本文選題：皰疹病毒 + 神經(jīng)病理性痛　； 參考：《第四軍醫(yī)大學(xué)》2011年博士論文

【摘要】：帶狀皰疹后神經(jīng)痛(postherpetic neuralgia; PHN)是帶狀皰疹后期最常見和最嚴重的并發(fā)癥,這種劇烈的神經(jīng)痛通常在皮疹消退后一個月產(chǎn)生,并且維持三個月以上的時間。PHN是一種慢性神經(jīng)病理性痛,像其他類型神經(jīng)病理性痛(外傷,炎癥,糖尿病等引起)的疼痛癥狀和體征,主要表現(xiàn)為受損皮區(qū)產(chǎn)生自發(fā)痛,異常疼痛和痛覺過敏。PHN通常持續(xù)遷延,其發(fā)病率與老齡化密切相關(guān),并且臨床上證實各種鎮(zhèn)痛藥(三環(huán)類抗抑郁藥、抗癲癇藥、阿片類藥、非甾體類抗炎藥等)對于PHN的鎮(zhèn)痛效果甚微。PHN可導(dǎo)致失眠、焦慮、抑郁,甚至是生活和工作能力的喪失。PHN嚴重地影響著帶狀皰疹病人的預(yù)后和生活質(zhì)量,然而目前針對PHN尚無有效的預(yù)防和治療措施,其主要原因是對于PHN的發(fā)病機制未能明確闡述。傳統(tǒng)觀點認為,周圍神經(jīng)纖維和末梢的炎癥和變性是PHN發(fā)病的主要原因。然而越來越多的學(xué)者認為,PHN的形成并不只是由周圍神經(jīng)的病變所致,PHN的形成可能與中樞神經(jīng)系統(tǒng)(脊髓和大腦)病變有著密切關(guān)系。 傳統(tǒng)觀念認為疼痛發(fā)生和疼痛調(diào)控只有神經(jīng)元及其纖維發(fā)揮作用,然而近來上述觀點已發(fā)生明顯變化,中樞神經(jīng)系統(tǒng)膠質(zhì)細胞(星形膠質(zhì)細胞和小膠質(zhì)細胞)的激活對于神經(jīng)病理性痛的誘導(dǎo)和維持同樣至關(guān)重要。在外傷或炎癥引起的神經(jīng)病理性痛模型上,脊髓背角星形膠質(zhì)細胞或小膠質(zhì)細胞都有不同程度的激活,激活的膠質(zhì)細胞會釋放大量的各種活性分子(包括神經(jīng)營養(yǎng)因子、促炎性細胞因子和化學(xué)物質(zhì)等)。這些活性分子可能會作用于神經(jīng)突觸終末和神經(jīng)元胞體上相應(yīng)的自身受體,從而增強突觸信號傳遞,最終導(dǎo)致疼痛信號過度的放大以及痛覺過敏的形成。目前,在帶狀皰疹后神經(jīng)痛(PHN)病理生理機制方面的研究,并沒有中樞膠質(zhì)細胞方面的報道。由于①PHN本身就是一種神經(jīng)病理性痛,②PHN的形成與中樞神經(jīng)系統(tǒng)的病變有著密切關(guān)系,③中樞膠質(zhì)細胞激活導(dǎo)致病理性痛是近年研究的熱點;本課題預(yù)測脊髓中樞膠質(zhì)細胞的激活和活性分子的過多釋放極有可能參與了帶狀皰疹后神經(jīng)痛的產(chǎn)生和維持�；诖吮菊n題以PHN大鼠模型為工具,綜合運用行為學(xué)、形態(tài)學(xué)、分子生物學(xué)和電生理學(xué)等神經(jīng)科學(xué)研究方法,進行了開拓性實驗。 本課題研究結(jié)果如下: ①將水痘-帶狀皰疹病毒(varicella-zoster virus; VZV)注入SD大鼠的足底,形成了顯著的機械性痛覺過敏(von Frey絲檢測),而假接種組(Mock infected)和空白對照組(Naive)沒有出現(xiàn)機械性痛覺過敏;對VZV注射組系統(tǒng)給予抗病毒治療,結(jié)果對機械性痛覺過敏并沒有任何影響。以上結(jié)果提示VZV感染導(dǎo)致了PHN,并且PHN并非由病毒的急性活躍性復(fù)制引起。 ②星形膠質(zhì)細胞的特異標(biāo)記物為膠質(zhì)原纖維酸性蛋白(glial fibrillary acidic protein; GFAP),而小膠質(zhì)細胞的特異標(biāo)記物為白細胞分化抗原11b(CD11b; OX-42)。免疫熒光組織化學(xué)染色顯示,與對照組相比,在水痘-帶狀皰疹病毒(VZV)引起的帶狀皰疹后神經(jīng)痛(PHN)大鼠模型,脊髓背角GFAP的染色密度出現(xiàn)了顯著增加,并且增加的染色密度主要集中在脊髓背角淺層。Western blot檢測顯示,PHN大鼠模型脊髓背角GFAP的蛋白表達水平具有顯著提升,并且GFAP的表達水平與痛覺閾值的降低密切相關(guān)。熒光實時定量PCR檢測顯示,PHN大鼠模型脊髓背角GFAP的mRNA核酸表達水平同樣具有顯著提升。另外,在所有大鼠,脊髓背角OX42的表達沒有任何變化。以上結(jié)果提示在PHN時,脊髓背角星形膠質(zhì)細胞而不是小膠質(zhì)細胞處于激活狀態(tài),并且有可能對于痛覺信息的調(diào)控至關(guān)重要。 ③在PHN大鼠模型痛覺的形成期和維持期,經(jīng)鞘內(nèi)給予星形膠質(zhì)細胞抑制劑LAA (L-α-aminoadipate)能夠劑量依賴地產(chǎn)生鎮(zhèn)痛作用,而小膠質(zhì)細胞抑制劑Minocycline對于痛覺閾值沒有任何影響。將LAA與三環(huán)類抗抑郁藥,抗癲癇藥,嗎啡等傳統(tǒng)鎮(zhèn)痛藥相結(jié)合,結(jié)果對于PHN產(chǎn)生明顯的協(xié)同鎮(zhèn)痛作用。以上結(jié)果提示脊髓星形膠質(zhì)細胞而非小膠質(zhì)細胞的激活誘導(dǎo)和維持了PHN,LAA與傳統(tǒng)鎮(zhèn)痛藥相結(jié)合使用,可以減少用藥量,提高藥效,減低副作用。 ④細胞外單電位電生理檢測顯示,與對照組相比,在PHN大鼠模型,脊髓背角廣動力閾神經(jīng)元出現(xiàn)了顯著的放電頻率增加,即疼痛的中樞敏化。在PHN大鼠模型,經(jīng)鞘內(nèi)給予星形膠質(zhì)細胞抑制劑LAA (L-α-aminoadipate)能夠明顯抑制放電頻率的增加,而小膠質(zhì)細胞抑制劑Minocycline對于放電頻率沒有任何影響。以上結(jié)果提示PHN狀態(tài)下脊髓星形膠質(zhì)細胞的激活導(dǎo)致了脊髓背角神經(jīng)元的中樞敏化。 ⑤與對照組相比,在PHN大鼠模型,背根神經(jīng)節(jié)和脊髓背角誘生型一氧化氮合酶(inducible nitric oxide synthase; iNOS)的表達出現(xiàn)了顯著上調(diào),并且鞘內(nèi)給予L-NIL (iNOS抑制劑)或者PTIO (NO清除劑)均能抑制GFAP的表達上調(diào)。以上結(jié)果提示PHN狀態(tài)下脊髓背角iNOS的激活以及NO的過多生成導(dǎo)致了星形膠質(zhì)細胞的激活。 ⑥Western blot檢測顯示,與對照組相比,PHN大鼠模型脊髓背角一些細胞因子,如白介素-1β(IL-1β)、腫瘤壞死因子-α(TNF-α);磷酸化MAPK類分子,如磷酸化p38(p-p38)和磷酸化Jun氨基末端激酶(p-JNK)都有顯著的表達上調(diào)。在PHN大鼠模型,經(jīng)鞘內(nèi)給予細胞因子清除劑pentoxifylline或者白介素-1受體拮抗劑(IL-1ra)均能產(chǎn)生明顯的鎮(zhèn)痛作用。在PHN大鼠模型,脊髓背角IL-1β的蛋白表達變化與GFAP的蛋白表達變化過程相一致;免疫熒光雙重標(biāo)記顯示,IL-1β免疫陽性產(chǎn)物只是定位于GFAP陽性細胞而非NeuN或OX42陽性細胞。在PHN大鼠模型,經(jīng)鞘內(nèi)給予星形膠質(zhì)細胞抑制劑LAA能夠明顯降低IL-1β的過度表達。以上結(jié)果提示PHN狀態(tài)下脊髓星形膠質(zhì)細胞過多生成釋放IL-1β,IL-1β導(dǎo)致了痛覺過敏的產(chǎn)生。 ⑦Western blot檢測顯示,與對照組相比,PHN大鼠模型脊髓背角磷酸化的NMDA受體NR1亞單位(P-NR1)的表達水平具有顯著提升。在PHN大鼠模型,經(jīng)鞘內(nèi)給予NMDA受體拮抗劑AP5或者MK-801均能產(chǎn)生明顯的鎮(zhèn)痛作用。在PHN大鼠模型,脊髓背角P-NR1的表達變化與IL-1β或GFAP的蛋白表達變化過程相一致;免疫熒光雙重標(biāo)記顯示,P-NR1免疫陽性產(chǎn)物和IL-1RI免疫陽性產(chǎn)物只是定位于NeuN陽性細胞而非GFAP或OX42陽性細胞,并且P-NR1免疫陽性產(chǎn)物和IL-1RI免疫陽性產(chǎn)物互相之間完全雙標(biāo)。在PHN大鼠模型,經(jīng)鞘內(nèi)給予星形膠質(zhì)細胞抑制劑LAA,細胞因子清除劑pentoxifylline或者IL-1受體拮抗劑IL-1ra均能明顯降低P-NR1的過度表達。以上結(jié)果提示過多生成的IL-1β作用于神經(jīng)元上自身受體IL-1RI促進了NMDA受體NR1亞單位的磷酸化,最終導(dǎo)致了痛覺過敏和中樞敏化。 ⑧脊髓背角膜片鉗全細胞記錄顯示,在PHN痛覺過敏狀態(tài)下,星形膠質(zhì)細胞過多生成的IL-1β作用于突觸前末梢上自身受體,從而促進了谷氨酸的釋放。另外,IL-1β作用于突觸后胞體上的自身受體,從而增強了谷氨酸的突觸后興奮性效應(yīng)。以上結(jié)果提示IL-1β作用于神經(jīng)元突觸前或突觸后能夠引起興奮性效應(yīng)。 綜合以上所有研究結(jié)果,本人得出的最終結(jié)論是:在PHN時,脊髓背角激活的星形膠質(zhì)細胞過度生成和釋放IL-1β。一方面,IL-1β作用于突觸前末梢上自身受體IL-1RI從而觸發(fā)了致痛物質(zhì)谷氨酸的過多釋放;另一方面, IL-1β作用于突觸后神經(jīng)元上自身受體IL-1RI,從而激發(fā)胞內(nèi)信號轉(zhuǎn)導(dǎo)和NMDA受體NR1亞單位的磷酸化,NR1亞單位的磷酸化會致使NMDA受體的分子構(gòu)象發(fā)生變化,使NMDA受體對于其配體谷氨酸的反應(yīng)性增強,進一步使突觸后神經(jīng)元細胞膜的去極化更明顯。以上IL-1β突觸前和突觸后的綜合效應(yīng)就是:最終導(dǎo)致動作電位過多的產(chǎn)生和痛覺信號過度的放大,引發(fā)中樞敏化和痛覺過敏。 本課題首次提出脊髓星形膠質(zhì)細胞的激活導(dǎo)致了PHN的產(chǎn)生和維持,深入探討了PHN時星形膠質(zhì)細胞合成釋放的活性分子IL-1β導(dǎo)致脊髓背角神經(jīng)元中樞敏化的具體分子機制,為更深入地認識PHN的發(fā)病機制開辟了新途徑,為PHN的預(yù)防治療提供了一個新的靶點。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R752.12

【共引文獻】

相關(guān)期刊論文 前4條

1 浦少鋒;杜冬萍;;小膠質(zhì)細胞上P2X受體在神經(jīng)病理性疼痛中的作用[J];上海醫(yī)學(xué);2009年06期

2 張玉輝;郝飛;樸花;袁博;張楠楠;李愛萍;;ATP誘導(dǎo)大鼠原代培養(yǎng)海馬神經(jīng)元神經(jīng)毒性及對GAP-43表達的影響[J];神經(jīng)解剖學(xué)雜志;2011年05期

3 蔣勁,朱宏騫,胡廣詢,羅施南;阿霉素神經(jīng)干注射治療帶狀皰疹后神經(jīng)痛療效觀察[J];實用疼痛學(xué)雜志;2005年03期

4 劉桂冬;丁健青;肖勤;陳生弟;;P2Y6受體與免疫炎癥(英文)[J];Neuroscience Bulletin;2009年03期

相關(guān)博士學(xué)位論文 前1條

1 孫雅潔;水通道4在神經(jīng)病理性疼痛中的作用及可能機制[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2013年

相關(guān)碩士學(xué)位論文 前4條

1 張楠楠;阻斷P2X_7受體抑制ATP介導(dǎo)的BV-2小膠質(zhì)細胞凋亡[D];大連醫(yī)科大學(xué);2010年

2 白倩;SNL大鼠鞘內(nèi)注射利多卡因鎮(zhèn)痛的實驗研究[D];鄭州大學(xué);2013年

3 蘆睿;異丙酚預(yù)處理對大鼠心肺復(fù)蘇后腦損傷的保護作用及機制[D];第四軍醫(yī)大學(xué);2013年

4 黃海艷;右美托咪啶對SNL模型大鼠的鎮(zhèn)痛效應(yīng)及機制研究[D];中南大學(xué);2013年

，

本文編號：2050728