Analgesic effect of gastrin-releasing peptide in the dorsal horn.

Itch and pain are both unpleasant, but they are discrete sensations. Both of these sensations are transmitted by C-fibers and processed in laminae I-II of the dorsal horn. To examine whether pruriception modulates pain, we first confirmed the activation of cells in the itch-related circuits that were positive for gastrin-releasing peptide (GRP) and GRP receptor (GRPR) using a paw formalin injection model. This pain model with typical biphasic pain behavior increased c-Fos but did not affect the expressions of GRP and GRPR mRNAs in the dorsal horn. Using c-Fos expression as a marker for activated cells, we confirmed that formalin injection increased the number of cells double-labeled for c-Fos and GRP or GRPR in the dorsal horn. The emergence of these neurons indicates the activation of itch-related circuits by acute pain signals. The effect of an antagonist for a GRPR was examined in the paw formalin injection model. Intrathecal chronic antagonization of spinal GRPR enhanced the onset of phase II of paw formalin injection-induced pain behavior. Exogenous intrathecal GRP infusion to the paw-formalin injection model not only showed significant reduction of pain behavior but also increased c-Fos in the inhibitory neurons in the dorsal horn. The anti-nociceptive effect of spinal GRP infusion was observed in the peripheral inflammation model (complete Freund's adjuvant injection model). In this study we suggest that painful stimuli activated itch-related neuronal circuits and uncovered the spinal activation of the itch-induced analgesic effect on acute and established inflammatory pain.

Recombinant interleukin-4 alleviates mechanical allodynia via injury-induced interleukin-4 receptor alpha in spinal microglia in a rat model of neuropathic pain.

Glial cells play important roles in the development and maintenance of neuropathic pain. In particular, activated microglia in the spinal cord facilitate the hyper-excitability of dorsal horn neurons after peripheral nerve injury via pro-inflammatory molecules. In this study, we investigated the possible involvement of the anti-inflammatory cytokine, interleukin-4 (IL-4), in neuropathic pain. We did not detect the expression of IL-4 mRNA in the rat dorsal root ganglion or spinal cord; however, peripheral nerve injury induced the expression of IL-4 receptor (IL-4R) alpha mRNA in the spinal cord. A histological analysis revealed that nerve injury induced IL-4R alpha mRNA in activated spinal microglia ipsilateral to the injury site. Additionally, the increases in IL-4R alpha were coincident with the increased expression of phosphorylated signal transducer and activator of transcription 6 (pSTAT6) in spinal microglia. Intrathecal administration of recombinant IL-4 suppressed mechanical hypersensitivity in neuropathic rats, and the analgesic effect of IL-4 was accompanied by further enhancement of pSTAT6 expression in spinal microglia. Taken together, these results suggest that the adaptive responses of microglia to nerve injury involve both inflammatory and anti-inflammatory signaling, including IL-4R alpha and pSTAT6. These findings support that utilizing the endogenous anti-nociceptive activity of IL-4R alpha may modify the cell lineage of pro-nociceptive microglia, thus providing a novel therapeutic strategy for neuropathic pain.

Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord after peripheral nerve injury.

BACKGROUND: LTB4 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. RESULTS: Bath 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-ß-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. CONCLUSION: Our 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.

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

BACKGROUND: Artemin, 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. RESULTS: We 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 Ca(2+) influx. Artemin-induced p38 MAPK pathway regulated the TRPV1 channel expression, however TRPA1 upregulation by artemin is not mediated through p38 MAPK. CONCLUSIONS: These 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.

Transition to persistent orofacial pain after nerve injury involves supraspinal serotonin mechanisms.

The orofacial region is a major focus of chronic neuropathic pain conditions characterized by primary hyperalgesia at the site of injury and secondary hyperalgesia outside the injured zone. We have used a rat model of injury to the maxillary branch (V2) of the trigeminal nerve to produce constant and long-lasting primary hyperalgesia in the V2 territory and secondary hyperalgesia in territories innervated by the mandibular branch (V3). Our findings indicate that the induction of primary and secondary hyperalgesia depended on peripheral input from the injured nerve. In contrast, the maintenance of secondary hyperalgesia depended on central mechanisms. The centralization of the secondary hyperalgesia involved descending 5-HT drive from the rostral ventromedial medulla and the contribution of 5-HT3 receptors in the trigeminal nucleus caudalis (Vc), the homolog of the spinal dorsal horn. Electrophysiological studies further indicate that after nerve injury spontaneous responses and enhanced poststimulus discharges in Vc nociresponsive neurons were time-dependent on descending 5-HT drive and peripheral input. The induction phase of secondary hyperalgesia involved central sensitization mechanisms in Vc neurons that were dependent on peripheral input, whereas the maintenance phase of secondary hyperalgesia involved central sensitization in Vc neurons conducted by a delayed descending 5-HT drive and a persistence of peripheral inputs. Our results are the first to show that the maintenance of secondary hyperalgesia and underlying central sensitization associated with persistent pain depend on a transition to supraspinal mechanisms involving the serotonin system in rostral ventromedial medulla-dorsal horn circuits.

Resistin-like molecule ß is abundantly expressed in foam cells and is involved in atherosclerosis development.

OBJECTIVE: Resistin-like molecule (RELM) ß is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELMß and thus investigated the role of RELMß in the development of atherosclerosis. APPROACH AND RESULTS: It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELMß. RELMß knockout ((-/-)) and wild-type mice were mated with apolipoprotein E-deficient background mice. RELMß(-/-) apolipoprotein E-deficient mice exhibited less lipid accumulation in the aortic root and wall than RELMß(+/+) apolipoprotein E-deficient mice, without significant changes in serum lipid parameters. In vitro, RELMß(-/-) primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide-induced nuclear factor-κB classical pathway activation and inflammatory cytokine secretion than RELMß(+/+), whereas stimulation with RELMß upregulated inflammatory cytokine expressions and increased expressions of many lipid transporters and scavenger receptors in PCPMs. Flow cytometric analysis revealed inflammatory stimulation-induced RELMß in F4/80(+) CD11c(+) PCPMs. In contrast, the expressions of CD11c and tumor necrosis factor were lower in RELMß(-/-) PCPMs, but both were restored by stimulation with recombinant RELMß. CONCLUSIONS: RELMß is abundantly expressed in foam cells within plaques and contributes to atherosclerosis development via lipid accumulation and inflammatory facilitation.

COX2 expression plays a role in spinal cord injury-induced neuropathic pain.

BACKGROUND CONTEXT: Elucidating the mechanism of neuropathic pain (NeP) is crucial as it can result in motor dysfunction and negatively impact quality of life in patients with spinal cord injury (SCI). Although it has been reported that cyclooxygenase 2 (COX2) is involved in NeP in rat models of peripheral nerve injury and that COX2 inhibitors can alleviate NeP, these mechanisms after SCI have not been fully investigated. PURPOSE: The purpose is to investigate whether the thoracic SCI affects the expression of mRNAs for COX1 and COX2 in the lumbar spinal cord, and the effect of COX2 inhibitor on its behavior. STUDY DESIGN: Male Sprague-Dawley (SD) rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon (IH) impactor device. SCI rats received COX2 inhibitors (50 µg/day) on days 5 and 6 after SCI. METHODS: Male SD rats underwent T10 laminectomy under mixed anesthesia, and IH impactors were applied to the same site to create a rat SCI model. Rats that underwent only laminectomy were designated as sham. Lumbar spinal cord at the L4-5 level was harvested at 3, 5, 7, 14, and 28 days after SCI, and COX2 and COX1 were quantified by reverse-transcription PCR (RT-PCR). COX2 expression, expression site, and expression time were determined by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISHH) at the same time points. The expression site and time of COX2 expression were also examined at the same time point by ISHH. On 5th and 6th day after SCI, saline and COX2 inhibitor (50 µg/day) were administered into the subarachnoid space as a single dose, and the two groups were compared in terms of mechanical withdrawal latency using the dynamic plantar esthesiometer, which is an automated von Frey-type system. RESULTS: COX2 was significantly increased at 5 and 7 days after SCI, but no significant difference in COX1 was observed after SCI by RT-PCR. ISHH targeting COX2 showed clear expression of COX2 in spinal cord vascular endothelial cells at 5 and 7 days after SCI. COX2 expression was almost abolished at day 14 and 28. Behavioral experiments showed that pain was significantly improved from day 2 after COX2 inhibitor administration compared to the saline group, with improvement up to day 14 after SCI, but no significant difference was observed after day 21. CONCLUSIONS: The present findings suggest that thoracic SCI increased COX2 in vascular endothelial cells in the lumbar spinal cord and that the administration of COX2 inhibitor significantly alleviated mechanical hypersensitivity of the hind-paw following the thoracic SCI. Therefore, endothelial cell derived COX2 in the lumbar spinal cord may be involved in the induction of neuropathic pain in the SCI model rats. CLINICAL SIGNIFICANCE: The findings in the present study regarding the induction of endothelial COX2 and the effect of its inhibitor on the mechanical hypersensitivity suggest that endothelial cell-derived COX2 is one of the focuses for the treatment for neuropathic pain in the acute phase of SCI.

Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration.

Peripheral nerve injury activates spinal glial cells, which may contribute to the development of pain behavioral hypersensitivity. There is growing evidence that activated microglia show dynamic changes in cell morphology; however, the molecular mechanisms that underlie the modification of the membrane and cytoskeleton of microglia are not known. Here, we investigated the phosphorylation of ezrin, radixin, and moesin (ERM) proteins in the spinal cord after peripheral nerve injury. ERM is known to function as membrane-cytoskeletal linkers and be localized at filopodia- and microvilli-like structures. ERM proteins must be phosphorylated at a specific C-terminal threonine residue to be in the active state. The nature of ERM proteins in the spinal cord of animals in a neuropathic pain model has not been investigated and characterized. In the present study, we observed an increase in the phosphorylated ERM in the spinal microglia following spared nerve injury. The intrathecal administration of lysophosphatidic acid induced the phosphorylation of ERM proteins in microglia along with the development of mechanical pain hypersensitivity. Intrathecal administration of ERM antisense locked nucleic acid suppressed nerve injury-induced tactile allodynia and decreased the phosphorylation of ERM, but not the Iba1 staining pattern, in spinal glial cells. These findings suggest that lysophosphatidic acid induced the phosphorylation of ERM proteins in spinal microglia and may be involved in the emergence of neuropathic pain. These findings may underlie the pathological mechanisms of nerve injury-induced neuropathic pain.

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. The mixture 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.

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

BACKGROUND: Platelet-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. METHODS: Using 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). RESULTS: RT-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. CONCLUSIONS: Our 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.

Leukotrienes in nociceptive pathway and neuropathic/inflammatory pain.

The purpose of this review is to summarize the recent studies examining the expression of leukotrienes (LTs) and their receptors in nociceptive pathways, and their crucial roles in pathological pain conditions. 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 known to be important factors in a variety of local and systemic diseases and allergic/inflammatory diseases. We examined whether LTs were implicated in neuropathic pain following peripheral nerve injury. Using the SNI model in rats, we investigated the expression of LT synthases and receptors mRNAs in the spinal cord and the roles on the pain behaviors. We found the expression of 5-lipoxygenase (5-LO), FLAP and the cysteinyl leukotrienes (CysLT1) mRNAs in spinal microglia, LTA4h and LTC4s mRNAs in both spinal neurons and microglia, and BLT1 mRNA in spinal neurons. Administration of the 5-LO inhibitor or the receptor antagonists suppressed mechanical allodynia. Our findings suggest that the increase of LT synthesis in spinal microglia produced via p38 mitogen-activated protein kinase (MAPK) plays a role in the generation of neuropathic pain. We also examined the expression and roles on pain behaviors of LT receptors in the dorsal root ganglion (DRG) using a peripheral inflammation model. The data indicate CysLT2 expressed in DRG neurons may play a role as a modulator of P2X3, and contribute to the potentiation of the neuronal activity following peripheral inflammation. This review summarizes the hypothesis that LTs might work in the spinal cord and primary afferent in pathological pain conditions.

Aberrant Axo-Axonic Synaptic Reorganization in the Phosphorylated L1-CAM/Calcium Channel Subunit α2δ-1-Containing Central Terminals of Injured c-Fibers in the Spinal Cord of a Neuropathic Pain Model.

In the dorsal horn of the spinal cord, peripheral nerve injury induces structural and neurochemical alterations through which aberrant synaptic signals contribute to the formation of neuropathic pain. However, the role of injured primary afferent terminals in such plastic changes remain unclear. In this study, we investigated the effect of nerve injury on the morphology of cell adhesion molecule L1-CAM [total L1-CAM (tL1-CAM)]-positive primary afferent terminals and on the synaptic contact pattern in the dorsal horn. In the confocal images, the tL1-CAM-positive terminals showed morphologic changes leading to the formation of hypertrophic varicosities in the c-fiber terminal. These hypertrophic varicosities in the dorsal horn were co-labeled with phosphorylated (Ser1181) L1-CAM (pL1-CAM) and shown to store neurotransmitter peptides, but not when co-labeled with the presynaptic marker, synaptophysin. Quantitative analyses based on 3D-reconstructed confocal images revealed that peripheral nerve injury reduced dendritic synaptic contacts but promoted aberrant axo-axonic contacts on the tL1-CAM-positive hypertrophic varicosities. These tL1-CAM-positive varicosities co-expressed the injury-induced α2δ-1 subunit of the calcium channel in the dorsal horn. Administration of the anti-allodynic drug, pregabalin, inhibited accumulation of α2δ-1 and pL1-CAM associated with a reduction in hypertrophic changes of tL1-CAM-positive varicosities, and normalized injury-induced alterations in synaptic contacts in the dorsal horn. Our findings highlight the formation of aberrant spinal circuits that mediate the convergence of local neuronal signals onto injured c-fibers, suggesting that these hypertrophic varicosities may be important contributors to the pathologic mechanisms underlying neuropathic pain.

Localization of prostaglandin E2 synthases and E-prostanoid receptors in the spinal cord in a rat model of neuropathic pain.

Prostaglandin E2 (PGE2) is a lipid mediator which plays a role in the generation of inflammatory and neuropathic pain. In the peripheral nervous system, PGE2 sensitizes nociceptive afferent neurons through E-prostanoid (EP) receptors. In the central nervous system, PGE2 modulates pain sensitivity and contributes to the development of neuropathic pain. However, the distribution of PGE2 and EP receptors in the spinal cord remains unclear. In the present study, we examined the expression of PGE2 synthases (microsomal PGE synthase [mPGES]-1, mPGES-2, and cytosolic PGE synthase [cPGES]) and EP receptors (EP1-4) in a rat model of neuropathic pain. We identified that mPGES-1 mRNA was upregulated in spinal endothelial cells after nerve injury and exhibited co-localization with cyclooxygenase-2 (COX-2). We detected that mPGES-2 mRNA and cPGES mRNA were expressed in spinal neurons and noted that their expression level was not affected by nerve injury. With respect to EP receptors, EP2 mRNA and EP4 mRNA were expressed in spinal neurons in the dorsal horn. EP3 mRNA was expressed in motor neurons, whereas EP1 mRNA was not detected in the spinal cord. Intrathecal injection of tumor necrosis factor alpha (TNFα) upregulated mPGES-1 mRNA in blood vessels in the spinal cord. Intrathecal injection of a TNFα-neutralizing antibody partially inhibited the upregulation of mPGES-1 mRNA after nerve injury. These results indicate that PGE2 is synthesized by COX-2/mPGES-1 in spinal endothelial cells after nerve injury. These results suggest that in neuropathic pain condition, endothelial cell-derived PGE2 may act on EP2 and EP4 receptors on spinal neurons and modulate pain sensitivity.

Role of Rho-associated coiled-coil containing protein kinase in the spinal cord injury induced neuropathic pain.

BACKGROUND CONTEXT: Spinal cord injury (SCI) can lead to increased phosphorylation of p38 in spinal cord microglia. This is one of the main causes for the development of persistent pain. Recently, we reported our study on the activation of p38 mitogen-activated protein kinases (MAPK) in spinal microglia, which has been considered the key molecule for the onset and maintenance of neuropathic pain after peripheral nerve injury, using a rat model. We also reported that the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) pathway mediates p38 activation in spinal microglia in peripheral nerve injury. But the precise mechanisms of neuropathic pain induced by SCI are still unclear. PURPOSE: This study aimed to examine the activation of microglia and the p38 MAPK expression in the lumbar spinal cord after thoracic SCI in rats, and the correlation to the therapeutic effect of ROCK inhibitor ripasudil in rats with SCI. STUDY DESIGN: Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon impactor device. SCI rats received ROCK inhibitor ripasudil (24 nmol/day or 240 nmol/day) from just before SCI to 3 days after SCI. METHODS: The mechanical threshold in the rat's hind paws was measured over four weeks. Morphology of microglia and phosphorylation of p38 (p-p38) in the lumbar spinal cord and were analyzed using immunohistochemistry. RESULTS: The p-p38 positive cell and Iba1 (a maker of microglia) positive area were significantly increased at the lumbar spinal dorsal horn (L4-5) 3 days and 7 days after SCI compared with the sham-control (p<.05), whereas phosphorylated p38 was co-localized with microglia. Three days after SCI, the intensity of phosphorylated p38 and Iba1 immunoreactive cells in the dorsal horn was significantly lower in the ripasudil treated groups than in the saline group. However, administration of ROCK inhibitor did not affect the numbers of microglia. Moreover, the withdrawal threshold of the ripasudil-treated rats was significantly higher than that of the saline-injected rats on 14 days and 28 days after SCI. CONCLUSIONS: Our results suggest that activation of ROCK in spinal cord microglia is likely to have an important role in the activation of p38 MAPK, which has been considered as a key molecule that switches on neuropathic pain after SCI. Inhibition of ROCK signaling may offer a means in developing a novel neuropathic pain treatment after SCI. It may help patients with neuropathic pain after SCI. CLINICAL SIGNIFICANCE: The findings in the present study regarding intracellular mechanisms suggest that modulation of ROCK signaling may be a focus for novel treatment for neuropathic pain after SCI.

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.

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.

Effects of manual acupuncture with sparrow pecking on muscle blood flow of normal and denervated hindlimb in rats.

INTRODUCTION: In clinical practice, it has been thought that acupuncture might serve to wash out pain-generating metabolic end-products by improving blood circulation in muscles. We investigated the effects of manual acupuncture (MA) on muscle blood flow (MBF) of normal and denervated hindlimbs in rats. METHOD: Sprague-Dawley rats (n=100) anaesthetised with urethane (1.2g/kg ip) were used. Manual acupuncture with sparrow pecking (SP) at different doses (1, 10 or 30 pecks) was given to the right ventral hindlimb muscles (tibial anterior and extensor digitorum longus muscles) or the right dorsal hindlimb muscles (gastrocnemius, plantaris and soleus muscles). MBF with or without MA was measured using the radiolabelled microsphere technique. The blood pressure was recorded through the right common carotid artery until MBF measurement started. Denervation of hindlimb was conducted by cutting the sciatic and femoral nerves. RESULTS: In normal rats, significantly increased MBF after MA were observed only in muscles which were penetrated by an acupuncture needle. The size of the increase depended on the number of times of pecking and seemed to be sustained at least until 60 minutes after MA. However, the increase was observed after both acute and chronic denervation. On the other hand, the mean arterial blood pressure (MAP) did not change significantly before, during or after MA. CONCLUSION: These results suggest that MA could increase muscle blood flow locally in a dose-dependent manner and that this increase may be caused by local vasodilators, as well as the axon reflex. A further study is needed to elucidate the mechanism.

Upregulation of calcium channel alpha-2-delta-1 subunit in dorsal horn contributes to spinal cord injury-induced tactile allodynia.

BACKGROUND CONTEXT: Spinal cord injury (SCI) commonly results not only in motor paralysis but also in the emergence of neuropathic pain (NeuP), both of which can impair the quality of life for patients with SCI. In the clinical field, it is well known that pregabalin, which binds to the voltage-gated calcium channel alpha-2-delta-1 (α2δ-1) subunit has therapeutic effects on NeuP after SCI. A previous study has demonstrated that SCI increased α2δ-1 in the L4-L6 dorsal spinal cord of SCI rats by Western blot analysis and that the increase of α2δ-1 was correlated with tactile allodynia of the hind paw. However, the detailed feature of an increase in α2δ-1 protein in the spinal dorsal horn and the mechanism of pregabalin effect on SCI-induced NeuP have not been fully examined. PURPOSE: This study aimed to examine the detailed distribution of α2δ-1 expression in the lumbar spinal cord after thoracic SCI in rats and the correlation of the therapeutic effect of pregabalin in SCI rats. STUDY DESIGN: Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using the IH impactor device. Spinal cord injury rats received pregabalin (30 mg/kg) once a day for 2 weeks over a 4-week period after SCI. METHODS: The mechanical threshold in the rat hind paw was measured over 4 weeks. Alpha-2-delta-1 expression in the lumbar spinal cord and in the dorsal root ganglion (DRG) was analyzed using immunohistochemistry and in situ hybridization histochemistry. RESULTS: A significant reduction of the withdrawal threshold of mechanical stimuli to the hind paw was observed for 2 weeks and continued at least 4 weeks after SCI. In the control rats, expression of α2δ-1 immunoreactivity was detected mainly in laminae I and II in the lumbar dorsal horn. Thoracic SCI significantly increased α2δ-1 immunoreactivity in laminae I and II in the lumbar dorsal horn 4 weeks after SCI; however, thoracic SCI did not affect the expression of α2δ-1 mRNA in the L4 and L5 DRGs. Meanwhile, the signal intensity of α2δ-1 mRNAs in the lumbar spinal cord increased from Day 7 and continued for at least 28 days after SCI. Cellular analysis showed that SCI increased the number of α2δ-1-expressing cells in laminae I and II. The tactile allodynia of the hind paw in the SCI rats was reversed after pregabalin treatment and was maintained for 21 days. This administration of pregabalin decreased the α2δ-1 immunoreactivity significantly in the lumbar dorsal horn of thoracic SCI rats at 28 days after SCI. CONCLUSIONS: The present study results suggest that an increase of α2δ-1 in the L4 and L5 dorsal horns after thoracic SCI is derived from the increase in the expression in lumbar spinal neurons. This increase may be involved in the development of NeuP in the hind paws and the therapeutic effect of pregabalin on central NeuP after SCI.

Serum leptin and adiponectin are positively associated with bone mineral density at the distal radius in patients with type 2 diabetes mellitus.

There have been several reports about associations of serum leptin or adiponectin with bone mineral density and biochemical markers of bone turnover. However, the precise roles of adipocytokines in bone metabolism have not been fully elucidated. We investigated the associations of serum level of leptin or adiponectin with bone mineral density, serum osteocalcin, and urinary N-terminal telopeptide of type I collagen (NTX) in 40 Japanese patients with type 2 diabetes mellitus. Bone mineral density was measured by using dual-energy x-ray absorptiometry at different sites (distal radius, femoral neck, and lumbar spine) and was expressed as z score. Multiple regression analysis revealed that there were significant positive correlations between serum leptin or adiponectin level and z score at the distal radius, but not at the femoral neck or the lumbar spine. Although no correlation was observed between serum leptin and serum osteocalcin, there was a significant negative correlation between serum leptin and urinary NTX, a marker of bone resorption. No correlation was observed between serum adiponectin and serum osteocalcin or urinary NTX. These results indicate that leptin and adiponectin may have a protective effect on bone metabolism in patients with type 2 diabetes mellitus.