胶质细胞间缝隙连接在神经病理性疼痛中的作用机制

发布时间：2018-03-16 17:06

本文选题：神经病理性疼痛　切入点：胶质细胞　出处：《南京大学》2014年博士论文　论文类型：学位论文

【摘要】：[目的] 在外周神经损伤后,位于脊髓背角的感觉神经元兴奋性增强,成为传递和调控痛觉的重要位点。而背角神经元周围的星形胶质细胞在神经元兴奋性增强,痛觉敏感传递,尤其是神经病理性疼痛的维持中也起到重要作用。虽然已知星形胶质细胞之间信号传递的一个重要途径是缝隙连接,但脊髓胶质细胞间缝隙连接在维持神经病理性疼痛中的作用并不清楚,尤其是特异性表达在星形胶质细胞之间的缝隙连接蛋白43(Connexin43,Cx43)在神经病理性疼痛中的作用。甘珀酸(Carbenoxolone, CBX)是非特异性缝隙连接阻滞剂,可非特异性阻滞缝隙连接的功能。利用小干扰RNA (Small interfering RNA, siRNA)技术可以特异性降低星形胶质细胞间缝隙连接蛋白Cx43的表达。本研究旨在观察用药物(如甘珀酸)阻滞缝隙连接功能和siRNA下调脊髓Cx43蛋白表达能否抑制神经病理性疼痛或降低脊髓背角广动力范围神经元的活性。 [材料与方法] 1.建立大鼠脊神经结扎(Spinal nerve ligation, SNL)神经病理性疼痛模型后,使用甘珀酸(CBX)鞘内注射。首先在大鼠持续异氟烷吸入麻醉下,单次注射不同剂量的CBX,采用清醒动物行为学观察大鼠机械痛敏和热痛敏的变化。其次,进一步观察和对比相同剂量的CBX、甘草酸(Glycyrrhizic acid, GCA,是CBX的类似物,但不阻滞缝隙连接)、甲氟喹(Mefloquine, MFQ,是特异性神经元缝隙连接阻滞剂)对大鼠机械痛敏的不同影响。最后,检测连续注射CBX对大鼠机械痛敏是否有长期影响。 2.建立大鼠SNL神经病理性疼痛模型后,动物在麻醉状态下在体记录脊髓背角广动力范围(Wide dynamic range, WDR)神经元的活性,并观察记录脊髓表面给予CBX药物处理后,WDR神经元对外周机械性刺激反应的变化。此外,在WDR神经元上记录反复电刺激外周感受野所诱发的C纤维传导的动作电位增强的变化,并做出windup曲线。观察脊髓表面应用CBX对WDR神经元C-componer动作电位windup的影响。 3.免疫荧光方法检测和比较假手术组与SNL组腰段脊髓胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP,星形胶质细胞标志)、Cx43和OX42(小胶质细胞标志)蛋白的表达分布。 4.构建Cx43siRNA和Control siRNA,将大鼠分为假手术组、Cx43siRNA组和Control siRNA组。观察鞘内反复注射siRN垢,SNL大鼠机械痛敏的变化。同时采用Western blot检测双侧SNL大鼠腰段脊髓Cx43, Cx36,和GFAP蛋白表达水平的变化。 [结果] 1.在大鼠SNL术后2-3周,鞘内注射CBX (0.1μig、0.5μg、5μg、25μg、50μg,10μl注射量)可剂量依赖性地抑制SNL诱导的机械痛敏。鞘内注射CBX5μg也明显抑制了大鼠热痛敏。与对照组相比,在相同剂量(0.81nmol、8.1nmol、81nmol)下,CBX明显抑制了大鼠机械痛敏,而GCA和MFQ无明显镇痛作用。虽然GCA在最大剂量81nmol下似乎对机械痛敏有抑制作用,但大鼠表现出明显药物副作用。连续注射CBX(25μg/天,连续3天)过程中,对大鼠机械痛敏的抑制作用表现出了明显的累加效应,即在第2天和第3天给药前所测得的机械缩足阈值均明显比第1天给药前高。 2.脊髓表面应用CBX可以明显减少SNL大鼠WDR神经元对递增机械刺激的反应,并明显抑制WDR神经元所表现的对电刺激诱导C-component的windup现象。 3.与假手术组相比,免疫荧光实验显示大鼠腰段脊髓背角星形胶质细胞(GFAP)和小胶质细胞(OX42)在SNL后均明显激活；脊髓背角Cx43表达在SNL后明显减少。星形胶质细胞标志GFAP与Cx43的表达有共同定位,而小胶质细胞标志OX42与Cx43无明显共同定位。 4.在大鼠SNL术后14-17天,鞘内注射Cx43siRNA明显抑制了SNL诱导的大鼠机械痛敏,并下调了腰段脊髓Cx43的表达,而对GFAP表达无明显影响。 [结论] 1.鞘内注射CBX可以剂量依赖性的抑制SNL诱导的机械性痛敏,其机制可能通过抑制胶质细胞间的缝隙连接功能。 2.脊髓表面应用CBX可以抑制WDR神经元对递增机械刺激的反应,也可以减少电刺激诱导WDR神经元C-component的windup,表明CBX对机械性痛敏的抑制作用可能由于其对脊髓WDR神经元兴奋性的抑制作用。 3.神经损伤后,位于脊髓的星形胶质细胞和小胶质细胞均被激活,但Cx43表达下降。而SNL术后14-17天,采用鞘内注射Cx43siRN进一步下调Cx43表达后,机械性痛敏明显减轻。这些结果表明神经损伤后,虽然Cx43蛋白表达量减少,但缝隙连接的功能或通透性可能增强了。 4.综上所述,本研究发现脊髓胶质细胞之间缝隙连接的功能增强,尤其是Cx43所构成的缝隙连接,可能在维持SNL诱导的神经病理性疼痛中起到重要作用。这些新发现为神经病理性疼痛的机制研究提供了新线索,也为临床治疗神经病理性疼痛提供了重要靶点。
[Abstract]:[Objective]
In peripheral nerve injury, located in the dorsal horn of the spinal cord excitability of sensory neurons increased, become an important site for transmission and modulation of pain. And around the dorsal horn neurons astrocytes increase in neuronal excitability, pain sensitive transfer, especially the maintenance of neuropathic pain also play an important role. Although an important way the signal transduction between known astrocytes are gap junctions, but the spinal glial cell gap junctions is not clear in the maintenance of neuropathic pain in the role, especially the specific expression of gap between glial cell connexin 43 (Connexin43, Cx43) in neuropathic pain. The effect of carbenoxolone (Carbenoxolone, CBX) non specific gap junction blockers can block the non-specific gap junction function. Using small interfering RNA (siRNA Small interfering RNA) technology In order to reduce the specific expression of astrocyte gap junction protein Cx43. The purpose of this study was to observe the drug (such as carbenoxolone) block gap junction function and down-regulation of siRNA expression of Cx43 protein in the spinal cord can inhibit neuropathic pain or reduce spinal dorsal horn wide dynamic range of neuronal activity.
[materials and methods]
Established in 1. rats with spinal nerve ligation (Spinal nerve, ligation, SNL) model of neuropathic pain, the use of carbenoxolone (CBX) intrathecal injection. First in rats anesthetized with isoflurane, a single injection of different doses of CBX, the conscious animal behavior changes of rats were observed mechanical and thermal hyperalgesia. Secondly, further observation and comparison of the same dose of CBX, glycyrrhizic acid (Glycyrrhizic acid, GCA CBX, is similar, but not to block the gap junction), mefloquine (Mefloquine, MFQ, is a specific neuronal gap junction blocker) effect on mechanical hyperalgesia in rats. Finally, whether there is long term effects of detection of continuous injection of CBX on mechanical pain rat sensitivity.
2. establish the SNL rat model of neuropathic pain after animal under anesthesia in vivo recording of spinal dorsal horn wide dynamic range (Wide dynamic, range, WDR) neurons, and observe and record the spinal cord surface given CBX after treatment, changes of WDR neurons to the peripheral mechanical stimulus response. In addition, the WDR neurons recorded repeated electrical stimulation of action potential changes of peripheral receptive fields induced by C fiber conduction enhancement, and make windup curve. To observe the effect of application of CBX on the WDR C-componer spinal cord neurons action potential windup.
3. immunofluorescence method was used to detect and compare the expression and distribution of Glial fibrillary acidic protein (GFAP), astrocyte markers, Cx43 and OX42 (microglia markers) protein in lumbar spinal cord of sham operation group and SNL group.
4. construction of Cx43siRNA and Control siRNA, the rats were divided into sham operation group, Cx43siRNA group and Control siRNA group. Observation of intrathecal repeated injections of siRN scale, SNL changes the mechanical hyperalgesia in rats. At the same time using Western blot to detect rat bilateral SNL lumbar spinal cord Cx43, Cx36, and the expression of GFAP protein.
[results]
In 1. rats 2-3 weeks after SNL, intrathecal injection of CBX (0.1 Ig, 0.5 g, 5 g, 25 g, 50 g, 10 L injection volume) dose dependently inhibited SNL induced mechanical hyperalgesia. Intrathecal injection of CBX5 g also significantly inhibited the rats of thermal hyperalgesia. Compared with the control group at the same dose (0.81nmol, 8.1nmol, 81nmol), the mechanical hyperalgesia in rats was inhibited by CBX, GCA and MFQ had no significant analgesic effect. Although the GCA in the maximum dose of 81nmol seems to have inhibitory effect on mechanical hyperalgesia, but the rats showed obvious the side effects of the drug. The continuous injection of CBX (25 g/ day for 3 consecutive days) in the process of mechanical hyperalgesia in rats of inhibition showed significant additive effect, namely in the second and third days before the administration of the measured mechanical withdrawal thresholds were significantly more than first days before administration high.
2., the application of CBX on the spinal cord surface can significantly reduce the response of WDR neurons to incremental mechanical stimulation in SNL rats, and significantly inhibit the windup phenomenon of WDR induced by electrical stimulation.
3. compared with sham operation group, immunofluorescence experiments showed that rat spinal cord dorsal horn astrocytes (GFAP) and microglia (OX42) after SNL were significantly activated; spinal Cx43 expression decreased obviously after SNL. Astrocyte marker expression of GFAP and Cx43 are Co located, and small glial cell marker OX42 and Cx43 were Co located.
4., on the 14-17 day after SNL, intrathecal injection of Cx43siRNA significantly inhibited SNL induced mechanical hyperalgesia in rats, and down regulated the expression of Cx43 in lumbar spinal cord, but had no significant effect on GFAP expression.
[Conclusion]
The 1. intrathecal injection of CBX can inhibit SNL induced mechanical pain sensitization in a dose-dependent manner, and its mechanism may be mediated by the inhibition of gap junction function between glial cells.
2., the application of CBX on the spinal cord can inhibit the response of WDR neurons to incremental mechanical stimulation, and also reduce the windup of WDR neurons induced by electrical stimulation, indicating that the inhibitory effect of CBX on the mechanical pain sensitivity may be due to its inhibitory effect on the excitability of the spinal WDR neurons.
3. after nerve injury in spinal cord astrocytes and microglia were activated, but decrease the expression of Cx43 and SNL. After 14-17 days, the intrathecal injection of Cx43siRN by down-regulation of Cx43 expression after mechanical hyperalgesia significantly reduced. These results indicate that after nerve injury, although the expression of Cx43 protein decreased. But the gap junction function or permeability may enhance.
4. in summary, this study found that the gap junction function between spinal cord glial cells increased, especially the gap formed by the Cx43 connection, may play an important role in the maintenance of neuropathic pain induced by SNL. These new findings for the mechanism study of neuropathic pain and provided new clues, but also provides an important target for neuropathic pain in clinic the treatment.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741;R614

【共引文献】