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Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensations. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

DOI： 10.1016/j.neuropharm.2020.108025

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DOI： 10.1177/1744806918796409

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEDERATION AMER SOC EXP BIOL  

Transcriptional and post-translational regulations are important in peripheral nerve injury-induced neuropathic pain, but little is known about the role of post-transcriptional modification. Our objective was to determine the possible effect of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyze post-transcriptional RNA editing, in tactile allodynia, a hallmark of neuropathic pain. Seven days after L5 spinal nerve transection (SNT) in adultmice, we found an increase in ADAR2 expression and a decrease in ADAR3 expression in the injured, but not in the uninjured, dorsal root ganglions (DRGs). These changes were accompanied by elevated levels of editing at the D site of the serotonin (5-hydroxytryptamine) 2C receptor (5-HT2CR), at the I/V site of coatomer protein complex subunit alpha(COPA), and at the R/G site of AMPA receptor subunit GluA2 in the injured DRG. Compared to Adar2(+/+)/Gria2(R/R) littermate controls, Adar2(+/+)/Gria2(R/R) mice completely lacked the increased editing of 5-HT2CR, COPA, and GluA2 transcripts in the injured DRG and showed attenuated tactile allodynia after SNT. Furthermore, the antidepressant fluoxetine inhibited neuropathic allodynia after injury and reduced the COPAI/V site editing in the injured DRG. These findings suggest that ADAR2 is a mediator of injury-induced tactile allodynia and thus a potential therapeutic target for the treatment of neuropathic pain.-Uchida, H., Matsumura, S., Okada, S., Suzuki, T., Minami, T., Ito, S. RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury.

DOI： 10.1096/fj.201600950R

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Itch and pain are intimately related and may share similar peripheral and central mechanisms and pathways. However, it has been believed that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Although we previously demonstrated that phosphorylation of GluN2B subunits of the NMDA receptor at Tyr1472 is important for central sensitization in a neuropathic pain model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of GluN2B (Y1472F-KI), the role of NMDA receptors in itch transmission remains unknown. Here, we demonstrated that the scratching behaviors elicited by various pruritogens applied to the cheek and c-fos expression in the region innervated by the trigeminal nerve were markedly attenuated in the Y1472F-KI mice. The c-fos immunoreactivity was co-localized with the receptor of gastrin-releasing peptide (GRP). Scratching behaviors evoked by chloroquine were inhibited by the NMDA receptor antagonists D-AP5 and CP101,606 and by the Src kinase inhibitor PP2. Direct activation of the trigeminal region by intracisternal administration of NMDA and GRP induced robust scratching behaviors, both of which were reduced by the GRP receptor antagonist RC-3095. Taken together, the data obtained in this present study are the first to demonstrate that phosphorylation of GluN2B subunit at Tyr1472 is important for trigeminal transmission of itch and suggest that the NMDA receptor activation occurs upstream of the GRP-GRP receptor pathway.

DOI： 10.1111/ejn.13337

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Adenosine deaminase acting on RNA (ADAR) enzymes, ADAR1 and ADAR2, mediates adenosine-to-inosine RNA editing, and their mRNA expressions are altered during developmental, physiological, and pathophysiological processes in the nervous system. The present study attempted to investigate the involvement of epigenetic modifying enzymes, such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC), in the regulation of ADAR1 and ADAR2 mRNA expressions in neuronal cells. Using human neuronal SH-SY5Y cells, we found that the DNMT inhibitor 5-aza-2-deoxycytidine led to an increase in ADAR2, but not ADAR1, mRNA expression in a concentration-dependent and time-dependent manner. However, treatment with HDAC inhibitor trichostatin A elicited an increase in ADAR2 mRNA expression and a decrease in ADAR1 mRNA expression, and these changes were blocked by actinomycin D, a transcription inhibitor. Taken together, these findings suggest that ADAR1 and ADAR2 expressions are subject to different regulations by DNMT and HDAC enzymes in neuronal SH-SY5Y cells.

DOI： 10.1097/WNR.0000000000000474

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

Neuropathic pain is often insensitive to morphine. Our previous study has demonstrated that neuronrestrictive silencer factor represses mu opioid receptor (MOP) gene expression in the dorsal root ganglion (DRG) via histone hypoacetylation-mediated mechanisms after peripheral nerve injury, thereby causing loss of peripheral morphine analgesia. Here, we showed that histone deacetylase (HDAC) inhibitors, such as trichostatin A and valproic acid, restored peripheral and systemic morphine analgesia in neuropathic pain. Also, these agents blocked nerve injury-induced MOP down-regulation in the DRG. These results suggest that HDAC inhibitors could serve as adjuvant analgesics to morphine for the management of neuropathic pain. (c) 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.

DOI： 10.1016/j.jphs.2015.07.040

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

Treatment of fibromyalgia is an unmet medical need. To develop novel therapies for the treatment of fibromyalgia, we explored pain therapeutic actions of existing pharmaceuticals, which inhibit the somatic symptoms frequently observed in fibromyalgia patients. This study first examined the therapeutic actions of pilocarpine, which inhibits dry-eye and dry-mouth symptoms, using an experimental fibromyalgia-like chronic pain model produced by intermittent cold stress (ICS) in mice. A single intraperitoneal and intracerebroventricular, but not intrathecal, pilocarpine administration attenuated ICS-induced thermal hyperalgesia and mechanical allodynia, and this action was abolished by muscarinic antagonist pirenzepine (i.c.v.). Treatment with 1-10 mg/kg donepezil (i.p.), which can easily penetrate into the brain, also showed similar therapeutic effects. Importantly, we found that both pilocarpine and donepezil produced antihyperalgesic effects via supraspinal action. Furthermore, repeated donepezil treatments completely cured the ICS-induced hyperalgesia and allodynia even after the cessation of drug treatments. Acute and chronic treatments of these cholinomimetics had no effects on the nociceptive threshold in control animals. By contrast, the lack of morphine (i.c.v.) analgesia initially observed in the ICS model remained in ICS model mice treated with long-term donepezil. Collectively, these findings suggest that stimulation of the muscarinic cholinergic system effectively inhibits some mechanisms underlying chronic pain in the ICS model, but does not inhibit the lack of descending pain-inhibitory mechanisms, which are driven by central morphine.

DOI： 10.1124/jpet.114.222414

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Language：English   Publisher：BENTHAM SCIENCE PUBL LTD  

Neuropathic pain is characterized by complicated combination of positive (e.g., hyperalgesia and allodynia) and negative (e. g., hypoesthesia and hypoalgesia) symptoms, and is often refractory to conventional pharmacological agents, including morphine. Although the molecular mechanisms for positive symptoms are extensively studied, those for negative symptoms are poorly understood. There is convincing evidence that altered gene expression within peripheral and central nervous systems is a key mechanism for neuropathic pain; however, its transcriptional mechanisms are poorly understood. Epigenetic modifications, such as DNA methylation and histone modifications (e.g., acetylation, methylation, and phosphorylation), are known to cause stable gene expression via chromatin remodeling. These mechanisms have a role not only in the determination of developmental cell fates, but also in the physiological and pathological processes in nervous system. Moreover, epigenetic therapies using epigenetic modifying compounds are progressively advanced in the treatments of diverse diseases, including cancer and neurological diseases. Importantly, there is emerging evidence that a variety of genes undergo epigenetic regulation via DNA methylation and histone modifications within peripheral and central nervous systems, thereby contributing to the alterations in both pain sensitivity and pharmacological efficacy in neuropathic pain. In this review, we will highlight the epigenetic gene regulation underlying neuropathic pain, especially focusing on the negative symptoms. Moreover, we will discuss whether epigenetic mechanisms can serve as a potential target to treat neuropathic pain.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Paclitaxel, which is widely used for the treatment of solid tumors, causes neuropathic pain via poorly understood mechanisms. Previously, we have demonstrated that lysophosphatidic acid (LPA) and its receptors (LPA(1) and LPA(3)) are required for the initiation of peripheral nerve injury induced neuropathic pain. The present study aimed to clarify whether LPA and its receptors could mediate paclitaxel-induced neuropathic pain.
Results: Intraperitoneal administration of paclitaxel triggered a marked increase in production of LPA species (18:1-, 16:0-, and 18:0-LPA) in the spinal dorsal horn. Also we found significant activations of spinal cytosolic phospholipase A(2) and calcium independent phospholipase A2 after the paclitaxel treatment. The paclitaxel-induced LPA production was completely abolished not only by intrathecal pretreatment with neurokinin 1 (NK1) or N-methyl-(D)-aspartate (NMDA) receptor antagonist, but also in LPA(1) receptor deficient (Lpar1(-/-)) and LPA3 receptor deficient (Lpar3(-/-)) mice. In addition, the pharmacological blockade of NK1 or NMDA receptor prevented a reduction in the paw withdrawal threshold against mechanical stimulation after paclitaxel treatments. Importantly, the paclitaxel-induced mechanical allodynia was absent in Lparl(-/-) and Lpar3(-/-) mice.
Conclusions: These results suggest that LPA(1) arid LPA(3) receptors mediated amplification of spinal LPA production is required for the development of paclitaxel-induced neuropathic pain.

DOI： 10.1186/1744-8069-10-71

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-I alpha expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-I alpha, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-I alpha; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-I alpha activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 mu M to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-I alpha was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.

DOI： 10.1007/s00216-013-7535-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Brain-derived neurotrophic factor (BDNF) is known to be up-regulated in the dorsal root ganglion (DRG) after peripheral nerve injury, and to contribute to neuropathic pain. Here, we found that thermal hyperalgesia and mechanical allodynia at day 7 post-injury were inhibited only when anti-BDNF antibody was intrathecally administrated at day 2 post-injury. Consistent with behavioral results, Western blot analysis showed that the expression levels of BDNF protein in the spinal dorsal horn were markedly induced during early stage post-injury. Moreover, the maximal increase in BDNF mRNA expression in the DRG was observed at day 1 post-injury, and significantly elevated levels were sustained for at least 14 days. Four of five BDNF mRNA transcripts were up-regulated after nerve injury, and the most inducible transcript was exon I. Using a chromatin immunoprecipitation (ChIP) assay, we found that nerve injury promotes histone H3 and H4 acetylation, transcriptionally active modifications, at BDNF promoter I at day 1 post-injury, and the levels of histone acetylation remain elevated for at least 7 days. Taken together, our findings suggest that an initial increase in BDNF exon I expression controlled by epigenetic mechanisms might have a crucial role in the development of neuropathic pain. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2013.02.053

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

Background: Fibromyalgia (FM) is characterized by chronic widespread pain, which is often refractory to conventional painkillers. Numerous clinical studies have demonstrated that antidepressants are effective in treating FM pain. We previously established a mouse model of FM-like pain, induced by intermittent cold stress (ICS).
Results: In this study, we find that ICS exposure causes a transient increase in plasma corticosterone concentration, but not in anxiety or depression-like behaviors. A single intrathecal injection of an antidepressant, such as milnacipran, amitriptyline, mianserin or paroxetine, had an acute analgesic effect on ICS-induced thermal hyperalgesia at post-stress day 1 in a dose-dependent manner. In addition, repeated daily antidepressant treatments during post-stress days 1-5 gradually reversed the reduction in thermal pain threshold, and this recovery was maintained for at least 7 days after the final treatment. In addition, relief from mechanical allodynia, induced by ICS exposure, was also observed at day 9 after the cessation of antidepressant treatment. In contrast, the intravenous administration of these antidepressants at conventional doses failed to provide relief.
Conclusions: These results suggest that the repetitive intrathecal administration of antidepressants permanently cures ICS-induced FM pain in mice.

DOI： 10.1186/1744-8069-7-69

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Although neuropathic pain is frequently observed in demyelinating diseases such as Guillain-Barre syndrome and multiple sclerosis, the molecular basis for the relationship between demyelination and neuropathic pain behaviors is poorly understood. Previously, we found that lysophosphatidic acid receptor (LPA(1)) signaling initiates sciatic nerve injury-induced neuropathic pain and demyelination.
Results: In the present study, we have demonstrated that sciatic nerve injury induces marked demyelination accompanied by myelin-associated glycoprotein (MAG) down-regulation and damage of Schwann cell partitioning of C-fiber-containing Remak bundles in the sciatic nerve and dorsal root, but not in the spinal nerve. Demyelination, MAG down-regulation and Remak bundle damage in the dorsal root were abolished in LPA(1) receptor-deficient (Lpar1(-/-)) mice, but these alterations were not observed in sciatic nerve. However, LPA-induced demyelination in ex vivo experiments was observed in the sciatic nerve, spinal nerve and dorsal root, all which express LPA(1) transcript and protein. Nerve injury-induced dorsal root demyelination was markedly attenuated in mice heterozygous for autotaxin (atx(+/-)), which converts lysophosphatidylcholine (LPC) to LPA. Although the addition of LPC to ex vivo cultures of dorsal root fibers in the presence of recombinant ATX caused potent demyelination, it had no significant effect in the absence of ATX. On the other hand, intrathecal injection of LPC caused potent dorsal root demyelination, which was markedly attenuated or abolished in atx(+/-) or Lpar1(-/-) mice.
Conclusions: These results suggest that LPA, which is converted from LPC by ATX, activates LPA(1) receptors and induces dorsal root demyelination following nerve injury, which causes neuropathic pain.

DOI： 10.1186/1744-8069-6-78

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

P>The present study demonstrates a novel high-affinity neuronal target for endocrine disrupting chemicals (EDCs), which potentially cause psychological disorders. EDCs competitively inhibited the binding of bovine serum albumin-conjugated progesterone to recombinant human microtubule-associated protein 2C (rhMAP2C) with an inhibition constant at picomolar levels. In the rhMAP2C-stimulated tubulin assembly assay, agonistic enhancement was observed with dibutyl phthalate and pentachlorphenol and pregnenolone, while an inverse agonistic effect was observed with 4-nonylphenol. In contrast, progesterone and many of the EDCs, including bisphenol A, antagonized the pregnenolone-induced enhancement of rhMAP2C-stimulated tubulin assembly. These agonistic and inverse agonistic actions were not observed in tubulin assembly stimulated with delta 1-71 rhMAP2C, which lacks the steroid-binding site. Using a dark-field microscopy, pregnenolone and pentachlorphenol were observed to generate characteristic filamentous microtubules in a progesterone- or bisphenol A-reversible manner. In cultured hippocampal neurons, similar agonist-antagonist relationships were reproduced in terms of dendritic outgrowth. Fluorescent recovery after photobleaching of hippocampal neurons showed that pregnenolone and agonistic EDCs enhanced, but that 4-nonylphenol inhibited the MAP2-mediated neurite outgrowth in a progesterone- or antagonistic EDC-reversible manner. Furthermore, none of the examined effects were affected by mifepristone or ICI-182,786 i.e. the classical progesterone and estrogen receptor antagonists. Taken together, these results suggest that EDCs cause a wide variety of significant disturbances to dendritic outgrowth in hippocampal neurons, which may lead to psychological disorders following chronic exposure during early neuronal development.

DOI： 10.1111/j.1471-4159.2010.06847.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We previously reported that lysophosphatidic acid (LPA) initiates nerve injury-induced neuropathic pain and its underlying mechanisms. In addition, we recently demonstrated that intrathecal injection of LPA induces de novo LPA production through the action of autotaxin (ATX), which converts lysophosphatidylcholine to LPA. Here, we examined nerve injury-induced de novo LPA production by using a highly sensitive biological titration assay with B103 cells expressing LPA(1) receptors. Nerve injury caused high levels of LPA production in the ipsilateral sides of the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve, or sciatic nerve. Nerve injury-induced LPA production reached its maximum at 3 h after injury, followed by a rapid decline by 6 h. The LPA production was significantly attenuated in ATX heterozygous mutant mice, whereas the concentration and activity of ATX in cerebrospinal fluid were not affected by nerve injury. On the other hand, the activities of cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent phospholipase A(2) (iPLA(2)) were enhanced, with peaks at 1 h after injury. Both de novo LPA production and neuropathic pain-like behaviors were substantially abolished by intrathecal injection of arachidonyl trifluoromethyl ketone, a mixed inhibitor of cPLA(2) and iPLA(2), or bromoenol lactone, an iPLA(2) inhibitor, at 1 h after injury. However, administration of these inhibitors at 6 h after injury had no significant effect on neuropathic pain. These findings provide evidence that PLA(2)- and ATX-mediated de novo LPA production in the early phase is involved in nerve injury-induced neuropathic pain.

DOI： 10.1124/jpet.109.164830

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

P>Lysophosphatidic acid receptor (LPA(1)) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA(1) receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA(1) receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.

DOI： 10.1111/j.1471-4159.2010.06664.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Peripheral nerve injury causes a variety of alterations in pain-related gene expression in primary afferent, which underlie the neuronal plasticity in neuropathic pain. One of the characteristic alterations is a long-lasting down-regulation of voltage-gated potassium (K-v) channel, including K(v)4.3, in the dorsal root ganglion (DRG). The present study showed that nerve injury reduces the messenger RNA (mRNA) expression level of K(v)4.3 gene, which contains a conserved neuron-restrictive silencer element (NRSE), a binding site for neuron-restrictive silencer factor (NRSF). Moreover, we found that injury causes an increase in direct NRSF binding to K(v)4.3-NRSE in the DRG, using chromatin immunoprecipitation (Chip) assay. Chip assay further revealed that acetylation of histone H4, but not H3, at K(v)4.3-NRSE is markedly reduced at day 7 post-injury. Finally, the injury-induced K(v)4.3 downregulation was significantly blocked by antisense-knockdown of NRSF. Taken together, these data suggest that nerve injury causes an epigenetic silencing of K(v)4.3 gene mediated through transcriptional suppressor NRSF in the DRG. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2009.12.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Na(v)1.8 sodium channel and mu-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Na(v)1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Na(v)1.8 gene expressions, C-fiber hypoesthesia, and the losses of peripheral morphine analgesia and Na(v)1.8-selective blocker-induced hypoesthesia. Together, these data suggest that NRSF causes pathological and pharmacological dysfunction of C-fibers, which underlies the negative symptoms in neuropathic pain.

DOI： 10.1523/JNEUROSCI.5541-09.2010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Following stroke or traumatic damage, neuronal death via both necrosis and apoptosis causes loss of functions, including memory, sensory, perception, and motor skills. As necrosis has the nature to expand, while apoptosis stops the cell death cascade in the brain, necrosis is considered to be a promising target for rapid treatment for stroke. We identified the nuclear protein, prothymosin alpha (ProT alpha) from the conditioned medium of serum-free culture of cortical neurons as a key protein-inhibiting necrosis. In the culture of cortical neurons in the serum-free condition without any supplements, ProT alpha inhibited the necrosis, but caused apoptosis. In the ischemic brain or retina, ProT alpha showed a potent inhibition of both necrosis and apoptosis. By use of anti-brain-derived neurotrophic factor or anti-erythropoietin IgG, we found that ProT alpha inhibits necrosis, but causes apoptosis, which is in turn inhibited by ProT alpha-induced neurotrophins under the condition of ischemia. From the experiment using anti-ProT alpha IgG or antisense oligonucleotide for ProT alpha, it was revealed that ProT alpha has a pathophysiological role in protecting neurons in stroke.

DOI： 10.1111/j.1749-6632.2010.05466.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: We previously reported that intrathecal injection of lysophosphatidylcholine (LPC) induced neuropathic pain through activation of the lysophosphatidic acid (LPA)-1 receptor, possibly via conversion to LPA by autotaxin (ATX).
Results: We examined in vivo LPA-induced LPA production using a biological titration assay with B103 cells expressing LPA(1) receptors. Intrathecal administration of LPC caused time-related production of LPA in the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve or sciatic nerve. LPC-induced LPA production was markedly diminished in ATX heterozygotes, and was abolished in mice that were deficient in LPA(3), but not LPA(1) or LPA(2) receptors. Similar time-related and LPA(3) receptor-mediated production of LPA was observed following intrathecal administration of LPA. In an in vitro study using spinal cord slices, LPA-induced LPA production was also mediated by ATX and the LPA(3) receptor. Intrathecal administration of LPA, in contrast, induced neuropathic pain, which was abolished in mice deficient in LPA(1) or LPA(3) receptors.
Conclusion: These findings suggest that feed-forward LPA production is involved in LPA-induced neuropathic pain.

DOI： 10.1186/1744-8069-5-64

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Lysophosphatidic acid (LPA) signaling, through LPA(1) receptor and its downstream RhoA, has been reported to initiate nerve injury-induced neuropathic pain. In the present study, we performed gene expression profiling of the dorsal root ganglion (DRG) to identify genes induced by intrathecal injection of LPA in a botulinum toxin C3 (BoNT/C3)-reversible manner. We selected and functionally characterized ephrinB1 from 82 identified genes as a potential gene involved in pain transmission, since ephrinB1 is implicated to modulate N-methyl-D-aspartate (NMDA) receptor functions in spinal pain transmission. The LPA-induced and BoNT/C3-reversible ephrinB1 gene expression was confirmed by quantitative real-time PCR. Furthermore, treatments with an antisense oligodeoxynucleotide for ephrinB1 largely abolished the LPA-induced thermal hyperalgesia and allodynia in response to mechanical or A beta-fiber-mediated electrical stimuli on day 1 after the injection. In addition, intrathecal treatment with a soluble ligand, ephrinB1-Fc, caused similar neuropathic pain-like behaviors in a manner that was reversible by the NMDA receptor antagonist MK-801. These results suggest that ephrinB1 plays a crucial role in LPA-induced neuropathic pain. In addition, the present study may provide a new strategy to identify unique neuropathic pain-related genes. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2008.11.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

1 Agonistic neurosteroids, including pregnenolone, dehydroepiandrosterone and its sulfate (DHEAS), caused rapid degranulation in measurements of beta-hexosaminidase (beta-HEX) release from a mast cell line, RBL-2H3. This degranulation was blocked by BSA-conjugated progesterone (PROG-BSA) or 17 beta-estradiol, both of which are antagonistic neurosteroids.
2 DHEAS-induced beta-HEX release was blocked by U-73122 or xestospongin C, but not by PTX or EGTA. DHEAS-induced beta-HEX release was also abolished by G(q/11)-AS, but not by G(q/11)-MS. Pharmacological analyses revealed that the neurosteroids stimulated a putative membrane receptor through activation of the novel G(q/11) and phospholipase C.
3 While representative endocrine-disrupting chemicals (EDCs) did not show any degranulation or nocifensive actions by themselves, they blocked the DHEAS-induced degranulation.
4 The binding of a PROG-BSA-fluorescein isothiocyanate conjugate (PROG-BSA-FITC) to cells was inhibited by neurosteroids and EDCs.
5 In the algogenic-induced biting and licking responses test, DHEAS caused agonistic nocifensive actions in a dose-dependent manner between 1 and 10 fmol (i.pl.). DHEAS-induced nocifensive actions were abolished by PROG-BSA or nonylphenol.
6 Taken together, these results suggest that a G(q/11)-coupled neurosteroid receptor may regulate the neuroimmunological activity related to sensory stimulation and that some EDCs have antagonistic actions for this receptor.

DOI： 10.1038/sj.bjp.0706213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The intraplantar injection of dehydroepiandrosterone sulfate (DHEAS), a representative neurosteroid, showed hyperalgesia in the Hargreaves' thermal or automatic paw-pressure mechanical nociception test. The DHEAS-induced hyperalgesia was abolished by diphenhydramine (DPH), a H-1 histamine (His) receptor antagonist, as well as the hyperalgesia induced by His or compound 48/80, a mast cell degranulating agent. The DHEAS-induced hyperalgesia was also blocked by progesterone (PROG), another type of neurosteroid and a putative neurosteroid receptor antagonist. Neither DPH nor PROG showed any changes in the thermal threshold. On the other hand, endocrine disrupting chemicals (EDCs) are known to disrupt reproductive system in wild-lives and humans through the disturbance of the endocrine homeostasis. In this study, the flexor responses induced by intraplantar injection of DHEAS were blocked by p,p'-DDE, an EDC as well as by PROG in the algogenics-induced nociceptive flexor responses test (ANF test) in mice. Similarly, p,p'-DDE blocked the DHEAS-induced hyperalgesia in Hargreaves' thermal nociception test. Besides the hyperalgesic actions, DHEAS increased vascular permeability as measured with Evans blue plasma extravasation. Consistent with behavioral studies, it was blocked by DPH, PROG, and p,p'-DDE. These results suggest that DHEAS has significant hyperalgesic and vasodilatory actions through histamine release, and these actions were reversible by PROG and an EDC. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0197-0186(02)00135-3

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The parathyroid hormone 2 (PTH2) receptor's anatomical distribution suggests that, among other functions, it may be involved in modulation of nociception. We localized PTH2 receptor protein to spinal cord lamina 11 and showed that it is synthesized by subpopulations of primary sensory neurons and intrinsic spinal cord dorsal horn neurons. Tuberoinfundibular peptide of 39 residues (TIP39) selectively activates the PTH2 receptor. Intraplantar microinjection of TIP39 caused a paw-withdrawal response and intrathecal injection caused scratching, biting, and licking, a nocifensive response. Intrathecal administration of a TIP39 antibody decreased sensitivity in tail-flick and paw-pressure assays. Intrathecal administration of TIP39 potentiated responses in these assays. We determined the sequence of TIP39's precursor and found that mRNA encoding TIP39 and TIP39-like immuno reactivity is concentrated in two brainstem areas, the subparafascicular area and the caudal paralemniscal nucleus. Cells in these areas project to the superficial dorsal horn of the spinal cord. Our data suggest that TIP39 released from supraspinal fibers potentiates aspects of nociception within the spinal cord.

DOI： 10.1073/pnas.042416199

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PubMed

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