大鼠甲醛炎性痛过程中脊髓后角诱导型一氧化氮合酶阳性细胞的构成

发布时间：2018-05-13 14:36

本文选题：甲醛炎性痛 + 胶质细胞　；参考：《河北医科大学》2007年硕士论文

【摘要】： 目的:一氧化氮(nitric oxide,NO)是调节疼痛的重要介质,NO合酶(NO synthase,NOS)是生成NO的限速酶。已有证据表明,外周伤害性刺激可诱导脊髓NOS表达增多,产生大量NO参与疼痛的产生与维持。我室既往应用免疫组化和NADPH-d等方法研究表明,甲醛炎性痛时,脊髓后角nNOS和iNOS表达增多,进一步证实了NO在外周炎性痛和痛过敏的产生和维持中的作用。但是,这些NOS阳性细胞的细胞构成尚不清楚。越来越多的证据表明,脊髓胶质细胞的激活与病理性痛和痛觉过敏的产生和维持有密切关系。例如,可逆性胶质细胞代谢抑制剂可以抑制许多疼痛模型中的自发痛和痛觉过敏的产生和维持。在许多类型的神经病理性痛,如脊神经损伤(冷冻,结扎)、坐骨神经结扎等神经病理性痛,以及甲醛、酵母多糖等引起的炎性痛过程中,均可见脊髓星形胶质细胞和小胶质细胞增殖活化。这些增殖活化的胶质细胞可释放多种化学物质和细胞因子促进痛和痛觉过敏的产生和维持。由此可见,除神经元以外,脊髓胶质细胞可能也参与甲醛炎性痛时脊髓NOS表达的上调。因此,本实验采用免疫组织化学双重标记GFAP(星形胶质细胞特异性标记蛋白)和iNOS、以及连续切片免疫组化对比观察OX42(小胶质细胞特异性标记蛋白)和iNOS的表达等方法,观察大鼠足底注射甲醛引起的急性炎性痛过程中,脊髓后角星形胶质细胞和小胶质细胞在脊髓iNOS表达增多中的作用,以期进一步阐明脊髓胶质细胞和NO在外周炎症性痛和痛过敏中的作用。方法:将30只雄性Sprague-Dawley大鼠随机分为生理盐水组和甲醛组,分别足底注射生理盐水和甲醛,每组按足底注射后取材时间的不同又分为1 h、24 h和48 h组(n=5)。各组于相应时间点断头取腰5脊髓节段(L5),石蜡切片上行iNOS/GFAP免疫组化双重染色。另取30只大鼠随机分为生理盐水组和甲醛组,每组按足底注射后取材时间的不同分为1 h、1 d、3 d、7 d和14 d组。各组于相应时间点灌注取材,冰冻连续切片,免疫组化对比观察L5脊髓后角OX42和iNOS的表达。对以上各组动物均于取材前行相应的行为学观测:1 h组动物于取材前行痛反应评分;其他组动物于取材前测定热辐射缩足反射潜伏期和机械缩足反射阈值。结果:大鼠右后足底注射5%甲醛后,注射足出现典型的双向性自发缩足反应,持续1 h以上。注射部位热辐射缩足潜伏期延长和机械刺激缩足反射阈值升高,而非注射足相应部位的热辐射缩足潜伏期缩短和机械刺激缩足反射阈值降低。注射后甲醛后1 h,24 h和48 h,在注射足同侧的L5脊髓后角和中央管周围均可观察到少量iNOS/GFAP双标细胞散在分布,各时间点之间无显著差异。连续切片免疫组化对比观察发现,足底注射甲醛后,L5脊髓后角iNOS与OX42阳性细胞从形态、分布、表达时相上均有显著差异,并未呈现明显一致的趋势,iNOS与OX42的表达没有显著的相关性。结论:大鼠足底注射甲醛后,注射部位对热和机械刺激感觉减退,而非注射足相应部位出现痛觉过敏现象。足底注射甲醛诱导脊髓后角iNOS表达上调的过程中,脊髓胶质细胞发挥一定的作用,但不起主要作用;脊髓后角表达上调的iNOS可能主要由神经元所介导。
[Abstract]:Objective: nitric oxide (NO) is an important mediator for regulating pain, and NO synthase (NO synthase (NOS) is a speed limiting enzyme for NO. The evidence shows that peripheral nociceptive stimulation can induce the increase of NOS expression in the spinal cord and produce a large number of NO involved in the production and maintenance of pain. The increase in the expression of nNOS and iNOS in the posterior horn of the spinal cord further confirms the role of NO in the production and maintenance of peripheral inflammatory and painful hypersensitivity, however, the cell composition of these NOS positive cells is not yet clear.
There is growing evidence that the activation of spinal glial cells is closely related to the formation and maintenance of pathological pain and hyperalgesia. For example, reversible glial metabolic inhibitors can inhibit the production and maintenance of pain and hyperalgesia in many of the pain models. In many types of neuropathic pain, such as spinal nerve injury (spinal nerve injury). The proliferation and activation of astrocytes and microglia in the spinal cord can be seen during the process of inflammatory pain caused by the ligature of the sciatic nerve and the ligature of the sciatic nerve. These proliferating and activated glial cells can release a variety of chemicals and cell factors to promote the production and maintenance of pain and hyperalgesia. It can be seen that in addition to neurons, spinal glial cells may also participate in upregulation of NOS expression in spinal cord during formalin induced pain.
Therefore, the experimental immunohistochemical double labeling GFAP (astrocyte specific labeling protein) and iNOS, and the continuous slice immunohistochemical staining were used to observe the expression of OX42 (microglia specific marker protein) and iNOS, and to observe the acute inflammatory pain during the acute inflammatory pain caused by the injection of formaldehyde in the foot of the rat, and the posterior horns of the spinal cord. The role of glial cells and microglia in the increase of iNOS expression in the spinal cord is expected to further elucidate the role of spinal glial cells and NO in peripheral inflammatory pain and pain allergy.
Methods: 30 male Sprague-Dawley rats were randomly divided into normal saline group and formaldehyde group. The normal saline and formaldehyde were injected into the plantar. Each group was divided into 1 h, 24 h and 48 h group (n=5) after the plantar injection. Each group was divided into 5 spinal segments at the corresponding time point (L5), and the paraffin section was performed by iNOS/GFAP immunohistochemistry. Dyed.
Another 30 rats were randomly divided into normal saline group and formaldehyde group. Each group was divided into 1 h, 1 D, 3 D, 7 d and 14 d groups according to the plantar injection time. Each group was perfused at corresponding time points, frozen continuous slices and immunohistochemical staining was used to observe the expression of OX42 and iNOS in the posterior horn of spinal cord by immunohistochemistry.
The animals in all of the above groups were observed before they were taken. 1 h groups were used to score the pain response before taking wood. The other animals measured the latent period of radiation contraction reflex and the threshold of mechanical contraction reflex before taking wood.
Results: after the injection of 5% formaldehyde in the right posterior foot of the rat, the injection foot had a typical two-way spontaneous contraction reaction, lasting more than 1 h. The latent period of heat radiation contraction in the injection site and the increase of the reflex threshold of mechanical stimulation were increased, but the latent period of heat radiation contraction in the corresponding part of the foot of the injection foot was shortened and the threshold of the mechanical stimulation contraction reflex was reduced. After 1 h, 24 h and 48 h after formalin, a small amount of iNOS/GFAP double standard cells scattered around the posterior horn of the L5 spinal cord and around the central canal were observed, and there was no significant difference between each time point.
The contrast observation of continuous slice immunohistochemical staining showed that after the injection of formaldehyde in the plantar, there were significant differences in the morphology, distribution and expression of iNOS and OX42 positive cells in the posterior horn of the L5 spinal cord, and did not show a significant consistent trend. There was no significant correlation between the expression of iNOS and OX42.
Conclusion: after the injection of formaldehyde in the foot of the rat, the sensation of heat and mechanical stimulation is reduced, and the hyperalgesia occurs in the corresponding part of the foot. The spinal glial cells play certain roles in the process of inducing the up-regulated expression of iNOS in the posterior horn of the spinal cord by the injection of formaldehyde. The expression of iNOS in the posterior horn of the spinal cord may be up-regulated. It is mainly mediated by neurons.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【参考文献】