Masamichi Okubo

Multiple P2Y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury.

A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Among the P2Y metabotropic receptors, we reported before that the P2Y12 receptor is upregulated in microglia following nerve injury and involved in the phosphorylation of p38 MAPK, and in the development of pain behavior. In this study, we examined the expression of P2Y6, P2Y13, and P2Y14 receptors in the spinal cord and whether these receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury. We found that spared nerve injury induced a dramatic increase of not only P2Y12, but also P2Y6, 13, and 14 receptor mRNA expression in spinal microglia. The increase continued for at least 2 weeks after injury. To determine whether p38 MAPK can induce the expression of P2Y receptors, we administered intrathecally the p38 MAPK inhibitor SB203580 and found that it significantly suppressed P2Y6, P2Y13, and P2Y14 but not P2Y12 mRNAs. Intrathecal injection of the specific P2Y6 antagonist MRS2578, specific P2Y13 antagonist MRS2211 or P2Y14 antisense LNA, attenuated mechanical pain hypersensitivity. Themixture of three antagonists for P2Y6, 12, and 13 showed a longer suppressive effect on pain behavior than the individual treatments. Our data demonstrate that ATP and other nucleotides may stimulate activated microglia with the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors following nerve injury and these receptors are involved in the development of neuropathic pain.

Leukotriene synthases and the receptors induced by peripheral nerve injury in the spinal cord contribute to the generation of neuropathic pain.

Leukotrienes (LTs) belong to a large family of lipid mediators, termed eicosanoids, which are derived from arachidonic acids and released from the cell membrane by phospholipases. LTs are involved in the pathogenesis of inflammatory diseases, such as asthma, rheumatoid arthritis, and peripheral inflammatory pain. In the present study, we examined whether LTs were implicated in pathomechanism of neuropathic pain following peripheral nerve injury. Using the spared nerve injury (SNI) model in rats, we investigated the expression of LT synthases (5‐lipoxygenase; 5‐LO, Five lipoxygenase activating protein; FLAP, LTA4 hydrolase; LTA4h and LTC4 synthase; LTC4s) and receptors (BLT1, 2 and CysLT1, 2) mRNAs in the rat spinal cord. Semi‐quantitative RT‐PCR revealed that 5‐LO, FLAP, LTC4s, BLT1, and CysLT1 mRNAs increased following SNI, but not CysLT2 mRNAs. Using double labeling analysis of in situ hybridization with immunohistochemistry, we observed that 5‐LO, FLAP, and CysLT1 mRNAs were expressed in spinal microglia. LTA4h and LTC4s mRNAs were expressed in both spinal neurons and microglia. BLT1 mRNA was expressed in spinal neurons. The p38 mitogen‐activated protein kinase inhibitor, but not MEK inhibitor, reduced the increase in 5‐LO in spinal microglia. Continuous intrathecal administration of the 5‐LO inhibitor or BLT1 and CysLT1 receptor antagonists suppressed mechanical allodynia induced by SNI. Our findings suggest that the increase of LT synthesis in spinal microglia produced via p38 MAPK plays a role in the generation of neuropathic pain.

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

Summary Although a limited number of L4 dorsal root ganglion neurons undergo minor injuries by L5 spinal nerve ligation, they are unlikely the major contributors to pathomechanisms. ABSTRACT 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.

Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats

Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats.

Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury

BackgroundPlatelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown.MethodsUsing the rat spared nerve injury (SNI) model, we investigated the expression of PAF synthases (LPCAT1 and 2) and PAF receptor (PAFr) mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR) and double-labeling analysis of in situ hybridization histochemistry (ISHH) with immunohistochemistry (IHC) were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086).ResultsRT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia.ConclusionsOur data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development 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. J. Comp. Neurol. 519:1597–1615, 2011.

Peripherally increased artemin is a key regulator of TRPA1/V1 expression in primary afferent neurons

BackgroundArtemin, a member of the glial cell line-derived neurotrophic factor family, is known to have a variety of neuronal functions, and has been the subject of attention because it has interesting effects, including bi-directional results in modulation in neuropathic and inflammatory pain. It has been shown that the overexpression of artemin is associated with an increase in the expression of TRP family channels in primary afferents and subsequent hyperalgesia, and an increase in neuronal activity. The purpose of this study was to examine the peripheral synthesis of artemin in inflammatory and neuropathic pain models, and to demonstrate the effects of long-term or repeated application of artemin in vivo on pain behaviors and on the expression of TRP family channels. Further, the regulatory mechanisms of artemin on TRPV1/A1 were examined using cultured DRG neurons.ResultsWe have demonstrated that artemin is locally elevated in skin over long periods of time, that artemin signals significantly increase in deep layers of the epidermis, and also that it is distributed over a broad area of the dermis. In contrast, NGF showed transient increases after peripheral inflammation. It was confirmed that the co-localization of TRPV1/A1 and GFRα3 was higher than that between TRPV1/A1 and TrkA. In the peripheral sciatic nerve trunk, the synthesis of artemin was found by RT-PCR and in situ hybridization to increase at a site distal to a nerve injury. We demonstrated that in vivo repeated artemin injections into the periphery changed the gene expression of TRPV1/A1 in DRG neurons without affecting GFRα3 expression. Repeated artemin injections also induced mechanical and heat hyperalgesia. Using primary cultured DRG neurons, we found that artemin application significantly increased TRPV1/A1 expression and Ca2+ influx. Artemin-induced p38 MAPK pathway regulated the TRPV1 channel expression, however TRPA1 upregulation by artemin is not mediated through p38 MAPK.ConclusionsThese data indicate the important roles of peripherally-derived artemin on the regulation of TRPV1/A1 in DRG neurons in pathological conditions such as inflammatory and neuropathic pain.

Participation of calcitonin gene related peptide released via axon reflex in the local increase in muscle blood flow following manual acupuncture

Objective The purpose of this study was to determine how calcitonin gene related peptide (CGRP) via axon reflex participates in increasing local muscle blood flow (MBF) following manual acupuncture (MA). Methods Male Sprague–Dawley rats (N=56, 270–350 g) were used. We examined (1) the effects of MA on MBF in the tibialis anterior (TA) muscle in normal rats; (2) the effects of MA on MBF in the TA injected with saline or hCGRP8-37 (low: 2×10−4 mol/litre; high: 2×10−3 mol/litre), a competitive CGRP receptor antagonist, in rats; and (3) the effects of MA on MBF in the TA in capsaicin-treated rats. The capsaicin-treated rats were injected with capsaicin dissolved in an ethanol solution within 24 h after birth (50 mg/kg subcutaneously). MA was applied to the right TA for 1 min. 51Cr-labelled microspheres (15 μm in diameter) were used to measure MBF. Results MA significantly increased MBF without changing arterial blood pressure in normal rats (p<0.05). MA also significantly increased MBF in saline-injected, low hCGRP8-37-injected and high hCGRP8-37-injected rats (p<0.001, 005 and 0.05, respectively). The increases in low and high hCGRP8-37-injected rats were lower than those in saline-injected rats, but the difference was not significant. However, MA did not significantly increase MBF in capsaicin-treated rats (p=0.38). Conclusions We obtained conflicting results, suggesting that the participation of CGRP released via axon reflex may be limited to a local increase in MBF following MA.

Leukotriene Enhances NMDA-Induced Inward Currents in Dorsal Horn Neurons of the Rat Spinal Cord after Peripheral Nerve Injury

BackgroundLTB4 is classified as a leukotriene (LT), a group of lipid mediators that are derived from arachidonic acid. It is recognized that leukotrienes are involved in the pathogenesis of many diseases, including peripheral inflammatory pain. However, little is known about the effects of leukotrienes on the spinal dorsal horn during neuropathic pain. Previously, we reported that there was increased expression of 5-lipoxygenase (5-LO) at spinal microglia, and the leukotriene B4 receptor 1 (BLT1), a high affinity receptor of LTB4, in spinal neurons in spared nerve injury (SNI) model rats. In the present study, we examined the effects of LTB4 on spinal dorsal horn neurons in both naïve and SNI model rats using patch-clamp methods.ResultsBath application of LTB4 did not change AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) or membrane potentials. However, we found that LTB4 enhanced the amplitude of NMDA receptor-mediated sEPSCs and significantly increased exogenous NMDA-induced inward currents in SNI model rats. This increase of inward currents could be inhibited by a selective LTB4 antagonist, U75302, as well as a GDP-β-S, a G-protein inhibitor. These results indicate that both increased LTB4 from spinal microglia or increased BLT1 in spinal neurons after peripheral nerve injury can enhance the activity of NMDA receptors through intracellular G-proteins in spinal dorsal horn neurons.ConclusionOur findings showed that LTB4, which may originate from microglia, can activate BLT1 receptors which are expressed on the membrane of spinal dorsal horn neurons during neuropathic pain. This glia-neuron interaction induces the enhancement of NMDA currents through intracellular G-proteins. The enhancement of NMDA receptor sensitivity of dorsal horn neurons may lead to central sensitization, leading to mechanical pain hypersensitivity.

Contributions of nitric oxide and prostaglandins to the local increase in muscle blood flow following manual acupuncture in rats.

Objective To investigate the contributions of nitric oxide (NO) and prostaglandins (PGs) to the increase in local muscle blood flow (MBF) observed following manual acupuncture (MA). Methods Male Sprague-Dawley rats (n=112; 250–310 g) were injected intraperitoneally with a non-selective NO synthase inhibitor (NG-nitro-L-arginine methyl ester hydrochloride: L-NAME; 10, 50 or 500 mg/kg), a non-selective cyclo-oxygenase inhibitor (indomethacin; 10, 50 or 500 mg/kg), a combination of L-NAME and indomethacin (500 mg/kg each) or saline only under urethane anaesthesia (1.2 g/kg). We used the sparrow pecking technique for 1 min with a stainless steel acupuncture needle (0.20×30 mm) as the acupuncture stimulation method. The stimulus point was on the right tibialis anterior muscle. 51Chromium-labelled microspheres were used for MBF measurement. Results MA increased MBF in the saline-injected group (p<0.001). This increase was partially inhibited by L-NAME in a dose-dependent manner (p>0.05, p<0.05 and p<0.001 for 10, 50 and 500 mg/kg, respectively). On the other hand, indomethacin did not suppress the increase (p>0.05 each for 10, 50 and 500 mg/kg). No significant difference was observed between the inhibitory effects of combined administration of L-NAME and indomethacin and single administration of L-NAME (p>0.05). Conclusions These results suggest that NO is a major factor in the MA-induced increase in MBF, while PGs do not contribute significantly to this increase. As complete inhibition was not achieved by administration of L-NAME±indomethacin, it appears that non-NO and non-PG vasodilators are additionally involved.