【作者】 刘乃红；

【导师】 张策；

【作者基本信息】 山西医科大学 ， 生理学， 2006， 硕士

【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。近年来的研究表明,脊髓胶质细胞的激活在慢性痛的发生发展中发挥重要作用。在此过程,脊髓小胶质细胞与星形胶质细胞均有激活,并且小胶质细胞在慢性痛敏的产生和诱导过程发挥重要作用,而星形胶质细胞对于痛敏状态的持续维持发挥作用。本研究室之前的观察已证实,外周第III组代谢型谷氨酸受体(mGluRs)在Formalin引起的急性炎性痛中发挥重要作用。由于另外的研究表明,脊髓胶质细胞上同样表达第III组mGluRs。本实验在大鼠脚掌注射CFA的基础上,通过痛行为观察(热辐射刺激缩足反射潜伏期,PWL)、对脊髓星形胶质细胞活化标志物(GFAP)的免疫组化检测,结合外周注射第III组mGluRs激动剂L-SOP,观察外周第III组mGluRs在CFA所致热痛敏及脊髓胶质细胞活化中的作用。实验在Sprague-Dawley雄性大鼠(90~100g)进行,分为免疫组化实验和行为学观察两部分。60只大鼠分为两组,第一组(予先给药组)在CFA注射前15分钟预先脚掌皮下给予第III组mGluRs的激动剂L-SOP (250nmol),然后在CFA注射后的第1,2,3,4天分别重复给予同样剂量的L-SOP。第二组(晚期给药组)在给予CFA后的第4,5,6,7,8天分别重复给予相同剂量的L-SOP。两组均在CFA注射后8小时,1天、4天,8天,14天随机选择六只大鼠,进行痛行为学观察及脊髓星形胶质细胞激活标志物GFAP的免疫组化检测,分别对比背角两侧星形胶质细胞的免疫组化反应,进行图像分析及统计学处理。行为学观察结果显示,大鼠足掌给予CFA(1:1,50μl)后,动物即刻出现痛行为反应,表现为PWL的缩短,4-8小时出现了热痛敏反应的高峰,观察至第14天仍未完全恢复正常。在早期给药组外周应用L-SOP(250nmol)后,PWL明显延长,与对照侧相比,8h、1天、4天具有显著性差异(P<0.05),在晚期给药组,PWL的延长在4天、8天与对照侧相比有统计学差异(P<0.05)。免疫组化结果显示,大鼠足掌给予CFA后,脊髓背角星形胶质细胞在4天后表达增加,并持续到14天以后。在外周早期给予L-SOP后,星形胶质细胞GFAP阳性表达明显降低,在4、8、14天GFAP阳性表达的抑制率分别为18.61%、24.45%、20.54%,与对照侧相比均有显著性差异(P<0.05)。在晚期给药组,GFAP阳性表达第8天时在用药侧有更多还原

【Abstract】 Injection of complete Freund’s adjuvant (CFA) to the hind paw of the rat is a widely used animal model of chronic inflammatory pain, after CFA administration, animal will display a hypersensitivity to thermal and mechanic stimuli, which can be characterized by a rapid reduction in paw withdrawal latency to thermal and mechanic stimuli. The thermal hyperalgesia peaks in 2 to 6 hr and persists more than 2 wks after peripheral injection of CFA.Recently, spinal astrocytes and microglia have been recognized as potential active participants in the initiation and maintenance of hyperalgesia. In this processes, microglial activation preceded astrocytic activation following peripheral inflammation, supporting a role of microglia in the initiation phase and astrocytes in maintaining long-term hyperalgesia (LTH). Glutamate is one of the predominant excitatory neurotransmitters. Activation of GluRs including iGluRs and mGluRs mediate nociceptive processing in both peripheral and central sensitization. Previous studies demonstrated that peripheral group III mGluRs play a critical role in the acute inflammatory pain induced by hindpaw administration of Formalin. Recently, studies showed that spinal glia express group III mGluRs.In the present study, CFA pain model was employed in which observation of nociceptive behavior in response of thermal stimuli, and immunohistochemical examination of activated marker of spinal astrocytes (GFAP) were carried out, combined with peripheral administration of L-SOP, an agonist of group III mGluRs, to determine the role of peripheral group III mGluRs in the spinal astrocytes activation and long-term hyperalgesia induced by CFA.Experiments were performed on adult male Sprague–Dawley rats with two parts of observations, behavioral examination and immunohistochamical assay. Sixty rats were divided into two groups, the first group or Pre-CFA treated group, L-SOP (250nmol, 25μL, Tocris) was injected into the plantar of the left hind paw 15 min prior to CFA injection and repeat the administration 1, 2, 3 and 4 days after CFA injection. The contralateral right hind paw of the rat received same volume of saline; the second group or Post-CFA treated group, L-SOP was injected into the plantar of the left hind paw 4 days after CFA injection and continued for another four days. The contralateral right hind paw of the same rat received same volume of saline. The nociceptive responses to thermal stimuli named paw withdraw latence was tested at 1d before CFA injection as a control and at 8h, 1d, 4d, 8d, and 14d post CFA injection, respectively. Rats更多还原