ポスター発表 (Poster Sessions)

痛覚、痒み、及びその障害 Pain, Itch and Their Disorders
P1-2-65 有痛性神経障害糖尿病ラットにおけるノルアドレナリン下行性疼痛制御系の機能不全 Dysfunction of the noradrenergic descending nociception regulatory system in rats with painful diabetic neuropathy ○木下淳^1,2, 高橋由香里¹, 渡部文子¹, 宇都宮一典², 加藤総夫¹ ○Jun Kinoshita^1,2, Yukari Takahashi¹, Ayako Watabe¹, Kazunori Utsunomiya², Fusao Kato¹ 慈恵医大神経科学研究部　神経生理¹, 慈恵医大　糖尿病・代謝・内分泌内科² Lab Neurophysiol, Dept Neurosci, Jikei Univ Sch Med, Tokyo¹, Dept Diabetes, Metab and Endocr of Intern Med, Jikei Univ Sch Med, Tokyo² Painful diabetic neuropathy (PDN), which primarily stems from peripheral neuropathy, involves functional as well as neurochemical modifications in the spinal and supraspinal areas belonging to the pain matrix (Morgado et al., 2011). Recent clinical studies indicate that serotonin and noradrenaline reuptake inhibitors such as duloxetine alleviates pain sensation in patients with PDN, which contrasts to the limited effects of selective serotonin reuptake inhibitors against PDN (Dworkin et al., 2007). We therefore challenged a hypothesis that the hyperalgesia in PDN involves dysfunction of central noradrenergic (NA) system associated with nociceptive transmission. To address this issue, we made streptozotocin (STZ)-induced diabetic model in male Wistar rats weighing 200-230 g, that showed hyperglycemia, mechanical allodynia and thermal hyperalgesia. Duloxetine (10 mg/kg, i.p.) significantly and markedly reduced these nocifensive behaviors in STZ-treated rats. Administration of N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 50 mg/kg, i.p.), which drastically eliminated nerve endings with dopamine-beta-hydroxylase in the lumber dorsal horn and significantly reduced noradrenaline content in the brain and the spinal cord, significantly eliminated this effect of duloxetine in STZ-treated rats. However, in the STZ-treated rats, the noradrenaline content in the brain and the spinal cord was rather higher than vehicle-treated ones. It is therefore speculated that in the animals with PDN might develop functional insufficiency in the noradrenaline-mediated regulation of the spinal cord nociceptive networks, resulting in hyperalgesic behaviors.	P1-2-66 炎症時におけるグリア細胞由来神経栄養因子による侵害受容性三叉神経節ニューロンの興奮性の修飾作用 Glial cell line-derived neurotrophic factor modulates the excitability of nociceptive trigeminal ganglion neurons via a paracrine mechanism following inflammation ○武田守¹, 高橋誠之¹, 原紀文¹, 松本茂二¹ ○Mamoru Takeda¹, Msayuki Takahashi¹, Norifumi Hara¹, Shigeji Matsumoto¹ 日本歯科大学生命歯学部生理学講座¹ Dept Physiol, Nippon Dental University, Tokyo¹ Previous our report indicated that acute application of glial cell line-derived neurotrophic factor (GDNF) enhances the neuronal excitability of adult rat small-diameter TRG neurons, which innervate the facial skin. This study investigated whether under in vivo conditions, GDNF modulates the excitability of nociceptive Aδ-trigeminal ganglion (TRG) neurons innervating the facial skin via a paracrine mechanism following inflammation. We used extracellular electrophysiological recording with multibarrel-electrodes in this study. Spontaneous Aδ-TRG neuronal activity was induced in control rats after iontophoretic application of GDNF into the trigeminal ganglia (TRGs). Noxious and non-noxious mechanical stimuli evoked Aδ-TRG neuronal firing rate were significantly increased by iontophoretic application of GDNF. The mean mechanical threshold of nociceptive TRG neurons was significantly decreased by GDNF application. The increased discharge frequency and decreased mechanical threshold induced by GDNF were antagonized by application of K252b. The number of Aδ-TRG neurons with spontaneous firings and their firing rates in rats with inflammation induced by Complete Freund's Adjuvant were significantly higher than control rats. The firing rates of Aδ-TRG spontaneous neuronal activity were significantly decreased by iontophoretic application of K252b in inflamed rats. K252b also inhibited Aδ-TRG neuron activity evoked by mechanical stimulation in inflamed rats. These results suggest that in vivo GDNF enhances the excitability of nociceptive Aδ-TRG neurons via a paracrine mechanism within TRGs following inflammation.
P1-2-67 TRPA1チャネル阻害剤の全身投与が痛覚感受機構および呼吸調節機構に及ぼす影響 Effects of systemic administration of TRPA1 channel antagonist on nociceptive and respiratory control systems ○岡田泰昌¹, 武田湖太郎², 小山田吉孝³ ○Yasumasa Okada¹, Kotaro Takeda², Yoshitaka Oyamada³, Mieczyslaw Pokorski¹ 独立行政法人国立病院機構村山医療センター・臨床研究センター・電気生理学¹, 独立行政法人国立病院機構村山医療センター・臨床研究センター・生体機能制御解析², 独立行政法人国立病院東京医療センター・呼吸器科³ Laboratory of Electrophysiology, Clinical Research Center, Murayama Medical Center¹, Laboratory of Biomedical Engineering, Clinical Research Center, Murayama Medical Center², Respatory Department, Tokyo Medical Center³ It has been reported that TRPA1 channel plays important roles in perception of chronic pain and in a respiratory response to hypoxia. Although pain sensation and respiratory control are coupled in the central nervous system and thus mutually interact, the roles of TRPA1 channel in nociceptive and respiratory control systems and their interaction have not been well understood. In order to clarify this issue, we have conducted in vivo experiments using TRPA1 channel antagonist in adult mice. Sensitivity of nociception and hypoxic (O2 concentration 13% and 7%), hyperoxic (O2 concentration 100%) and hypercapnic (CO2 concentraton 5% in O2 balance) ventilatory responses were analyzed before and after intraperitoneal injection of TRPA1 channel antagonist (HC-030031; 50 mg/kg and 250 mg/kg). Nociceptive and respiratory control function was assessed by hot-plate test and whole body plethysmography, respectively. HC-030031 did not affect nociceptive sensitivity. HC-030031 suppressed ventilatory augmentation in a dose-dependent manner in response to hypoxia. However, either hyperoxic or hypercapnic response was not affected by HC-030031. We suggest acute hypoxic ventilatory response is TRPA1 channel dependent but either acute pain or hyperoxic and hypercapnic ventilatory responses is not.	P1-2-68 成熟ラットの脊髄後角におけるオキシトシンの鎮痛作用機序 Cellular mechanisms for antinociception produced by oxytocin in the adult rat spinal dorsal horn ○蒋昌宇¹, 藤田亜美¹, 羅清甜¹, 康欽¹, 大坪瀬奈¹, 松下晋大¹, 熊本栄一¹ ○Chang-Yu Jiang¹, Tsugumi Fujita¹, Qing-Tian Luo¹, Qin Kang¹, Sena Ohtsubo¹, Akitomo Matsushita¹, Eiichi Kumamoto¹ 佐賀大学医学部生体構造機能学講座（神経生理学分野）¹ Dept Physiol, Saga Med Sch, Saga, Japan¹ Although several lines of evidence suggest that a posterior pituitary hormone oxytocin is involved in antinociception in the spinal dorsal horn, cellular mechanisms for this action have not yet been revealed. We previously reported that bath-applied oxytocin repitatively produces an inward current at -70 mV (EC₅₀ = 0.022 μM) without a change in glutamatergic spontaneous excitatory transmission and enhances spontaneous inhibitory transmission in spinal lamina II (substantia gelatinosa; SG) neurons. These oxytocin responses were mimicked by [Thr⁴,Gly⁷]-oxytocin, and inhibited by [d(CH₂)₅¹,Tyr(Me)²,Thr⁴,Orn⁸,des-Gly-NH₂⁹]-vasotocin; the oxytocin current was inhibited by intracellular GDP-β-S. The inward current was resistant to voltage-gated Na⁺-channel blocker tetrodotoxin (TTX), Ca²⁺-free and non-NMDA receptor antagonist, while the inhibitory transmission enhancements were depressed by TTX. The oxytocin inward current was seen in a SG neuron where endogenous analgesics, norepinephrine, serotonin or adenosine, produced an outward current at -70 mV. The present study further examined the action of oxytocin on synaptic transmission in SG neurons of adult rat spinal cord slices by using the blind whole-cell patch-clamp technique. Bath-applied oxytocin unaffected monosynaptically-evoked primary-afferent Aδ-fiber and C-fiber excitatory transmission. GABAergic and glycinergic spontaneous IPSC frequency increases produced by oxytocin were concentration-dependent with the EC₅₀ values of 0.024 and 0.038 μM, respectively. Although the oxytocin current was inhibited by vasopressin V1-receptor antagonist d(CH₂)₅[Tyr(Me)²,Arg⁸]-vasopressin, there was no correlation between changes in holding currents produced by vasopressin-receptor agonist [Arg⁸]-vasopressin and oxytocin in the same neuron. It is concluded that oxytocin produces a membrane depolarization, which results in spontaneous inhibitory transmission enhancement, possibly via oxytocin receptor activation.
P1-2-69 神経障害性疼痛の病態形成におけるマトリックスメタロプロテアーゼ12の関与 Involvement of matrix metalloprotease 12 in pathogenesis of neuropathic pain ○小林悠佳¹, 木口倫一¹, 深澤洋滋^1,2, 岸岡史郎¹ ○Yuka Kobayashi¹, Norikazu Kiguchi¹, Yohji Fukazawa^1,2, Shiroh Kishioka¹ 和歌山県立医・医・薬理¹, 関西医療大学・保健医療・解剖² Dept Pharmacol, Wakayama Med Univ, Wakayama, Japan¹, Dept anatomy, Kansai Univ Health Sci, Osaka, Japan² Increasing evidence indicates that several immune cells play a crucial role in neuroinflammation and the excessive activation of immune system results in eliciting neuropathic pain. We have already reported that some inflammatory cytokines and chemokines derived from macrophages and neutrophils are closely related to the initiation of neuropathic pain. However, the underlying mechanisms of neuropathic pain remain to be fully elucidated. Matrix metalloproteases (MMPs) are zinc-dependent endopeptidases and break down the extracellular matrix proteins. Activated immune cells released several types of MMPs, which participate in inflammation, tissue remodeling and cell activation. Recently, MMP2 and MMP9 in the spinal cord have been reported to be implicated in neuropathic pain. In this study, we focus on the role of MMP12 in neuroinflammation and neuropathic pain. Mice received partial sciatic nerve ligation (PSL). After sciatic nerve (SCN) injury, macrophages, T-lymphocytes and neutrophils infiltrated significantly in the injured site. By RT-PCR, the expressions of CD14 mRNA (macrophage marker), CD25 mRNA (T-lymphocyte marker) and myeloperoxidase (neutrophil marker) were increased in the injured SCN after PSL. The expressions of MMP9 and MMP12 were also increased in the injured SCN after PSL, while MMP2 mRNA was not increased. By immunohistochemistry, MMP12 was localized on infiltrating macrophages in the injured SCN. In cultured macrophages, the mRNA expression of MMP12 was increased by lipopolysaccharide treatment. PSL elicited tactile allodynia and thermal hyperalgesia which were evaluated by von Frey test and Hargreaves test, respectively. After the depletion of macrophages by the treatment of liposomed-clodronate, PSL-induced tactile allodynia and thermal hyperalgesia were suppressed. These results suggest that MMP12 derived from macrophages involved in pathogenesis of neuropathic pain and MMP12 may be novel therapeutic target for neuropathic pain.	P1-2-70 1,8-および1,4-シネオールは成熟ラット脊髄膠様質ニューロンのグルタミン酸作動性の自発性興奮性シナプス伝達を促進する 1,8-Cineole and 1,4-cineole enhance glutamatergic spontaneous excitatory transmission in adult rat substantia gelatinosa neurons ○藤田亜美¹, 徐年香¹, 蒋昌宇¹, 羅清甜¹, 康欽¹, 八坂敏一¹, 大坪瀬奈¹, 松下晋大¹, 熊本栄一¹ ○Tsugumi Fujita¹, Nian-Xiang Xu¹, Chang-Yu Jiang¹, Qing-Tian Luo¹, Qin Kang¹, Yasaka Toshiharu¹, Sena Ohtsubo¹, Akitomo Matsushita¹, Eiichi Kumamoto¹ 佐賀大・医・生体構造機能学¹ Dept Physiol, Saga Med Sch, Saga, Japan¹ 1,8-Cineole and 1,4-cineole are present in many plant essential oils. Their actions are different from each other, although their chemical structures are similar to each other. The substantia gelatinosa (SG; lamina II) of the spinal dorsal horn is thought to play an important role in modulating nociceptive transmission through primary-afferent neurons from the periphery to the central nervous system. The TRP channels are activated by various plant-derived chemicals such as capsaicin (TRPV1 agonist), allyl isothiocyanate (TRPA1 agonist) and (-)-menthol (TRPM8 agonist), resulting in an enhancement of glutamatergic spontaneous excitatory transmission in SG neurons. We have previously revealed that bath-applied 1,8- and 1,4-cineole reversibly enhance spontaneous excitatory transmission in the SG neurons. The present study examined a detail of the facilitatory actions of 1,8- and 1,4-cineole by applying the blind whole-cell patch-clamp technique to the SG neurons of adult rat spinal cord slices. The 1,8- and 1,4-cineole actions were repeated, concentration-dependent and largely presynaptic in origin. EC₅₀ values for 1,8- and 1,4-cineole in increasing spontaneous EPSC frequency were 2.5 and 0.18 mM, respectively. The 1,8- and 1,4-cineole activities were unaffected by a TRPV1 channel antagonist capsazepine while being inhibited by a TRPA1 channel antagonist HC-030031. Although 1,8-cineole has an ability to activate TRPM8 channels, the spontaneous EPSC frequency increase produced by this drug was much greater than that expected from TRPM8 activation, as judged from the facilitatory action of (-)-menthol which was examined in the same neuron. This idea remains to be confirmed by using TRPM8 channel antagonists. It is concluded that 1,4-cineole enhances the spontaneous release of L-glutamate onto SG neurons effectively more than 1,8-cineole, possibly through TRPA1 channel activation. These 1,8- and 1,4-cineole activities could contribute to nociception.
P1-2-71 In vivo カルシウムイメージングによる脊髄後角ニューロンの神経活動の三次元分布の解析 Three-dimensional distribution of sensory stimulation-evoked neuronal activity of spinal dorsal horn neurons analyzed by in vivo calcium imaging ○西田和彦¹, 松村伸治¹, 伊藤誠二¹ ○Kazuhiko Nishida¹, Shinji Matsumura¹, Seiji Ito¹ 関西医大・医化学¹ Dept Med Chem, Kansai Med Univ, Osaka¹ Spinal dorsal horn comprises heterogeneous interneurons as well as projection neurons, which form neuronal circuits crucial for processing of primary sensory information. Although in vivo patch clump recordings have provided the physiological properties of various spinal dorsal horn neurons, simultaneous monitoring of activities from many neurons is needed to obtain comprehensive view on spinal dorsal horn circuits. In the present study, we measured sensory stimulation-evoked calcium transients across broad area of superficial spinal dorsal horn in living mice, and analyzed three-dimensional distribution of neuronal activities. To label spinal dorsal horn with calcium indicators, we first established gene transfer of the ratiometric genetically encoded calcium indicator, Yellow Cameleon (YC), into spinal dorsal horn neurons by in utero electroporation. This method allowed us to introduce YC into a broad area of superficial spinal dorsal horn neurons, and YC expression can be observed until at least 10 weeks after electroporation. In vivo two-photon calcium imaging system was then established to measure noxious and non-noxious stimuli-evoked calcium transients of spinal dorsal horn neurons. We obtained activities from populations of superficial dorsal horn at single-cell resolution up to a depth of 150 μm and a length of 1.5 mm along rostrocaudal axis. Finally, the precise localization of these neurons was analyzed to reconstruct sensory stimulation-evoked neuronal activity maps. These analyses revealed that each type of sensory stimulation elicits overlapping but distinct subsets of spinal dorsal horn neurons. Our results show for the first time neuronal activation pattern of a broad area of spinal dorsal horn neurons upon sensory stimulation, thus facilitate deeper understanding of the neuronal circuitry underlying sensory perception.	P1-2-72 アストロサイトのSTAT3は神経損傷後のアストロサイト活性化および神経障害性疼痛に重要な因子である Astrocytic STAT3 is a crucial factor for reactive astrocytes after nerve injury and neuropathic pain ○高露雄太¹, 津田誠¹, 坂口瑛美¹, 齊藤秀俊¹, 岡野栄之², 井上和秀¹ ○Yuta Kohro¹, Makoto Tsuda¹, Emi Sakaguchi¹, Hidetoshi Tozaki-Saitoh¹, Hideyuki Okano², Kazuhide Inoue¹ 九州大学大学院　薬学研究院　薬理学分野¹, 慶應大・医・生理² Dept. Mol. Syst. Parmacol., Grad. Sch. Parm. Sci., Kyushu Univ., Fukuoka¹, Dept. Physiol., Sch. Med., Keio Univ., Tokyo² Neuropathic pain that is often a consequence of lesion or dysfunction in the nervous system is a debilitating chronic pain. Accumulating evidence indicates that spinal astrocytes as well as microglia become reactive states following peripheral nerve injury (PNI), which is a crucial role for neuropathic pain. However, the molecular mechanisms regulating genes associated with reactive states of astrocytes after PNI are largely unknown. We have previously shown that STAT3, a transcription factor which is activated predominantly in spinal astrocytes after PNI plays an important role in the maintenance of neuropathic pain. In the present study, to reveal the precise role of astrocytic STAT3 signaling, we used mice with conditional deletion of STAT3 in astrocytes (GFAP-Cre/STAT3^fl/fl: STAT3-cKO). We found that loss of astrocytic STAT3 prevented activation of STAT3 and upregulation of expression of genes related with reactive phenotypes of astrocytes following PNI. We also showed that some of genes related with reactive astrocytes were upregulated during the late phase of neuropathic pain. Moreover, STAT3-cKO mice displayed the resistance and shortened duration of PNI-induced tactile allodynia, a typical symptom of neuropathic pain. Together, our findings suggest that STAT3 is a key regulator of converting reactive states of astrocytes which contributes to chronic pain states.
P1-2-73 慢性疼痛による分界条床核内neurokinin 1 受容体mRNAの発現増加：Neurokin 1 受容体の不安様行動への関与 Chronic pain increases neurokinin 1 receptor mRNA expression in the bed nucleus of stria terminalis: Roles of neurokinin 1 receptor in the anxiety-like behavior ○眞嶋悠幾¹, 井川ありさ¹, 井手聡一郎¹, 津田誠², 井上和秀², 南雅文¹ ○Yuki Majima¹, Arisa Igawa¹, Soichiro Ide¹, Makoto Tsuda², Kazuhide Inoue², Masabumi Minami¹ 北海道大学院・薬・薬理¹, 九州大院・薬・薬理² Dept.Pharmacol.,Grad.Sch.Pharm.Sci., Hokkaido Univ., Sapporo, Japan¹, Dept Mol Syst Pharmacol, Grad Sch Pharmaceut Sci, Kyusyu Univ, Fukuoka, Japan² Pain is a complex experience composed of sensory and affective components. The bed nucleus of the stria terminalis (BNST) is a limbic structure involved in stress responses and regulating negative affective states such as aversion, anxiety and fear. We previously demonstrated that noradrenergic neurotransmission within the ventral part of BNST (vBNST) mediated the negative affective component of acute inflammatory pain. On the other hand, roles of the BNST in the negative affective component of chronic pain remain to be determined. Neurokinin 1 receptor (NK1R) and its endogenous agonist, substance P (SP) are thought to be involved in negative emotion, especially anxiety. Therefore, in the present study, we first examined NK1R mRNA expression within the BNST using a rat chronic neuropathic pain model (segmental spinal nerve ligation (SNL) model). Male Sprague-Dawley rats showed the increased anxiety-like behaviors in an elevated plus-maze (EPM) test 4 weeks after SNL surgery. Real-time RT-PCR revealed the enhanced expression of NK1R mRNA within the vBNST of SNL rats compared with those of the sham-operated control animals. Bilateral intra-vBNST injection of SP induced anxiety-like behaviors in the EPM test. SP at a dose of 0.1 pmol/side significantly decreased the time spent in open arms. These results suggest that activation of SP-NK1R neurotransmission within the vBNST may be involved in the negative affective component of chronic pain, especially chronic pain-induced anxiety.	P1-2-74 神経障害性疼痛マウスのM1型に極性化したマクロファージ/ミクログリアは、cAMP-response element binding proteinのリン酸化を高める Macrophage/Microglia polarized into M1 phenotype enhances phosphorylation of cAMP-response element binding protein in neuropathic pain model mice ○上勝也¹, 仙波恵美子¹ ○Katsuya Kami¹, Emiko Senba¹ 和歌山県立医科大学　医学部　解剖学第二講座¹ Dept Neurobiology&Anatomy, Wakayama Medical University, Wakayama¹ Microglia activated by peripheral nerve injury in the spinal dorsal horn play critical roles in the pathogenesis of neuropathic pain. In our recent study, analysis of macrophages/microglia (Mφ/Mg) polarization in the spinal cord following sciatic nerve injury indicated that almost all the Mφ/Mg in the superficial dorsal horn were polarized into M1 phenotype, suggesting considerable contribution of M1 phenotype, but not M2 in the development of neuropathic pain. On the other hand, phosphorylated cAMP-response element binding protein (pCREB) has been shown to promote anti-apoptotic survival signals and anti-inflammatory reaction in Mφ. In the present study, we examined the temporal and spatial expression patterns of pCREB in Mφ/Mg in the spinal cord of mice subjected to partial sciatic nerve ligation (PSL), and some of these mice were transplanted with bone marrows of EGFP-transgenic mice ahead of PSL. At day 3 to 28 post-operation, spinal cords of C57BL/6J mice and EGFP-chimeric mice subjected to PSL were analyzed using immunohistochemistry. Following PSL, significant increase of Iba-1⁺/CD11b⁺-Mφ/Mg was induced in the ipsilateral spinal dorsal horn, and intense pCREB immunoreactivities were detected in NeuN⁺-neurons and CD11b⁺-Mφ/Mg in the ipsilateral dorsal horn. At 7 days post-PSL, nuclear localization of pCREB was observed in about 94% of CD11b⁺-Mφ/Mg in the ipsilateral superficial dorsal horn, while those of sham-operated and naïve mice were maintained at lower levels. Furthermore, although intense pCREB expression was observed in CD68⁺- and CD86⁺-Mφ/Mg, CD206⁺- and EGFP⁺-Mφ/Mg showed the lack or lower level of pCREB expression. The present study has demonstrated for the first time that CREB is phosphorylated in Mφ/Mg in the dorsal horn of neuropathic pain model mice, and suggested that predominant expression of pCREB in M1 phenotype indicates its critical role in Mφ/Mg polarization. pCREB may also enhance their survival by activating anti-apoptotic signals.

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