椎管内给予NMDA受体激动剂NMDA激活大鼠脊髓后角小胶质细胞

发布时间：2018-05-01 06:39

  本文选题：NMDA + NMDA受体　； 参考：《河北医科大学》2012年硕士论文

【摘要】：目的：目前，病理性疼痛的产生机制已经从单独的神经元机制，转向了神经元与胶质细胞相互作用机制，尤其是小胶质细胞作为中枢神经系统定居的吞噬细胞和主要免疫反应细胞受到了广泛的关注。大量研究表明，在外周神经损伤和伤害性刺激后，脊髓后角的小胶质细胞被激活，合成和分泌多种细胞因子和趋化因子包括白介素(IL-1)、肿瘤坏死因子（TNF-a）、前列腺素（PGE2）和一氧化氮（NO）等，促进病理性疼痛和痛觉敏感的产生。给予小胶质细胞功能或代谢的抑制药物如米诺环素或一些受体的抑制剂，则可明显地对病理性疼痛和痛觉过敏产生抑制作用。这些研究结果表明脊髓小胶质细胞的活化在调节病理性疼痛和痛觉过敏的过程中发挥重要作用。然而，病理性疼痛过程中脊髓小胶质细胞活化机制仍然不十分清楚。 N-甲基-D-天门冬氨酸（N-methyl-D-aspartate，NMDA）受体是兴奋性氨基酸受体的一种亚型，属于离子型受体，广泛分布于中枢神经系统，在脊髓的小胶质细胞上也有分布。生理条件下，该受体参与调节神经系统生长、发育，学习、记忆等复杂的生理功能。病理情况下，激活的NMDA受体引起细胞内一系列级联放大效应，增强突触前和突出后神经元的兴奋性，从而参与介导伤害性信息的传递与整合，在病理性疼痛和痛觉过敏形成过程中发挥重要作用。 我室以往应用免疫印迹分析和免疫组化技术，观察到椎管内注射NMDA受体非竞争性拮抗剂MK-801可抑制蜜蜂毒疼痛模型引起的脊髓后角小胶质细胞的活化，初步证明NMDA受体在引起脊髓后角小胶质细胞激活中的作用。 本实验则采用免疫组化技术，观察椎管内给予NMDA受体选择性激动剂NMDA对脊髓小胶质细胞激活的影响，进一步研究NMDA及其受体在伤害性信息传入所致脊髓后角小胶质细胞激活中的作用，为阐明病理性疼痛及痛觉过敏过程中脊髓小胶质细胞激活的机制提供实验依据。 方法：雄性Sprague-Dawley大鼠95只，体重260-280g，随机分为以下各组(n=5)。 sham组：椎管内注射生理盐水10μl，于相应的时间点测定热辐射缩足潜伏期、热甩尾潜伏期和机械刺激缩足阈值后，取腰5脊髓节段，观察脊髓后角小胶质细胞CD11b/c（OX-42）的表达。 NMDA I组：椎管内注射NMDA溶液10μl （10nmol），分别于注射后1h、4h、8h、12h、1d、2d和3d测定热辐射缩足潜伏期、热甩尾潜伏期和机械刺激缩足阈值后，取腰5脊髓节段，观察脊髓后角小胶质细胞CD11b/c（OX-42）表达的时间变化特点，分析CD11b/c（OX-42）的表达与痛觉过敏的时间对应关系。 NMDA II组：椎管内注射NMDA溶液10μl。根据NMDA的剂量，进一步分为0.1nmol，1nmol和10nmol三个亚组。每组动物于椎管内注射后8h测定热辐射缩足潜伏期、热甩尾潜伏期和机械刺激缩足阈值后，取腰5脊髓节段，观察NMDA上调脊髓后角小胶质细胞CD11b/（cOX-42）的表达和导致痛觉过敏的剂量依赖性。 MK-801+NMDA组：首先椎管内注射NMDA受体抑制剂MK-801溶液10μl (50nmol)，15min后椎管内注射NMDA溶液10μl (10nmol)，注射NMDA8h测定热辐射缩足潜伏期、热甩尾潜伏期和机械刺激缩足阈值后，取腰5脊髓节段，观察MK-801对NMDA引起的脊髓后角小胶质细胞CD11b/c（OX-42）表达的上调和痛觉过敏的影响。 以上各组动物均于相应时间点多聚甲醛灌注固定，，取腰5（L5）脊髓节段，冰冻连续切片，免疫组化法观察L5脊髓后角CD11b/c（OX-42）表达，反映脊髓后角小胶质细胞激活的状态。 数据用均数±标准差（x±s）表示。应用SPSS统计软件13.0对实验数据进行单因素方差分析（One-WayANOVA），比较组间差异。有显著差异者用最小显著差法进行两两比较，以p0.05为差异有统计学意义。 结果： 1痛行为反应 1.1自发痛行为反应椎管内注射NMDA后大鼠可出现紧张性的咬、舔后足或尾、肢体扭动和吱叫等特征性的伤害性行为反应，而椎管内预先注射MK-801（MK-801+NMDA组）则可抑制上述表现。 1.2热缩足和热甩尾潜伏期在NMDA I组，椎管内注射NMDA（10nmol）后1h，大鼠的热缩足潜伏期开始缩短（p0.05），可持续到12h。热缩足潜伏期1d时恢复到sham水平，表明椎管内注射NMDA后早期就可出现热痛觉过敏，并且这种现象可持续一段时间。在NMDAII组，NMDA组的热缩足潜伏期缩短呈现一定的剂量依赖关系，表现为随着NMDA注射剂量的增大，热缩足潜伏期的缩短逐渐明显。MK-801+NMDA组与相同剂量NMDA组相比，热缩足潜伏期显著延长（p0.05），表明椎管内注射MK-801可抑制上述热痛觉过敏。热甩尾潜伏期的变化特点与热缩足潜伏期相似。 1.3机械缩足反射阈值在NMDA I组，大鼠椎管内注射NMDA后4h机械缩足反射阈值开始出现降低（p0.05），可延长至椎管内注射NMDA后1d。机械缩足反射阈值2d时恢复到sham水平，表明椎管内注射NMDA后早期也可以表现出机械痛觉过敏，并且这种表现可持续较长时间。在NMDA II组，NMDA组的机械缩足反射阈值降低呈现一定的剂量依赖关系，表现为随着NMDA注射剂量的增大，机械缩足反射阈值减小逐渐明显。MK-801+NMDA组与相同剂量NMDA组相比，机械缩足反射阈值明显增高（p0.05），表明椎管内注射MK-801可抑制上述机械痛觉过敏。 2脊髓后角CD11b/c（OX-42）表达变化与sham组相比，椎管内注射NMDA4h后，脊髓后角小胶质细胞CD11b/c（OX-42）的表达明显升高（p0.05），镜下观察小胶质细胞被激活，其特征为胞体明显变大，突起增粗变短，染色加深。上述CD11b/c（OX-42）的表达和小胶质细胞的形态变化在注射NMDA8h后达到高峰，1d后开始减弱，3d基本恢复到sham水平。随着大鼠椎管内给予NMDA剂量的增加（0.1nmol,1nmol和10nmol），脊髓后角CD11b/c（OX-42）表达逐渐升高，呈明显的剂量依赖性。预先椎管内注射NMDA受体阻断剂MK-801可显著抑制上述NMDA引起的CD11b/c（OX-42）表达的上调（p0.05）。 结论： 1椎管内注射NMDA受体特异性激动剂NMDA可以引起伤害性感受和痛觉过敏，这种伤害性行为可被NMDA受体非竞争性抑制剂MK-801在一定程度上所抑制。 2椎管内注射NMDA受体特异性激动剂NMDA可以引起脊髓后角小胶质细胞活化，这种变化呈现一定的剂量依赖性，且可被NMDA受体非竞争性抑制剂MK-801所抑制。 3这些结果提示，NMDA及其受体在伤害性信息传入所致脊髓后角小胶质细胞活化过程中发挥重要作用。
[Abstract]:Objective : At present , the mechanism of pathogenesis of pathological pain has shifted from the individual neuron mechanism to the interaction mechanism between neuron and glial cells , especially the phagocytes and main immune response cells settled by microglial cells as the central nervous system .

N - methyl - D - aspartate ( NMDA ) receptor is a subtype of excitatory amino acid receptor , and belongs to ionic receptor . It is widely distributed in the central nervous system and is distributed in the microglial cells of the spinal cord . Under physiological conditions , the receptor is involved in regulating nervous system growth , development , learning , memory and other complex physiological functions .

In the past , we have observed that MK - 801 , a non - competitive antagonist of NMDA receptor in spinal canal , can inhibit the activation of microglial cells in spinal dorsal horn caused by bee venom pain model and demonstrate the role of NMDA receptor in the activation of glial cells after spinal cord injury .

In this experiment , the effects of NMDA receptor selective agonist NMDA on the activation of microglial cells in spinal cord were observed by immunohistochemistry , and the role of NMDA and its receptors in the activation of spinal dorsal horn microglial cells was further studied .

Methods : 95 male Sprague - Dawley rats weighing 260 - 280 g were randomly divided into the following groups ( n = 5 ) .

sham group : 10 渭l of normal saline was injected into the spinal canal , the latent period of heat radiation , the latent period of thermal spin tail and the threshold of mechanical stimulation were measured at the corresponding time point .

In the group of NMDA I , 10 渭l ( 10 nmol ) of NMDA solution was injected into the spinal canal , and the latent period of heat radiation , latent period of heat - spin tail and the threshold of mechanical stimulation were measured at 1h , 4h , 8h , 12h , 1d , 2d and 3d after injection .

NMDA was injected into the spinal canal for 10 渭l . According to the dose of NMDA , the rats were further divided into three subgroups of 0.1 nmol , 1 nmol and 10 nmol . The latency of heat radiation , latency of thermal spin tail and the threshold of mechanical stimulation were measured at 8 h after injection .

MK - 801 + NMDA group : 10 渭l ( 50 nmol ) of NMDA receptor inhibitor MK - 801 was injected into the spinal canal , and 10 渭l ( 10 nmol ) of NMDA solution was injected into the spinal canal after 15min . After injection of NMDA8h , the latent period of heat radiation , the latency of thermal spin tail and the threshold of mechanical stimulation were measured .

5 ( L5 ) spinal cord segments and frozen continuous sections were harvested at the corresponding time points , and the expression of CD11a / c ( OX - 42 ) in L5 spinal cord was observed by immunohistochemical method , which reflected the activation status of microglial cells in the posterior horn of spinal cord .

The data was expressed by mean 卤 standard deviation ( x 卤 s ) . SPSS statistical software was applied to analyze the single - factor variance ( One - way ANOVA ) in the experimental data .

Results :

1 Pain response

1.1 After injection of NMDA into the spinal canal by spontaneous pain , the rats were subjected to stress - induced sexual behavior such as tension , licking , hindfoot or tail , limb torsion and squeaking , while MK - 801 ( MK - 801 + NMDA group ) was pre - injected into the spinal canal to inhibit the above expression .

1 . The latent period of heat - shrinkable foot and warm - tail tail in NMDA group was shorter than that in NMDA group ( P < 0.05 ) .

1 . The threshold of mechanical contraction reflex in NMDA group was decreased ( p . 05 ) after injection of NMDA into the spinal canal of NMDA group .

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