脊髓星形胶质细胞的激活及其细胞因子在带状疱疹后神经痛形成中的作用

发布时间：2018-06-22 00:34

本文选题：疱疹病毒 + 神经病理性痛　；参考：《第四军医大学》2011年博士论文

【摘要】：带状疱疹后神经痛(postherpetic neuralgia; PHN)是带状疱疹后期最常见和最严重的并发症,这种剧烈的神经痛通常在皮疹消退后一个月产生,并且维持三个月以上的时间。PHN是一种慢性神经病理性痛,像其他类型神经病理性痛(外伤,炎症,糖尿病等引起)的疼痛症状和体征,主要表现为受损皮区产生自发痛,异常疼痛和痛觉过敏。PHN通常持续迁延,其发病率与老龄化密切相关,并且临床上证实各种镇痛药(三环类抗抑郁药、抗癫痫药、阿片类药、非甾体类抗炎药等)对于PHN的镇痛效果甚微。PHN可导致失眠、焦虑、抑郁,甚至是生活和工作能力的丧失。PHN严重地影响着带状疱疹病人的预后和生活质量,然而目前针对PHN尚无有效的预防和治疗措施,其主要原因是对于PHN的发病机制未能明确阐述。传统观点认为,周围神经纤维和末梢的炎症和变性是PHN发病的主要原因。然而越来越多的学者认为,PHN的形成并不只是由周围神经的病变所致,PHN的形成可能与中枢神经系统(脊髓和大脑)病变有着密切关系。传统观念认为疼痛发生和疼痛调控只有神经元及其纤维发挥作用,然而近来上述观点已发生明显变化,中枢神经系统胶质细胞(星形胶质细胞和小胶质细胞)的激活对于神经病理性痛的诱导和维持同样至关重要。在外伤或炎症引起的神经病理性痛模型上,脊髓背角星形胶质细胞或小胶质细胞都有不同程度的激活,激活的胶质细胞会释放大量的各种活性分子(包括神经营养因子、促炎性细胞因子和化学物质等)。这些活性分子可能会作用于神经突触终末和神经元胞体上相应的自身受体,从而增强突触信号传递,最终导致疼痛信号过度的放大以及痛觉过敏的形成。目前,在带状疱疹后神经痛(PHN)病理生理机制方面的研究,并没有中枢胶质细胞方面的报道。由于①PHN本身就是一种神经病理性痛,②PHN的形成与中枢神经系统的病变有着密切关系,③中枢胶质细胞激活导致病理性痛是近年研究的热点;本课题预测脊髓中枢胶质细胞的激活和活性分子的过多释放极有可能参与了带状疱疹后神经痛的产生和维持。基于此本课题以PHN大鼠模型为工具,综合运用行为学、形态学、分子生物学和电生理学等神经科学研究方法,进行了开拓性实验。本课题研究结果如下: ①将水痘-带状疱疹病毒(varicella-zoster virus; VZV)注入SD大鼠的足底,形成了显著的机械性痛觉过敏(von Frey丝检测),而假接种组(Mock infected)和空白对照组(Naive)没有出现机械性痛觉过敏;对VZV注射组系统给予抗病毒治疗,结果对机械性痛觉过敏并没有任何影响。以上结果提示VZV感染导致了PHN,并且PHN并非由病毒的急性活跃性复制引起。 ②星形胶质细胞的特异标记物为胶质原纤维酸性蛋白(glial fibrillary acidic protein; GFAP),而小胶质细胞的特异标记物为白细胞分化抗原11b(CD11b; OX-42)。免疫荧光组织化学染色显示,与对照组相比,在水痘-带状疱疹病毒(VZV)引起的带状疱疹后神经痛(PHN)大鼠模型,脊髓背角GFAP的染色密度出现了显著增加,并且增加的染色密度主要集中在脊髓背角浅层。Western blot检测显示,PHN大鼠模型脊髓背角GFAP的蛋白表达水平具有显著提升,并且GFAP的表达水平与痛觉阈值的降低密切相关。荧光实时定量PCR检测显示,PHN大鼠模型脊髓背角GFAP的mRNA核酸表达水平同样具有显著提升。另外,在所有大鼠,脊髓背角OX42的表达没有任何变化。以上结果提示在PHN时,脊髓背角星形胶质细胞而不是小胶质细胞处于激活状态,并且有可能对于痛觉信息的调控至关重要。 ③在PHN大鼠模型痛觉的形成期和维持期,经鞘内给予星形胶质细胞抑制剂LAA (L-α-aminoadipate)能够剂量依赖地产生镇痛作用,而小胶质细胞抑制剂Minocycline对于痛觉阈值没有任何影响。将LAA与三环类抗抑郁药,抗癫痫药,吗啡等传统镇痛药相结合,结果对于PHN产生明显的协同镇痛作用。以上结果提示脊髓星形胶质细胞而非小胶质细胞的激活诱导和维持了PHN,LAA与传统镇痛药相结合使用,可以减少用药量,提高药效,减低副作用。 ④细胞外单电位电生理检测显示,与对照组相比,在PHN大鼠模型,脊髓背角广动力阈神经元出现了显著的放电频率增加,即疼痛的中枢敏化。在PHN大鼠模型,经鞘内给予星形胶质细胞抑制剂LAA (L-α-aminoadipate)能够明显抑制放电频率的增加,而小胶质细胞抑制剂Minocycline对于放电频率没有任何影响。以上结果提示PHN状态下脊髓星形胶质细胞的激活导致了脊髓背角神经元的中枢敏化。 ⑤与对照组相比,在PHN大鼠模型,背根神经节和脊髓背角诱生型一氧化氮合酶(inducible nitric oxide synthase; iNOS)的表达出现了显著上调,并且鞘内给予L-NIL (iNOS抑制剂)或者PTIO (NO清除剂)均能抑制GFAP的表达上调。以上结果提示PHN状态下脊髓背角iNOS的激活以及NO的过多生成导致了星形胶质细胞的激活。 ⑥Western blot检测显示,与对照组相比,PHN大鼠模型脊髓背角一些细胞因子,如白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α);磷酸化MAPK类分子,如磷酸化p38(p-p38)和磷酸化Jun氨基末端激酶(p-JNK)都有显著的表达上调。在PHN大鼠模型,经鞘内给予细胞因子清除剂pentoxifylline或者白介素-1受体拮抗剂(IL-1ra)均能产生明显的镇痛作用。在PHN大鼠模型,脊髓背角IL-1β的蛋白表达变化与GFAP的蛋白表达变化过程相一致;免疫荧光双重标记显示,IL-1β免疫阳性产物只是定位于GFAP阳性细胞而非NeuN或OX42阳性细胞。在PHN大鼠模型,经鞘内给予星形胶质细胞抑制剂LAA能够明显降低IL-1β的过度表达。以上结果提示PHN状态下脊髓星形胶质细胞过多生成释放IL-1β,IL-1β导致了痛觉过敏的产生。 ⑦Western blot检测显示,与对照组相比,PHN大鼠模型脊髓背角磷酸化的NMDA受体NR1亚单位(P-NR1)的表达水平具有显著提升。在PHN大鼠模型,经鞘内给予NMDA受体拮抗剂AP5或者MK-801均能产生明显的镇痛作用。在PHN大鼠模型,脊髓背角P-NR1的表达变化与IL-1β或GFAP的蛋白表达变化过程相一致;免疫荧光双重标记显示,P-NR1免疫阳性产物和IL-1RI免疫阳性产物只是定位于NeuN阳性细胞而非GFAP或OX42阳性细胞,并且P-NR1免疫阳性产物和IL-1RI免疫阳性产物互相之间完全双标。在PHN大鼠模型,经鞘内给予星形胶质细胞抑制剂LAA,细胞因子清除剂pentoxifylline或者IL-1受体拮抗剂IL-1ra均能明显降低P-NR1的过度表达。以上结果提示过多生成的IL-1β作用于神经元上自身受体IL-1RI促进了NMDA受体NR1亚单位的磷酸化,最终导致了痛觉过敏和中枢敏化。 ⑧脊髓背角膜片钳全细胞记录显示,在PHN痛觉过敏状态下,星形胶质细胞过多生成的IL-1β作用于突触前末梢上自身受体,从而促进了谷氨酸的释放。另外,IL-1β作用于突触后胞体上的自身受体,从而增强了谷氨酸的突触后兴奋性效应。以上结果提示IL-1β作用于神经元突触前或突触后能够引起兴奋性效应。综合以上所有研究结果,本人得出的最终结论是:在PHN时,脊髓背角激活的星形胶质细胞过度生成和释放IL-1β。一方面,IL-1β作用于突触前末梢上自身受体IL-1RI从而触发了致痛物质谷氨酸的过多释放;另一方面, IL-1β作用于突触后神经元上自身受体IL-1RI,从而激发胞内信号转导和NMDA受体NR1亚单位的磷酸化,NR1亚单位的磷酸化会致使NMDA受体的分子构象发生变化,使NMDA受体对于其配体谷氨酸的反应性增强,进一步使突触后神经元细胞膜的去极化更明显。以上IL-1β突触前和突触后的综合效应就是:最终导致动作电位过多的产生和痛觉信号过度的放大,引发中枢敏化和痛觉过敏。本课题首次提出脊髓星形胶质细胞的激活导致了PHN的产生和维持,深入探讨了PHN时星形胶质细胞合成释放的活性分子IL-1β导致脊髓背角神经元中枢敏化的具体分子机制,为更深入地认识PHN的发病机制开辟了新途径,为PHN的预防治疗提供了一个新的靶点。
[Abstract]:Herpes zoster neuralgia (postherpetic neuralgia; PHN) is the most common and most serious complication of herpes zoster at the late stage of herpes zoster, which usually occurs one month after the eruption of the rash, and for more than three months,.PHN is a chronic neuropathic pain, like other types of neuropathic pain (trauma, inflammation, sugar). The symptoms and signs of pain caused by urinary diseases are mainly manifested in spontaneous pain in the damaged skin area, abnormal pain and hyperalgesia,.PHN usually persists, its incidence is closely related to aging, and it is clinically proved that the analgesics of various analgesics (tricyclic antidepressants, antiepileptic drugs, opioids, non steroidal anti-inflammatory drugs, etc.) for the analgesia of PHN The poor effect of.PHN can cause insomnia, anxiety, depression, and even the loss of life and work ability,.PHN seriously affects the prognosis and quality of life of herpes zoster patients. However, there is no effective prevention and treatment for PHN. The main reason is that the pathogenesis of PHN is not clearly explained. Inflammation and denaturation of fiber and endings are the main causes of PHN. However, more and more scholars believe that the formation of PHN is not only caused by peripheral nerve lesions, but the formation of PHN may be closely related to the central nervous system (spinal cord and brain).
Traditional ideas suggest that pain and pain control only play a role in neurons and their fibers. However, these views have changed significantly recently. The activation of glial cells (astrocytes and microglia) in the central nervous system is equally important for the induction and maintenance of neuropathic pain. The astrocytes or microglia of the dorsal horn of the spinal cord are activated in varying degrees in the model of the pathopain, and the activated glial cells release a large number of active molecules (including neurotrophic factors, pro-inflammatory cytokines and chemicals, etc.). These active molecules may act on the terminals of the synapses and the cell bodies of the neurons. The corresponding self receptors, which enhance synaptic transmission, eventually lead to excessive amplification of pain signals and the formation of hyperalgesia. Currently, the pathophysiological mechanism of post herpetic neuralgia (PHN) is not reported in the central glial cell line. (1) PHN itself is a neuropathic pain, and PHN is a neuropathic pain. There is a close relationship between the formation and the pathological changes of the central nervous system. (3) the activation of the central glial cells and the pathological pain are the hot spots in recent years. This topic predicts that the activation of glial cells in the spinal cord and the excessive release of active molecules are very likely to be involved in the production and maintenance of post herpes zoster neuralgia. Based on this subject, PHN rats The model is a tool, and a pioneering experiment is carried out by using the methods of neuroscience, such as behavior, morphology, molecular biology and electrophysiology.
The results of this study are as follows:
(1) injection of varicella zoster virus (varicella-zoster virus; VZV) into the plantar of SD rats to form a significant mechanical hyperalgesia (von Frey), while the false inoculation group (Mock infected) and the blank control group (Naive) did not have mechanical hyperalgesia, and the VZV injection group was treated with antiviral therapy and the result was mechanical pain. Allergy did not have any effect. The above results suggest that VZV infection causes PHN, and PHN is not caused by the acute active replication of the virus.
The specific markers for astrocytes were glial fibrillary acidic protein (glial fibrillary acidic protein; GFAP), and the specific markers for microglia were leukocyte differentiation antigen 11b (CD11b; OX-42). Immunofluorescent histochemical staining showed that the herpes zoster caused by varicella zoster virus (VZV) was compared with the control group. In the rat model of posterior nerve pain (PHN), the staining density of GFAP in the dorsal horn of the spinal cord increased significantly, and the increase of the density was mainly focused on the.Western blot in the superficial layer of the dorsal horn of the spinal cord. The protein expression level of GFAP in the dorsal horn of the spinal cord of the PHN rat model was significantly enhanced, and the expression level of GFAP was closely related to the reduction of the threshold of pain. Fluorescence real-time quantitative PCR detection showed that the mRNA nucleic acid expression level of GFAP in the spinal dorsal horn of the PHN rat model also increased significantly. In addition, there was no change in the expression of OX42 in the dorsal horn of the spinal cord in all rats. The results suggested that the astrocytes in the dorsal horn of the spinal cord were activated in the spinal dorsal horn rather than the microglia at PHN. It is essential to regulate pain information.
(3) in the formation and maintenance period of the PHN rat model, the astrocyte inhibitor LAA (L- alpha -aminoadipate) can produce a dose dependent analgesic effect in a dose-dependent manner, while the microglia inhibitor Minocycline has no effect on the pain threshold. The traditional analgesic effect of LAA and tricyclic antidepressants, antiepileptic drugs, morphine, and so on Combined with the drug, the results showed a significant synergistic analgesic effect on PHN. The results suggested that the activation of astrocytes instead of microglia induced and maintained PHN. The combination of LAA and traditional analgesics could reduce the dosage, improve the efficacy and reduce the side effects.
(4) single potential electrophysiological test showed that compared with the control group, there was a significant increase in the discharge frequency of the spinal dorsal horn neurons in the PHN rat model, that is, the central sensitization of the pain. In the PHN rat model, the astrocyte inhibitor LAA (L- a -aminoadipate) could obviously inhibit the increase of the discharge frequency in the rat model. The microglia inhibitor Minocycline has no effect on the discharge frequency. These results suggest that the activation of astrocytes in the spinal cord of the PHN state leads to the central sensitization of the dorsal horn neurons of the spinal cord.
5. Compared with the control group, the expression of inducible nitric oxide synthase (iNOS) in the dorsal root ganglion and the dorsal horn of the spinal cord was significantly up-regulated in the PHN rat model, and the expression of L-NIL (iNOS inhibitor) or PTIO (NO scavenger) in the sheath could inhibit the up-regulated expression of GFAP. The above results suggested the spinal cord in the PHN state. Activation of dorsal horn iNOS and overproduction of NO lead to astrocyte activation.
6. Western blot test showed that compared with the control group, some cytokines, such as interleukin -1 beta (IL-1 beta), tumor necrosis factor - alpha (TNF- alpha), and phosphorylated MAPK molecules, such as phosphorylated p38 (p-p38) and phosphorylated Jun amino terminal kinase (p-JNK), were significantly up-regulated in the PHN rat model. Cytokine scavenger pentoxifylline or IL-1ra -1 receptor antagonist (IL-1ra) can produce obvious analgesic effects. In PHN rat model, the protein expression of IL-1 beta in the dorsal horn of the spinal cord is in accordance with the changes in the protein expression of GFAP; the immunofluorescence double labeling shows that the positive products of IL-1 beta are only located in GFAP positive cells. Non NeuN or OX42 positive cells. In the PHN rat model, the administration of astrocyte inhibitor LAA in the sheath can significantly reduce the overexpression of IL-1 beta. The above results suggest that the astrocytes in the spinal cord of the spinal cord in PHN state are too much to release IL-1 beta, and IL-1 beta leads to the production of hyperalgesia.
Western blot detection showed that compared with the control group, the expression level of NMDA receptor NR1 subunit (P-NR1) in the dorsal horn of the spinal cord of the PHN rat model increased significantly. In the PHN rat model, the NMDA receptor antagonist AP5 or MK-801 could produce obvious analgesic effect in the PHN rat model. The expression of the dorsal horn of the spinal cord in the PHN rat model was changed. The expression of IL-1 beta or GFAP was consistent with protein expression, and immunofluorescence double labeling showed that P-NR1 immunoreactive products and IL-1RI immunoreactive products were only located in NeuN positive cells, not GFAP or OX42 positive cells, and P-NR1 immunoreactive products and IL-1RI immunoreactive products were completely double labeled with each other. In PHN rat model, The astrocyte inhibitor LAA in the sheath, the cytokine scavenger pentoxifylline or the IL-1 receptor antagonist IL-1ra can significantly reduce the overexpression of P-NR1. The above results suggest that the excessive IL-1 beta activity on the neuronal self receptor IL-1RI promotes the phosphorylation of the NR1 subunit of NMDA receptor and ultimately leads to the hyperalgesia. Sensitization and central sensitization.
The whole cell record of the spinal dorsal horn patch patch patch clamp shows that under PHN hyperalgesia, the excessive IL-1 beta generated by astrocytes acts on the receptor on the tip of the synapse and promotes the release of glutamate. In addition, IL-1 beta acts on the postsynaptic receptor on the postsynaptic cell, thereby enhancing the postsynaptic excitatory effect of glutamate. The above results suggest that IL-1 beta can induce excitatory effects on neurons before or after synapses.
In all the results, the final conclusion is that the astrocytes activated by the dorsal horn of the spinal cord excessively generate and release IL-1 beta at PHN. On the one hand, IL-1 beta acts on the receptor IL-1RI on the tip of the synapse and triggers the excessive release of glutamate in the pain causing substance; on the other hand, IL-1 beta acts on the postsynaptic neurons. The receptor IL-1RI can stimulate the intracellular signal transduction and the phosphorylation of the NMDA receptor NR1 subunit. The phosphorylation of the NR1 subunit will cause the changes in the molecular conformation of the NMDA receptor, which enhances the response of the NMDA receptor to its ligand glutamate, and further depolarizing the membrane of the postsynaptic neurons. Above the IL-1 beta synapse The combined effect after synapse is that it leads to excessive action potential and excessive amplification of pain signals, leading to central sensitization and hyperalgesia.
It is the first time that the activation of astrocytes in the spinal cord leads to the production and maintenance of PHN. The specific molecular mechanism of the active molecule IL-1 beta of astrocytes, which is released by astrocytes at PHN, leads to the central sensitization of the dorsal horn neurons of the spinal cord, which opens a new way for understanding the pathogenesis of PHN and for the prevention and treatment of PHN. A new target is provided.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R752.12

【共引文献】