外周NMDA受体在CFA引起的星形胶质细胞活化和长时程痛敏中的作用

发布时间：2018-03-20 17:35

本文选题：完全弗氏佐剂　切入点：星形胶质细胞　出处：《山西医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 大鼠脚掌注射完全弗氏佐剂(CFA)是一个被广泛应用的慢性炎性痛模型。在注射CFA后,动物会迅速出现一个对热刺激或机械刺激敏感的状态,表现为伤害性热刺激引起的缩足反射潜伏期明显缩短。由CFA导致的热痛敏的高峰出现在注射后2-6小时,并持续约2周左右。新近的研究表明,胶质细胞包括小胶质细胞和星形胶质细胞的激活与外周炎症或神经损伤后产生的长时程痛敏反应密切相关。研究结果还显示,在外周炎症引起的胶质细胞激活过程,小胶质细胞的激活早于星形胶质细胞,提示小胶质细胞在长时程痛敏的起始阶段而星形胶质细胞在维持阶段发挥主要作用,即伤害性刺激引起了小胶质细胞的活化并参与诱导了痛敏反应,与此同时小胶质细胞又通过某种机制引起了星形胶质细胞的活化,从而使痛敏反应持续维持形成了长时程痛敏。大量实验已证明,外周NMDA受体的激活在慢性炎症引发的机械痛敏和热痛敏中发挥重要作用。本实验在外周注射CFA引起慢性炎症的基础上,通过痛行为观察(热辐射刺激缩足反射潜伏期)、对星形胶质细胞活化增殖的免疫组化检测(GFAP阳性表达),结合外周注射NMDA受体拮抗剂MK-801,试图证实外周NMDA受体在CFA所致的长时程痛敏及脊髓星形胶质细胞活化中的作用。实验在Sprague-Dawley雄性大鼠(100-120g)进行。实验动物分为两组(n=30),每只大鼠双侧脚掌均注射等量CFA。第一组是预先给药组,在注射CFA前15分钟预先在左侧脚掌皮下注入MK-801 (50nmol,25μl),而右侧脚掌给予等量生理盐水(NS),并且在CFA注射后的第1天、第2天、第3天、第4天分别给予原剂量的MK-801或NS。第二组是晚期给药组,在注射CFA后第4天左侧脚掌皮下注入MK-801 (50 nmol),而右侧给予等量生理盐水(NS),并重复给予相同剂量的MK-801和NS持续四天。在注射CFA后的8h、1d、4d、8d、14d各组动物随机选择六只大鼠,通过测定热辐射刺激缩足反射潜伏期进行痛反应的行为学观察,行为检测结束后处死动物进行GFAP表达的免疫组化检测,对比背角两侧星形胶质细胞的免疫组化反应,并进行图像分析和统计学处理。预实验结果显示,注射CFA侧即刻发生热痛敏,第4小时达热痛敏高峰,持续时间超过14天;而免疫组化观察显示星形胶质细胞激活发生在4天之后,并持续超过14天。在预先给药组,脚掌注射NMDA受体拮抗剂MK-801后行为学热辐射刺激缩足反射潜伏期显著延长,在第1d、4d、8d具有显著性差异,第4天的抑制幅度最大达35.72%;而免疫组化测定的GFAP阳性表达水平较单独注入CFA侧明显降低,在第4天、第8天、
[Abstract]:Rat foot injection with complete Freund's adjuvant is a widely used model of chronic inflammatory pain. After injection of CFA, animals quickly develop a state that is sensitive to thermal or mechanical stimuli. The latent period of reflex induced by nociceptive heat stimulation was significantly shortened. The peak of thermal pain sensitivity caused by CFA appeared 2-6 hours after injection and lasted about 2 weeks. The activation of glial cells, including microglia and astrocytes, is closely related to the long-term pain response after peripheral inflammation or nerve injury. The activation of microglia is earlier than that of astrocytes, suggesting that microglia play a major role in the initiation of long-term pain sensitivity and astrocytes play a major role in the maintenance phase. That is, nociceptive stimulation induces the activation of microglia and participates in the induction of pain-sensitive reactions, while microglial cells induce the activation of astrocytes through some mechanism. Thus, the sustained maintenance of the hypersensitivity resulted in the formation of long-term pain sensitivity, which has been proved by a large number of experiments. The activation of peripheral NMDA receptors plays an important role in mechanical and thermal pain sensitivity induced by chronic inflammation. Through the observation of pain behavior (thermal radiation stimulates the latent period of foot contraction reflex, the positive expression of GFAP in astrocytes activated and proliferated by immunohistochemistry, combined with peripheral injection of NMDA receptor antagonist MK-801, we try to confirm the peripheral NMDA receptor in CFA. The role of long-term hyperalgesia and activation of astrocytes in spinal cord. The experiment was carried out in Sprague-Dawley male rats (n = 100 ~ 120 g). The experimental animals were divided into two groups. Each rat was injected with the same amount of CFAon on both sides of the foot. The first group was given the drug in advance. MK-801 was subcutaneously injected into the left foot's soles 15 minutes before injection of CFA, while the right foot was given the same amount of normal saline, and on the first day, the second day, the third day after CFA injection, the left foot was subcutaneously injected with MK-801 50 nmol-25 渭 L, and the right foot was given the same amount of normal saline on the first, second, third day after CFA injection. On the 4th day, MK-801 or NS were given respectively. The second group was treated with advanced administration. The left foot was subcutaneously injected with MK-801 50 nmola on the 4th day after CFA injection, while the right side was given the same amount of normal saline and the same dose of MK-801 and NS for 4 days. Six rats in each group were randomly selected for 14 days after the injection of CFA. The behavior of pain response was observed by measuring the latent period of reflex induced by thermal radiation, and the expression of GFAP was detected by immunohistochemical staining of astrocytes on both sides of the dorsal horn after the end of the behavioral test, and the immunohistochemical reaction of astrocytes on both sides of the dorsal horn was compared. And image analysis and statistical processing. The results of pre-test showed that the hyperthermia was immediately occurred in the CFA side and reached the peak at the 4th hour, which lasted for more than 14 days, while the activation of astrocytes occurred after 4 days by immunohistochemistry. After injection of NMDA receptor antagonist MK-801, the latency of paw reflex was significantly prolonged after injection of MK-801, and there was a significant difference on the 1st day, 4th day, 8d, between the two groups. On the 4th day, the maximum inhibition was 35.72%, while the positive expression of GFAP was significantly lower than that of CFA alone, and on the 4th day, the 8th day, the positive expression level of GFAP was significantly lower than that of the control group on the 4th day and 8th day.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R33

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