inflamed tissue and found that the pERK labelling occurred through the P2X3 receptors in the terminals. This activity-dependent activation of the ERK signal pathway may be useful for identifying which DRG neurons are involved in transmission of noxious stimuli under normal and pathological conditions.

The possible involvement of fibroblast growth factor receptor (FGFR) activation in the dorsal root ganglion (DRG) was examined following peripheral nerve injury in the rat. Ligation of the sciatic nerve down-regulated FGFR2, -3 and -4 mRNA; however, the expression of FGFR1 mRNA showed no change. Activation of FGFR was examined by immunohistochemistry using an antibody of the phosphorylated form of FGFR1-4. Ligation of the sciatic nerve produced phosphorylation of FGFR in the L4 and 5 DRG ipsilateral to the injury, starting at 3 days after the lesion and persisting for more than 30 days. Substantial activation of FGFR was observed, mainly in unmyelinated small DRG neurons that co-expressed phosphorylated p38 mitogen-activated protein kinase (MAPK). Continuous intrathecal infusion of the FGFR1 inhibitor, 3-[3-(2-carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone, reduced p38 MAPK phosphorylation in the DRG and pain-related behaviors in the partial sciatic nerve model rat without affecting on the activation of spinal glia cells (microglia and astrocyte). In the injured small DRG neurons, activation of FGFR1 may contribute to the generation of neuropathic pain by activating p38 MAPK.

The cell adhesion molecule L1 (L1-CAM) plays important functional roles in the developing and adult nervous systems. Here we show that peripheral nerve injury induced dynamic post-transcriptional alteration of L1-CAM in the rat dorsal root ganglia (DRGs) and spinal cord. Sciatic nerve transection (SCNT) changed the expression of L1-CAM protein but not L1-CAM mRNA. In DRGs, SCNT induced accumulation of the L1-CAM into the surface of somata, which resulted in the formation of immunoreactive ring structures in a number of unmyelinated C-fiber neurons. These neurons with L1-CAM-immunoreactive ring structures were heavily colocalized with phosphorylated p38 MAPK. Western blot analysis revealed the increase of full-length L1-CAM and decrease of fragments of L1-CAM after SCNT in DRGs. Following SCNT, L1-CAM-immunoreactive profiles in the dorsal horn showed an increase mainly in pre-synaptic areas of laminae I–II with a delayed onset and colocalized with growth-associated protein 43. In contrast to DRGs, SCNT increased the proteolytic 80-kDa fragment of L1-CAM and decreased full-length L1-CAM in the spinal cord. The intrathecal injection of L1-CAM antibody for the extracellular domain of L1-CAM inhibited activation of p38 MAPK and emergence of ring structures of L1-CAM immunoreactivity in injured DRG neurons. Moreover, inhibition of extracellular L1-CAM binding by intrathecal administration of antibody suppressed the mechanical allodynia and thermal hyperalgesia induced by partial SCNT. Collectively, these data suggest that the modification of L1-CAM in nociceptive pathways might be an important pathomechanism of neuropathic pain.

The expression of plasma membrane Ca 2+ -ATPase, a calcium pump located in cell membrane regulating intracellular Ca 2+ levels by Ca 2+ extrusion from cells, was examined in dorsal root ganglion neurons in naive rats and after spinal nerve ligation.The mRNAs and proteins in plasma membrane Ca 2+ -ATPase 1-3 were expressed in all size neurons with intense labeling in medium to large neurons. After spinal nerve ligation, these three isoforms showed downregulation of their expression. In contrast, plasma membrane Ca 2+ -ATPase 4 was expressed mainly in small neurons, and the number and signal intensity were significantly increased after spinal nerve ligation. These data suggest that plasma membrane Ca 2+ -ATPase isoforms have a distinct pattern of expression and regulation by axotomy in dorsal root ganglion neurons in normal and pathological conditions.

Proteinase-activated receptor (PAR) 2 is expressed in a subset of primary afferent neurons and is involved in inflammatory nociception. The P2X3 ion channel is localized on nociceptors of sensory neurons. Using immunohistochemistry, we showed that many P2X3s are co-expressed with the PAR2 in rat dorsal root ganglia neurons. Nocifensive behavior induced by alphabeta-methylene adenosine 5'-triphosphate (ATP) injection to the hind paw was significantly augmented after the application of PAR2 agonists. Fos expression induced by the alphabeta-methylene ATP injection in dorsal horn neurons was also increased after the pre-application of PAR2 agonists. These findings indicate that PAR2 agonists may potentiate the sensitivity of P2X3 ion channel to noxious stimuli, and the interaction between PAR2 and P2X3 may be an important mechanism underlying inflammatory pain.

Neurotrophic factors, which support neuronal survival and growth during development of the nervous system, have been shown to play significant roles in the transmission of physiologic and pathologic pain. Brain-derived neurotrophic factor (BDNF), synthesized in the primary sensory neurons, is anterogradely transported to the central terminals of the primary afferents in the spinal dorsal horn, where it is involved in the modulation of painful stimuli. In models of inflammatory and neuropathic pain, BDNF synthesis is greatly increased in different populations of dorsal root ganglion (DRG) neurons. Furthermore, it is now known that the activation of mitogen-activated protein kinases occurs in these sensory neurons and contributes to persistent inflammatory and neuropathic pain by regulating BDNF expression. The recent discovery that BDNF upregulation in the DRG and spinal cord contributes to chronic pain hypersensitivity indicates that blocking BDNF in sensory neurons could provide a fruitful strategy for the development of novel analgesics.

Two cold-sensitive transient receptor potential (TRP) channels, TRPA1 and TRPM8, have been identified and considered interesting because of their possible roles in thermosensation, nociception and other functions. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase occurred in primary afferent neurons in response to noxious heat stimulation of the peripheral tissue, i.e. activity-dependent activation of extracellular signal-regulated protein kinase and p38 in dorsal root ganglion neurons. In the present study, we investigated the phosphorylation of extracellular signal-regulated protein kinase, p38, and c-Jun N-terminal kinase in the rat dorsal root ganglion by cold stimulation using immunohistochemistry. Cold stimuli (28-4 degrees C) were applied by immersion of the hind paw into a water bath (six times of 10 s stimulation and 10 s interval, total 2 min). Noxious cold stimulation induced phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38, but not phosphorylated-c-Jun N-terminal kinase, in small to medium diameter sensory neurons with a peak at 2 min after stimulation. We found that a cold stimulation at 4 degrees C showed a marked increase in the number of activated neurons. Furthermore, double staining for phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38 showed no colocalization in the dorsal root ganglion neurons. We then performed double-labeling experiments for TRPA1 and TRPM8 mRNA and phosphorylation of mitogen-activated protein kinase. The majority of phosphorylated-extracellular signal-regulated protein kinase-positive neurons also expressed TRPM8 mRNA, whereas phosphorylated-p38 heavily colocalized with TRPA1 mRNA after noxious cold stimulation. Our data suggest that the noxious, but not innocuous, cold stimulation in vivo induced differential activation of extracellular signal-regulated protein kinase and p38 pathways in each subpopulation containing TRPA1 or TRPM8 in dorsal root ganglion.

A number of rat neuropathy models have been developed to simulate human neuropathic pain conditions, such as spontaneous pain, hyperalgesia, and allodynia. In the present study, to determine the relative importance of injury site (proximal or distal to the primary afferent neurons) and injury type (motor or sensory), we examined pain-related behaviors and changes of brain-derived neurotrophic factor expression in the dorsal root ganglion in sham-operated rats, and in the L5 dorsal rhizotomy, L5 ventral rhizotomy, L5 dorsal rhizotomy+ventral rhizotomy, and L5 spinal nerve transection models. L5 ventral rhizotomy and spinal nerve transection produced not only mechanical and heat hypersensitivity, but also an increase in brain-derived neurotrophic factor mRNA/protein in the L5 dorsal root ganglion at 7 days after surgery. In contrast, rats in the L5 dorsal rhizotomy and dorsal rhizotomy+ventral rhizotomy groups did not show both pain behaviors at 7 days after surgery, despite brain-derived neurotrophic factor upregulation in medium- and large-size neurons in the L5 dorsal root ganglion. On the other hand, L5 spinal nerve transection, but not dorsal rhizotomy, dorsal rhizotomy+ventral rhizotomy or ventral rhizotomy, increased the expression of brain-derived neurotrophic factor in the L4 dorsal root ganglion at 7 days after surgery. Taken together, these findings suggest that the upregulation of brain-derived neurotrophic factor expression in the L4 and L5 dorsal root ganglion neurons may be, at least in part, involved in the pathophysiological mechanisms of neuropathic pain and that the selective nerve root injury models may be useful for studying the underlying mechanisms of chronic pain after nerve injury.

We have previously found that tissue type and urokinase type plasminogen activators (tPA and uPA) are induced in dorsal root ganglia (DRG) neurons after peripheral axotomy and that tPA plays crucial roles in generating neuropathic pain. Here we examined whether the plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2) mRNA, endogenous inhibitors of tPA and uPA, are induced in the DRG following sciatic nerve transection. L4 and L5 DRG sections were examined using in situ hybridization histochemistry. The results showed that both PAI-1 and PAI-2 mRNA were up-regulated in DRG neurons within 1 day, and peaked at 1-3 days, after injury. Reduction of these mRNA was observed from 7 days after injury. The precise expression patterns of PAI-1 and PAI-2 mRNA at 3 days after axotomy revealed that PAI-1 mRNA was observed in predominantly small neurons, while much of the PAI-2 mRNA was expressed in large neurons. Double-labeling analysis of these mRNAs with activated transcription factor 3, known as an injury marker, revealed that most PAI-1 and PAI-2 mRNAs was induced in injured neurons. Co-expression of PAI-1, 2 with tPA and uPA in DRG neurons suggests that these inhibitors may act in an autocrine manner to modulate extracellular proteolytic activity after nerve injury.

To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small- to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.

Alterations in the intracellular signal transduction pathway in primary afferents may contribute to pain hypersensitivity. We demonstrated that very rapid phosphorylation of p38 mitogen-activated protein kinase occurred in dorsal root ganglion (DRG) neurons that were participating in the transmission of noxious signals. Capsaicin injection induced phosphorylated-p38 (p-p38) in small-to-medium diameter sensory neurons with a peak at 2 min after capsaicin injection. Furthermore, we examined the p-p38 labeling in the DRG after noxious thermal stimuli and found a stimulus intensity-dependent increase in labeled cell size and the number of activated neurons. Most of these p-p38-immunoreactive (IR) neurons were small- and medium-sized neurons, which coexpressed transient receptor potential ion channel TRPV1 and phosphorylated-extracellular signal-regulated protein kinase. Intrathecal administration of the p38 inhibitor, FR167653, reversed the thermal hyperalgesia produced by the capsaicin injection. Inhibition of p38 activation was confirmed by the decrease in the number of p-p38-IR neurons in the DRG following capsaicin injection. Taken together, these findings suggest that the activation of p38 pathways in primary afferents by noxious stimulation in vivo may be, at least in part, correlated with functional activity, and further, involved in the development of thermal hyperalgesia.

The mechanism of mechanical hyperalgesia in inflammation might involve a 'mechanochemical' process whereby stretch evokes the release of adenosine 5'-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (alphabetame-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after alphabetame-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or 2'- (or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.

To investigate the intracellular signal transduction pathways involved in regulating the gene expression of brain-derived neurotrophic factor (BDNF) in primary afferent neurons, we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons after peripheral inflammation and sciatic nerve transection. Peripheral inflammation induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons. The treatment of the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126 reversed the pain hypersensitivity and the increase in phosphorylated-ERK (p-ERK) and BDNF in DRG neurons induced by complete Freund's adjuvant. On the other hand, axotomy induced the activation of ERK mainly in medium-and large-sized DRG neurons and in satellite glial cells. U0126 suppressed the axotomy-induced autotomy behavior and reversed the increase in p-ERK and BDNF. The intrathecal application of nerve growth factor (NGF) induced an increase in the number of p-ERK-and BDNF-labeled cells, mainly small neurons, and the application of anti-NGF induced an increase in p-ERK and BDNF in some medium-to-large-diameter DRG neurons. The activation of MAPK in the primary afferents may occur in different populations of DRG neurons after peripheral inflammation and axotomy, respectively, through alterations in the target-derived NGF. These changes, including the changes in BDNF expression, might be involved in the pathophysiological changes in primary afferent neurons.

Neuropathic pain models, such as the chronic constriction injury (CCI) model, are partial nerve injury models where there exist both intact and injured peripheral axons. Recent studies suggested that dorsal root ganglion (DRG) neurons with intact axons also show the alteration of excitability and gene expression and might have some role in the pathophysiological mechanisms of neuropathic pain. The incidence of pain-related behavior after the CCI is unstable and variable. In the present study, we used activating transcription factor 3 (ATF3) expression as a neuronal injury marker, and analyzed a relationship between the number of axotomized neurons and the incidence of pain-related behavior. We divided all rats into three groups according to the percentage of ATF3-immunoreactive (IR) neurons, group 1 (12.5%), group 2 (12.5-25%), and group 3 (25%). We found that rats in groups 2 and 3 showed thermal hyperalgesia, whereas only the rats in group 2 developed tactile allodynia from the third day to the fourteenth day after surgery. Rats in group 1 did not show thermal hyperalgesia or tactile allodynia. The DRG neurons in group 2 contained ATF3-IR neurons mainly in medium- and large-sized neurons. In order to investigate brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid(A)-receptor (GABA(A)-R) regulation in both intact and injured primary afferent neurons after the CCI, we used a double-labeling method with immunohistochemistry and in situ hybridization, as well as double immunofluorescent staining. The CCI induced an increased number of BDNF-labeled neurons in the ipsilateral DRG and the increase in BDNF expression was observed mainly in small- and medium-sized neurons that were mainly ATF3-negative. On the other hand, the number of GABA(A)-Rgamma2 subunit mRNA-positive neurons decreased in the ipsilateral DRG and GABA(A)-R- and ATF3-labeled neurons rarely overlapped. These changes in molecular phenotype in intact and injured primary afferents may be involved in the pathophysiological mechanisms of neuropathic pain produced by partial nerve injury.

A variety of molecules released by inflammatory reactions in the dorsal root and dorsal root ganglion (DRG) may play important roles in the pathology of neuronal abnormalities in lumbar disc herniation. In order to elucidate the pathophysiological mechanisms of painful radiculopathy, secondary to lumbar disc herniation, we evaluated pain-related behavior and the change of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRG and dorsal root using a rat model of lumbar disc herniation. In the nucleus pulposus (NP) group, the left L4/5 nerve roots were exposed after hemilaminectomies and autologous intervertebral discs, which were obtained from coccygeal intervertebral discs, were implanted on each of the exposed nerve roots without mechanical compression. Rats in the NP group, but not the sham-operated rats, developed mechanical allodynia on the ipsilateral hind paw for 1 day after surgery and showed a significant increase in the number of NGF-immunoreactive (IR) cells in the nerve root and DRG. NGF-IR cells in the nerve root and DRG included macrophages and Schwann cells, because these cells were labeled for NGF and ED-1 or glial fibrillary acid protein by dual immunostaining. A significant increase in the percentage of BDNF-IR neurons in the DRG was observed in the NP group at 3 days after surgery and the increase in BDNF mRNA expression was confirmed using in situ hybridization histochemistry and reverse transcription-polymerase chain reaction. We also injected NGF into the endoneurial space of the normal rat spinal nerve root and found that the NGF injection produced dose-dependent mechanical allodynia on the ipsilateral hind paw at 1 day after surgery and an increase in the percentage of BDNF-IR neurons in the DRG at 3 days after surgery compared to the group receiving saline injection. These findings suggest that in the lumbar disc herniation model, i.e. neuritis of the nerve root, increased NGF produced by the inflammatory responses in the dorsal root and DRG tissues may affect the production of BDNF in the DRG and may play important roles in the modulation of the dorsal horn neurons. These changes in neurotrophic factors in the primary afferents may be involved in the pathophysiological mechanisms of neuropathic pain produced by lumbar disc herniation.