|Degree Level: ||博士
|Issued Date: ||2008-06-03
|Degree Grantor: ||中国科学院上海生命科学研究院
|Place of Degree Grantor: ||上海生命科学研究院
|Alternative Title: ||The neuronal mechanisms of nociception induced by scorpion venom|
|Abstract: ||本论文工作采用药理学、行为学、组织病理学、免疫组化和免疫印迹等技术和方法对富含特异性钠离子通道调制剂的东亚钳蝎（Buthus martensi Karsch，BmK）毒液致痛相关的外周和中枢敏化的分子细胞机制进行了较系统的研究，主要结果如下：
大鼠经足底注射BmK粗毒和类样蝎毒素多肽BmK I，可诱发肥大细胞脱颗粒。BmK粗毒诱导的自发痛、双侧后足机械痛敏、原发性热痛敏和后足水肿以及脊髓浅层c-Fos的表达可被compound 48/80慢性耗竭肥大细胞以及外周共施加组胺受体H1或H2受体拮抗剂部分压抑。提示肥大细胞脱颗粒和组胺部分介导了BmK粗毒诱导的痛相关行为以及水肿反应。
大鼠经鞘内注射MK-801（NMDA受体拮抗剂），CNQX（非NMDA受体拮抗剂），DL-AP3（I 组mGluR受体拮抗剂），APDC（II组mGluR受体激动剂）等谷氨酸受体拮抗剂或激动剂研究了脊髓谷氨酸受体在BmK粗毒诱导的痛相关行为中的作用。药物前处理实验发现，BmK粗毒诱导的自发痛行为可被MK801和CNQX所压抑，被DL-AP3和APDC轻微压抑，原发性热痛敏可被MK-801或DL-AP3压抑，双侧机械痛敏可被CNQX或DL-AP3压抑，而仅镜像机械痛敏可被APDC压抑。药物后处理实验发现，原发性热痛敏可被MK-801, CNQX, DL-AP3 或APDC所逆转，而双侧机械痛敏仅被APDC所逆转。提示脊髓谷氨酸受体参与了BmK蝎毒诱导的不同痛相关行为。
|English Abstract: ||In the present study, the neuronal mechanisms of pain-related behaviors induced by the venom of BmK (Buthus martensi Karsch) were investigated using behavioral test, histopathological staining, immunohistochemical methods, immunobloting, etc. The results are presented as follows:
1. Mast cells degranulation and histamine are involved in BmK venom-induced pain-related behaviors and paw edema in rats
The degranulation of mast cells could be triggered by BmK venom in rat hindpaw. The chronic degranulation of mast cells using compound 48/80 relieved the spontaneous nociceptive responses, the primary thermal and bilateral mechanical hyperalgesia and the rat paw edema, as well as partially reduced c-Fos expression in superficial layers of bilateral spinal cord induced by BmK venom. In addition, individual peripheral co-administration of either 100 nmol chlorpheniramine or 100 nmol pyrilamine (histamine H1 receptor antagonist) or 500 nmol cimetidine (histamine H2 receptor antagonist) and BmK venom suppressed the spontaneous nociceptive responses, partially the primary thermal and bilateral mechanical hyperalgesia and rat paw edema induced by BmK venom. Thus, these results suggest that mast cells degranulation and histamine are involved in BmK venom-induced rat pain-related behaviors and paw edema.
2. Spinal glutamate receptors are involved in BmK venom-induced rat pain-related behaviors
MK-801 (a noncompetitive NMDA receptor antagonist), CNQX (a non-NMDA receptor antagonist), DL-AP3 (a group I metabotropic glutamate receptor antagonist) and APDC (a group II metabotropic glutamate receptor agonist) were employed to explore the role of spinal glutamate receptors in BmK venom-induced pain. Upon the intrathecal injection of glutamate receptor antagonists/agonist before BmK venom administration by 10 min, BmK venom-induced spontaneous nociceptive responses could be suppressed by all tested agents. Primary thermal hyperalgesia could be inhibited by MK-801 and DL-AP3, while bilateral mechanical hyperalgesia could be inhibited by CNQX and DL-AP3 and contrlateral mechanical hyperalgesia could be inhibited by APDC. Upon the intrathecal injection of glutamate receptor antagonists/agonist after BmK venom injection by 4.5 hour, primary thermal hyperalgesia could be partially reversed by all tested agents, while bilateral mechanical hyperalgesia could only be inhibited by APDC. The results suggest that the role of spinal glutamate receptors may be different on the various manifestations of BmK venom-induced rat pain-related behaviors.
3. Spinal nitric oxide (NO) contributes to BmK venom-induced pain-related behaviors and spinal c-Fos expression in rats
Immunohistochemistrical results showed that the number of neuronal NO synthase (nNOS) positive neurons in bilateral spinal cord significantly increased in superficial (I–II), deep (V–VI) dorsal horn laminae and the ventral gray laminae (VII–X), but not in the nucleus proprius (III and IV) of lumbar spinal cord, after unilateral intraplantar injection of BmK venom from 2 hour to 7 day. This increase on the ipsilateral side to BmK venom injection was always greater than that on the contralateral side. Western blotting results confirmed that spinal nNOS expression was significantly up-regulated after BmK venom administration. In addition, intrathecal delivery of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; a NOS inhibitor) before intraplantar injection of BmK venom by 10 min significantly attenuated spontaneous nociceptive responses and prevented the development of primary thermal hyperalgesia and bilateral mechanical hyperalgesia. Spinal c-Fos expression at 2 h following BmK venom injection could be partially inhibited by intrathecal injection of L-NAME. The results suggest that spinal NO is involved in various pain-related behaviors and c-Fos expression induced by BmK venom in rats.
4. Spinal glia activation contributes to BmK venom-induced rat pain-related behaviors
Glial fibrillary acidic protein (GFAP) immunoreactivity indicative astrocyte activation in bilateral spinal cord started to increase by 3 d, peaked at 7 d and gradually reversed 14 d following intraplantar injection of BmK venom. The up-regulation of GFAP expression was confirmed by western blotting analysis. In contrast, bilateral spinal increase of OX-42 immunoreactivity indicative of microglial activation began at 4 h, peaked 1 d and gradually reversed at 3 and 7 days after BmK venom administration. Pretreatment with either intrathecal injection of fluorocitrate or intraperitonially injection of minocycline, two glial activation inhibitors, suppressed the spontaneous nociceptive responses, and prevented the primary thermal and bilateral mechanical hyperalgesia induced by BmK venom in rats. Moreover, intraperitonially injection of minocycline partially inhibited spinal c-Fos expression induced by BmK venom but lack of effect on BmK venom-induced paw edema. The current study demonstrated that spinal astrocyte and microglial activation may contribute to BmK venom-induced pain-related behaviors in rats.
5. Spinal ERK activation contributes BmK venom-induced rat pain-related behaviors and c-Fos expression
Following intraplantar injection of BmK venom，extracellular signal-regulated kinases (ERK) were activated in ipsilateral L4-L5 spinal cord dorsal horn, which started at 2 min, peaked at 30-60 min and almost disappeared at 4 h following intraplantar injection of BmK venom. Intrathecal injection of U0126 (0.1, 1 and 10 μg), a widely used specific MAP kinase kinase (MEK) inhibitor, suppressed spontaneous nociceptive responses and reduced primary heat hyperalgesia and bilateral mechanical hyperalgesia induced by BmK venom. BmK venom-induced spinal c-Fos expression could be inhibited by U0126 dose-dependently. In addition, intrathecal injection of MK-801 or CNQX suppressed the BmK venom-induced ERK activation in spinal cord. Thus, activation of ERK in spinal cord dorsal horn contributes to BmK venom-induced rat pain-related behaviors and c-Fos expression.
Taken together, it was found that mast cell degranulation and histamine were involved in BmK venom-induced peripheral sensitization, while glutamate receptors, NO, glia activation and intracellular ERK signaling activation were involved in BmK venom-induced central sensitization.|
|Content Type: ||学位论文
|Appears in Collections:||神经所(总)_学位论文|
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