Hiroki Yamanaka

TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury

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.

Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats.

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.

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.

Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord

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. J. Comp. Neurol. 498:443–454, 2006.

Differential expression patterns of mRNAs for P2X receptor subunits in neurochemically characterized dorsal root ganglion neurons in the rat.

The ionotropic purine receptors, P2X receptors, are composed of an assembly of multiple P2X subunits. At present, seven subunits have been cloned and named “P2X1–7.” We examined the precise distribution of mRNAs for these subunits in the rat lumbar dorsal root ganglion (DRG) by in situ hybridization histochemistry (ISHH) using riboprobes and characterized their expression among some neuronal subpopulations by ISHH and immunohistochemistry. P2X1 was not expressed by DRG neurons. P2X2 mRNA was preferentially expressed by neurofilament (NF)‐200 negative small‐sized neurons expressing Ret, but not TrkA or TrkC mRNAs. P2X3 mRNA was mainly expressed by NF‐200‐negative neurons. Most P2X3‐positive neurons had Ret mRNA, and about a half of them coexpressed TrkA and TRPV1 mRNAs. P2X4 was the most ubiquitous subunit, evenly distributing among all examined neuronal subpopulations. P2X5 and P2X6 were expressed by about half of the neurons, and most of these neurons were NF‐200‐positive. P2X7 mRNA‐expressing neurons were quite rare. We further examined the coexpression of all pairs of P2X2‐P2X6 mRNAs in DRG neurons and found that: 1) P2X4 was always present in combination with the other subunits. 2) All TrkC neurons had three subunits, P2X4, P2X5, and P2X6, and made up 32% of the total neurons. 3) 12.5% of the total neurons had both P2X2 and P2X3. 4) 12.9% of the neurons had both P2X3 and P2X5. We determined the neuronal subpopulation‐specific distribution of P2X subunits in the DRG. These findings suggest possible combinations of subunits of native P2X receptor in DRG neurons. J. Comp. Neurol. 481:377–390, 2005.

Activation of p38 MAPK in primary afferent neurons by noxious stimulation and its involvement in the development of thermal hyperalgesia

&NA; 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 role of ERK signaling and the P2X receptor on mechanical pain evoked by movement of inflamed knee joint

Abstract 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.

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.

The effect of site and type of nerve injury on the expression of brain-derived neurotrophic factor in the dorsal root ganglion and on neuropathic pain behavior

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.

Expression of mRNA for four subtypes of the proteinase-activated receptor in rat dorsal root ganglia

Proteinase-activated receptors (PARs) are members of the superfamily of G-protein coupled receptors that initiate intracellular signaling by the proteolytic activity of extracellular serine proteases. Three member of this family (PAR-1, PAR-3, and PAR-4) are considered thrombin receptors, whereas PAR-2 is activated by trypsin and tryptase. Recently, activation of PAR-2 signal was identified as a pro-inflammatory factor that mediates peripheral sensitization of nociceptors. Activation of PAR-1 in the periphery is also considered to be a neurogenic mediator of inflammation that is involved in peptide release. Here, we investigated the expression of these four members of PARs in the adult rat dorsal root ganglia (DRG) using radioisotope-labeled in situ hybridization histochemistry. We detected mRNA for all subtypes of PARs in the DRG. Histological analysis revealed the specific expression patterns of the PARs. PAR-1, PAR-2, and PAR-3 mRNA was expressed in 29.0+/-4.0%, 16.0+/-3.2%, and 40.9+/-1.3% of DRG neurons, respectively. In contrast, PAR-4 mRNA was mainly observed in non-neuronal cells. A double-labeling study of PARs with NF-200 and alpha calcitonin gene-related peptide (CGRP) also revealed the distinctive expression of PARs mRNA in myelinated or nociceptive neurons. This study shows the precise expression pattern of PARs mRNA in the DRG and indicates that the cells in DRG can receive modulation with different types of proteinase-activated receptors.