Spinal glial cell line-derived neurotrophic factor infusion reverses reduction of Kv4.1-mediated A-type potassium currents of injured myelinated primary afferent neurons in a neuropathic pain model.

High frequency spontaneous activity in injured primary afferents has been proposed as a pathological mechanism of neuropathic pain following nerve injury. Although spinal infusion of glial cell line-derived neurotrophic factor reduces the activity of injured myelinated A-fiber neurons after fifth lumbar (L5) spinal nerve ligation in rats, the implicated molecular mechanism remains undetermined. The fast-inactivating transient A-type potassium current (IA) is an important determinant of neuronal excitability, and five voltage-gated potassium channel (Kv) alpha-subunits, Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, display IA in heterologous expression systems. Here, we examined the effect of spinal glial cell line-derived neurotrophic factor infusion on IA and the expression of these five Kv mRNAs in injured A-fiber neurons using the in vitro patch clamp technique and in situ hybridization histochemistry. Glial cell line-derived neurotrophic factor infusion reversed axotomy-induced reduction of the rheobase, elongation of first spike duration, and depolarization of the resting membrane potential. L5 spinal nerve ligation significantly reduced the current density of IA and glial cell line-derived neurotrophic factor treatment reversed the reduction. Among the examined Kv mRNAs, only the change in Kv4.1-expression was parallel with the change in IA after spinal nerve ligation and glial cell line-derived neurotrophic factor treatment. These findings suggest that glial cell line-derived neurotrophic factor should reduce the hyperexcitability of injured A-fiber primary afferents by IA recurrence. Among the five IA-related Kv channels, Kv4.1 should be a key channel, which account for this IA recurrence.

Role of nerve growth factor-tyrosine kinase receptor A signaling in paclitaxel-induced peripheral neuropathy in rats.

The mechanisms underlying paclitaxel-induced peripheral neuropathy remain unknown. Nerve growth factor (NGF) is a representative neurotrophic factor that maintains neuronal function, promotes survival, and mediates neuropathic pain. We investigated expression levels of NGF and its receptors in the dorsal root ganglia (DRG) and spinal dorsal horn (DH) following paclitaxel treatment. Intraperitoneal (I.P.) administration of paclitaxel induced significant mechanical hypersensitivity and cold allodynia in rats, significantly increased the expression of NGF and its receptor tyrosine kinase receptor A (trkA) in the DRG, and increased NGF expression in the DH. In contrast, paclitaxel treatment did not alter the mRNA levels of NGF or its receptors in the DRG, DH, sciatic nerve, or hindpaw skin. Moreover, expression of NEDD4-2, a negative regulator of trkA, was significantly increased in the DRG of paclitaxel-treated rats. Intrathecal (I.T.) administration of the tyrosine kinase receptor inhibitor k252a significantly alleviated mechanical hypersensitivity in paclitaxel-treated rats. Our results suggest that NGF-trkA signaling is involved in mechanical allodynia in paclitaxel-induced neuropathy.

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons.

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knockdown of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

Re-evaluation of the phenotypic changes in L4 dorsal root ganglion neurons after L5 spinal nerve ligation.

The L5 spinal nerve ligation (SNL) is a widely used animal neuropathic pain model. There are conflicting reports regarding the extent of injury to the L4 dorsal root ganglion (DRG) neurons in this model. If a significant number of these neurons were injured, the previously reported phenotypic and electrophysiological changes at this level are in need of re-evaluation by separating the injured neurons and the frankly spared ones. So, we immunostained activating transcription factor 3 (ATF3) and examined the change in expression of transcripts for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF) and several voltage-gated sodium channel α-subunits (Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) in the L4 DRG by comparing signal intensities of individual neurons using in situ hybridization histochemistry. ATF3-immunoreactivity was similarly observed in 4-6% of neuronal nuclei of the SNL and sham-operated ipsilateral L4 DRGs. Comparison between ATF3+ and ATF3- neurons in the SNL L4 DRG revealed that (1) whereas NPY induction occurred in ATF3+ cells, BDNF increased mainly in ATF3- neurons; (2) although ATF3+ neurons had higher Nav1.3 signals than ATF3- neurons, these signals were much lower than those of the L5 DRG neurons; and (3) ATF3+/N52- neurons selectively lost Nav1.8 and Nav1.9 mRNAs. Comparison of the total neuronal populations among naïve, SNL, and sham-operated rats revealed no significant differences for all examined Nav mRNAs. Because neuropathic pain behaviors were developed by rats with SNL but not the sham-operation, the small number of injured L4 neurons likely do not contribute to the pathomechanisms of neuropathic pain.

Increase of close homolog of cell adhesion molecule L1 in primary afferent by nerve injury and the contribution to neuropathic pain.

The L1 family of cell adhesion molecules (L1-CAMs) is known to be involved in various neuronal functions such as cell adhesion, axon guidance, and synaptic plasticity. We investigated the detailed expression/changes of a close homolog of the L1 cell adhesion molecule (CHL1) after nerve injury and the possible role on neuropathic pain using the rat spared nerve injury (SNI) model. SNI induced the expression of CHL1 in L4/5 DRG neurons, particularly in small-size injured neurons and in satellite cells. In the spinal cord, CHL1 immunoreactivity increased mainly in laminae I-II of the dorsal horn on the side ipsilateral to the nerve injury. Ultrastructural study clarified the fine localization of CHL1 in axons of primary afferents in the dorsal horn. CHL1 immunoreactivities were localized in the adherence such as axon-axon, axon-dorsal horn neurons (dendrite, soma), and axon-glial cells (astrocyte and microglia). Experimental inhibition of CHL1 adhesion by intrathecal administration of the antibody for CHL1 extracellular domain significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of CHL1 may be involved in the structural plasticity after peripheral nerve injury and have important roles in neuropathic pain.

Comparative study of voltage-gated sodium channel α-subunits in non-overlapping four neuronal populations in the rat dorsal root ganglion.

Voltage-gated sodium channel α-subunit (Nav) is the major determinant of neuronal electrophysiological characters. In order to compare the composition of Navs among neurochemically different neurons in the rat dorsal root ganglion (DRG), we examined the expression of Nav transcripts in four non-overlapping neuronal populations, with (+) or without (-) N52 immunoreactivity, a marker of neurons with myelinated axons, and TrkA mRNA identified by in situ hybridization histochemistry. Both N52-/TrkA+ and N52-/TrkA- populations had high levels of signals for Nav1.7, Nav1.8, and Nav1.9 mRNAs, but rarely expressed Nav1.1 or Nav1.6. There was no significant difference in these signals, suggesting that C-fiber peptidergic and non-peptidergic neurons have similar electrophysiological characters with regard to sodium currents. N52+/TrkA+ neurons (putative A-fiber nociceptors) had similar high levels of signals for Nav1.7 and Nav1.8, but a significantly lower level of Nav1.9 signals, as compared to N52- neurons. Although, almost no N52+/TrkA- neurons had Nav1.8 or Nav1.9, half of this population expressed Nav1.7 at similar levels to other three populations and the other half completely lacked this channel. These data suggest that Nav1.8 is a common channel for both C- and A-fiber nociceptors, and Nav1.9 is rather selective for C-fiber nociceptors. Nav1.7 is the most universal channel while some functionally unknown N52+/TrkA- subpopulation selectively lacks it.

Expression of leukotriene receptors in the rat dorsal root ganglion and the effects on pain behaviors.

BACKGROUND: Leukotrienes (LTs) belong to the large family of lipid mediators implicated in various inflammatory conditions such as asthma and rheumatoid arthritis. Four distinct types (BLT1, BLT2, CysLT1 and CysLT2) of G-protein-coupled receptors for LTs have been identified. Several studies have reported that LTs are involved in inflammatory pain, but the mechanism and the expression of LT receptors in the nociceptive pathway are unknown. RESULTS: We investigated the precise expression of these four types of LT receptors in the adult rat dorsal root ganglion (DRG) using reverse transcription-polymerase reaction (RT-PCR) and radioisotope-labeled in situ hybridization histochemistry (ISHH). We detected mRNAs for BLT1 and CysLT2 in the DRG, but not for BLT2 and CysLT1. CysLT2 mRNA was preferentially expressed by small sized DRG neurons (about 36% of total neurons), whereas BLT1 mRNA was expressed by non-neuronal cells. Double labeling analysis of CysLT2 with NF-200, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), transient receptor potential vanilloid subfamily 1 (TRPV1) and P2X3 receptor revealed that many CysLT2-labeled neurons were localized with unmyelinated and non-peptidergic neurons, and interestingly, CysLT2 mRNA heavily co-localized with TRPV1 and P2X3-positive neurons. Intraplantar injection of LTC4, a CysLT2 receptor agonist, itself did not induce the thermal hyperalgesia, spontaneous pain behaviors or swelling of hind paw. However, pretreatment of LTC4 remarkably enhanced the painful behaviors produced by alpha, beta-methylene adenosine 5'-triphosphate (αß-me-ATP), a P2X3 receptor agonist. CONCLUSIONS: These data suggests that CysLT2 expressed in DRG neurons may play a role as a modulator of P2X3, and contribute to a potentiation of the neuronal activity following peripheral inflammation.

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 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.

P2Y12 receptor upregulation in activated microglia is a gateway of p38 signaling and neuropathic pain.

Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A metabotropic ATP receptor, P2Y(12), has been shown to be expressed in spinal microglia constitutively and be involved in chemotaxis. Activation of p38 mitogen-activated protein kinase (MAPK) occurs in spinal microglia after nerve injury and may be related to the production of cytokines and other mediators, resulting in neuropathic pain. However, it remains unknown whether any type of P2Y receptor in microglia is involved in the activation of p38 MAPK and the pain behaviors after nerve injury. Using the partial sciatic nerve ligation (PSNL) model in the rat, we found that P2Y(12) mRNA and protein increased in the spinal cord and peaked at 3 d after PSNL. Double labeling studies revealed that cells expressing increased P2Y(12) mRNA and protein after nerve injury were exclusively microglia. Both pharmacological blockades by intrathecal administration of P2Y(12) antagonist and antisense knockdown of P2Y(12) expression suppressed the development of pain behaviors and the phosphorylation of p38 MAPK in spinal microglia after PSNL. The intrathecal infusion of the P2Y(12) agonist 2-(methythio) adenosine 5'-diphosphate trisodium salt into naive rats mimicked the nerve injury-induced activation of p38 in microglia and elevated pain behaviors. These data suggest a new mechanism of neuropathic pain, in which the increased P2Y(12) works as a gateway of the following events in microglia after nerve injury. Activation of this receptor by released ATP or the hydrolyzed products activate p38 MAPK pathway and may play a crucial role in the generation of neuropathic pain.

Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain.

Bradykinin is an inflammatory mediator that plays a pivotal role in pain and hyperalgesia in inflamed tissues by exciting and/or sensitizing nociceptors. TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain. Here, using electrophysiological, immunocytochemical and behavioural analyses, we showed a functional interaction of these two inflammation-related molecules in both heterologous expressing systems and primary sensory neurons. We found that bradykinin increased the TRPA1 currents evoked by allyl isothiocyanate (AITC) or cinnamaldehyde in HEK293 cells expressing TRPA1 and bradykinin receptor 2 (B2R). This potentiation was inhibited by phospholipase C (PLC) inhibitor or protein kinase A (PKA) inhibitor, and mimicked by PLC or PKA activator. The functional interaction between B2R and TRPA1, as well as the modulation mechanism, was also observed in rat dorsal root ganglia neurons. In an occlusion experiment, the PLC activator could enhance AITC-induced TRPA1 current further even in saturated PKA-mediated potentiation, indicating the additive potentiating effects of the PLC and PKA pathways. These data for the first time indicate that a cAMP-PKA signalling is involved in the downstream from B2R in dorsal root ganglia neurons in addition to PLC. Finally, subcutaneous pre-injection of a sub-inflammatory dose of bradykinin into rat hind paw enhanced AITC-induced pain behaviours, which was consistent with the observations in vitro. Collectively, these results represent a novel mechanism through which bradykinin released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury.

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha. Conversely, intrathecal injection of the TLR3 agonist polyinosine-polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine-polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.

Transforming growth factor-activated kinase 1 induced in spinal astrocytes contributes to mechanical hypersensitivity after nerve injury.

Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain.

Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain.

Proinflammatory agents trypsin and mast cell tryptase cleave and activate PAR2, which is expressed on sensory nerves to cause neurogenic inflammation. Transient receptor potential A1 (TRPA1) is an excitatory ion channel on primary sensory nerves of pain pathway. Here, we show that a functional interaction of PAR2 and TRPA1 in dorsal root ganglion (DRG) neurons could contribute to the sensation of inflammatory pain. Frequent colocalization of TRPA1 with PAR2 was found in rat DRG neurons. PAR2 activation increased the TRPA1 currents evoked by its agonists in HEK293 cells transfected with TRPA1, as well as DRG neurons. Application of phospholipase C (PLC) inhibitors or phosphatidylinositol-4,5-bisphosphate (PIP(2)) suppressed this potentiation. Decrease of plasma membrane PIP(2) levels through antibody sequestration or PLC-mediated hydrolysis mimicked the potentiating effects of PAR2 activation at the cellular level. Thus, the increased TRPA1 sensitivity may have been due to activation of PLC, which releases the inhibition of TRPA1 from plasma membrane PIP(2). These results identify for the first time to our knowledge a sensitization mechanism of TRPA1 and a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation.

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.

Roles of extracellular signal-regulated protein kinases 5 in spinal microglia and primary sensory neurons for neuropathic pain.

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.

Alteration of the cell adhesion molecule L1 expression in a specific subset of primary afferent neurons contributes to neuropathic pain.

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.

Intensity-dependent activation of extracellular signal-regulated protein kinase 5 in sensory neurons contributes to pain hypersensitivity.

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.

Suppression of the p75 neurotrophin receptor in uninjured sensory neurons reduces neuropathic pain after nerve injury.

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.

Agonist of proteinase-activated receptor 2 increases painful behavior produced by alpha, beta-methylene adenosine 5'-triphosphate.

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.

Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury.

Hypersensitivity to mechanical stimulation is a well documented symptom of neuropathic pain, for which there is currently no effective therapy. Src-family kinases (SFKs) are involved in proliferation and differentiation and in neuronal plasticity, including long-term potentiation, learning, and memory. Here we show that activation of SFKs induced in spinal cord microglia is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in SFK phosphorylation in the ipsilateral dorsal horn, and SFKs were activated in hyperactive microglia but not in neurons or astrocytes. Intrathecal administration of the Src-family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppressed nerve injury-induced mechanical hypersensitivity but not heat and cold hypersensitivity. Furthermore, PP2 reversed the activation of extracellular signal-regulated protein kinase (ERK), but not p38 mitogen-activated protein kinase, in spinal microglia. In contrast, there was no change in SFK phosphorylation in primary sensory neurons, and PP2 did not decrease the induction of transient receptor potential ion channel TRPV1 and TRPA1 in sensory neurons. Together, these results demonstrate that SFK activation in spinal microglia contributes to the development of mechanical hypersensitivity through the ERK pathway. Therefore, preventing the activation of the Src/ERK signaling cascade in microglia might provide a fruitful strategy for treating neuropathic pain.