本文選題：A型肉毒毒素 + 三叉神經(jīng)痛��； 參考：《鄭州大學(xué)》2016年博士論文

【摘要】：三叉神經(jīng)痛是臨床上最為痛苦和常見(jiàn)的神經(jīng)病理性疼痛之一。臨床表現(xiàn)為局限于三叉神經(jīng)分布區(qū)域的單側(cè)短暫性電擊樣或刀割樣劇痛。它的首選治療為口服藥物治療,在口服藥物治療無(wú)效的情況下可以考慮手術(shù)治療,然而有不少患者經(jīng)過(guò)多種治療后效果仍不理想。A型肉毒毒素是革蘭氏陽(yáng)性厭氧芽孢肉毒桿菌釋放的一種外毒素,廣泛應(yīng)用于肌張力障礙治療和美容領(lǐng)域。近年來(lái),它在疼痛治療方面的研究越來(lái)越多,2012年我們改進(jìn)了A型肉毒毒素治療三叉神經(jīng)痛的技術(shù)路線,在國(guó)際上首次用隨機(jī)、雙盲、安慰劑對(duì)照的試驗(yàn)方法證實(shí)了A型肉毒毒素能夠長(zhǎng)期有效的緩解三叉神經(jīng)痛的疼痛且不良反應(yīng)輕微。隨后的多項(xiàng)臨床研究也得出了相似的結(jié)論。但是A型肉毒毒素治療三叉神經(jīng)痛的機(jī)制仍不清楚。為此,我們建立大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)三叉神經(jīng)痛模型,探討A型肉毒毒素對(duì)三叉神經(jīng)痛的治療作用、作用部位和作用機(jī)制。為A型肉毒毒素治療三叉神經(jīng)痛和其他疼痛性疾病提供基礎(chǔ)理論依據(jù)。第一部分大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)建立三叉神經(jīng)痛模型的行為學(xué)和組織學(xué)研究目的建立大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)三叉神經(jīng)痛模型,模擬經(jīng)典三叉神經(jīng)痛,觀察術(shù)后大鼠的疼痛行為學(xué)和眶下神經(jīng)組織學(xué)改變。方法實(shí)驗(yàn)組大鼠經(jīng)口建立眶下神經(jīng)慢性縮窄環(huán)術(shù)三叉神經(jīng)痛模型,對(duì)照組只暴露眶下神經(jīng)而不結(jié)扎。用Von Frey filaments對(duì)兩組術(shù)前和術(shù)后每2天大鼠的疼痛學(xué)行為進(jìn)行評(píng)價(jià);采用HE染色和MBP免疫組織化學(xué)染色對(duì)術(shù)前和術(shù)后14天眶下神經(jīng)組織學(xué)改變進(jìn)行評(píng)價(jià)。結(jié)果實(shí)驗(yàn)組術(shù)后第4天出現(xiàn)一明顯的不應(yīng)期,同側(cè)面部疼痛閾值顯著上升,第6天疼痛閾值達(dá)到高峰,為24.6±8.6g,與術(shù)前相比有統(tǒng)計(jì)學(xué)差異(P0.05)。隨后疼痛閾值急劇下降,在術(shù)后第14天達(dá)到5.5±2.3g(P0.05),這一趨勢(shì)至少持續(xù)至術(shù)后34天。對(duì)照組術(shù)前和術(shù)后各時(shí)間點(diǎn)疼痛閾值相比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。實(shí)驗(yàn)組和對(duì)照組術(shù)前疼痛閾值分別為13.5±3.6g和12.6±5.5g,兩者相比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。兩組間疼痛閾值在術(shù)后第10天分別為5.5±3.2g和13.6±4.8g,組間比較有統(tǒng)計(jì)學(xué)差異(P0.05),這一差異至少持續(xù)至術(shù)后第34天。HE染色和MBP免疫組織化學(xué)染色表明實(shí)驗(yàn)組術(shù)后第14天眶下神經(jīng)存在明顯的脫髓鞘改變,而對(duì)照組與術(shù)前相比無(wú)顯著變化。結(jié)論大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)三叉神經(jīng)痛模型符合經(jīng)典三叉神經(jīng)痛的臨床特點(diǎn)和組織學(xué)改變。該模型簡(jiǎn)單易行,我們可以利用該模型探討三叉神經(jīng)痛的發(fā)病機(jī)制,研究新的治療方法。第二部分A型肉毒毒素對(duì)大鼠三叉神經(jīng)痛模型的抗傷害感受作用研究目的用A型肉毒毒素對(duì)三叉神經(jīng)痛模型進(jìn)行干預(yù),觀察大鼠疼痛行為學(xué)的改變。方法大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)建立三叉神經(jīng)痛模型術(shù)后第14天,實(shí)驗(yàn)組在手術(shù)同側(cè)面部須墊部皮下注射A型肉毒毒素(3U/Kg和10U/Kg),對(duì)照組在同樣部位注射等量的生理鹽水。用Von Frey filaments對(duì)各組術(shù)前和術(shù)后不同時(shí)間點(diǎn)的疼痛學(xué)行為進(jìn)行評(píng)價(jià)。結(jié)果A型肉毒毒素治療后第4天(術(shù)后第18天),3U組和10U組痛覺(jué)閾值均顯著升高,與對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P0.05),這一差異至少持續(xù)至治療后20天(術(shù)后34天)。10U組同一時(shí)間點(diǎn)疼痛閾值高于3U組,但兩組間痛覺(jué)閾值對(duì)比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論A型肉毒毒素須墊部皮下注射可以顯著升高大鼠三叉神經(jīng)痛模型的疼痛閾值,提高其抗傷害感受能力。第三部分A型肉毒毒素治療大鼠三叉神經(jīng)痛的作用部位研究目的在前兩部分研究的基礎(chǔ)上,通過(guò)對(duì)A型肉毒毒素的特異性標(biāo)記物c SNAP-25的檢測(cè),研究A型肉毒毒素治療三叉神經(jīng)痛的作用部位,探討軸突運(yùn)輸在A型肉毒毒素治療三叉神經(jīng)痛中的作用。方法大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)后13.5天,實(shí)驗(yàn)組在三叉神經(jīng)節(jié)內(nèi)注射軸突運(yùn)輸抑制劑秋水仙堿,對(duì)照組注射等量生理鹽水。術(shù)后14天在手術(shù)同側(cè)面部須墊部皮下注射A型肉毒毒素(10U/Kg)或生理鹽水溶液。觀察不同時(shí)間點(diǎn)各組大鼠對(duì)疼痛刺激的變化,探討軸突運(yùn)輸在A型肉毒毒素治療三叉神經(jīng)痛中的作用。用Western blot和免疫熒光對(duì)A型肉毒毒素的特異性標(biāo)志物c SNAP-25進(jìn)行檢測(cè),觀察A型肉毒毒素在三叉神經(jīng)痛模型大鼠的作用部位。結(jié)果實(shí)驗(yàn)組用秋水仙堿阻斷軸突運(yùn)輸降低了A型肉毒毒素的抗傷害感受作用,A型肉毒毒素治療后第4天實(shí)驗(yàn)組疼痛閾值顯著低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),這一差異持續(xù)至實(shí)驗(yàn)結(jié)束。A型肉毒毒素治療后7天,通過(guò)western-blot和免疫熒光檢測(cè)到大鼠三叉神經(jīng)脊束核尾側(cè)亞核c SNAP-25水平明顯增高。三叉神經(jīng)節(jié)注射秋水仙堿阻斷軸突運(yùn)輸后,可以阻斷其增高。Western blot表明三叉神經(jīng)節(jié)注射秋水仙堿阻斷軸突運(yùn)輸后三叉神經(jīng)脊束核尾側(cè)亞核c SNAP-25較對(duì)照組顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論秋水仙堿阻斷軸突運(yùn)輸后使A型肉毒毒素的抗傷害感受作用顯著減弱。A型肉毒毒素通過(guò)軸突運(yùn)輸至三叉神經(jīng)脊束核尾側(cè)亞核發(fā)揮作用。第四部分TRPs在A型肉毒毒素治療大鼠三叉神經(jīng)痛中的作用機(jī)制研究目的探討痛覺(jué)相關(guān)TRPs(TRPA1、TRPV1、TRPV2及TRPM8)在A型肉毒毒素對(duì)大鼠三叉神經(jīng)痛模型發(fā)揮抗傷害感受作用的機(jī)制。方法TRPA1、TRPV1、TRPV2和TRPM8是TRPs家族中可能與疼痛相關(guān)的離子通道,為了進(jìn)一步明確A型肉毒毒素對(duì)三叉神經(jīng)痛抗傷害感受的機(jī)制,采用Western blot對(duì)三叉神經(jīng)痛模型三叉神經(jīng)脊束核尾側(cè)亞核不同時(shí)間點(diǎn)的TRPA1、TRPV1、TRPV2和TRPM8蛋白表達(dá)進(jìn)行檢測(cè),觀察這些蛋白在三叉神經(jīng)痛模型中的動(dòng)態(tài)變化。同時(shí)用Western blot檢測(cè)A型肉毒毒素同側(cè)面部須墊部皮下注射(3U/Kg和10U/Kg)對(duì)三叉神經(jīng)痛模型三叉神經(jīng)脊束核尾側(cè)亞核中TRPA1、TRPV1、TRPV2和TRPM8蛋白表達(dá)的影響。結(jié)果Western blot表明三叉神經(jīng)脊束核尾側(cè)亞核中TRPA1和TRPV1的表達(dá)水平在眶下神經(jīng)慢性縮窄環(huán)術(shù)后第14天明顯增高(P0.05),在術(shù)后第28天仍顯著增高(P0.05)。TRPV2在術(shù)后第7天表達(dá)明顯增高(P0.05),術(shù)后第28天仍然持續(xù)增高(P0.05)。TRPM8在術(shù)后第7天開(kāi)始增高,在術(shù)后第14天達(dá)到高峰,術(shù)后第28天與對(duì)照組相比仍有統(tǒng)計(jì)學(xué)差異(P0.05)。A型肉毒毒素治療后第14天,Western blot表明大鼠三叉神經(jīng)脊束核尾側(cè)亞核中TRPA1和TRPV1的表達(dá)較對(duì)照組顯著降低(P0.05),A型肉毒毒素降低TRPA1和TRPV1的表達(dá)有劑量依賴的效果,10U組的TRPA1和TRPV1的表達(dá)與3U相比有統(tǒng)計(jì)學(xué)差異(P0.05)。A型肉毒毒素(10U/Kg)治療后第14天顯著降低了TRPV2的表達(dá)(P0.05),3U組TRPV2的表達(dá)水平與對(duì)照組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。與對(duì)照組相比,A型肉毒毒素(3U/Kg和10U/Kg)不影響TRPM8的表達(dá)(P0.05)。結(jié)論大鼠眶下神經(jīng)慢性縮窄環(huán)術(shù)后三叉神經(jīng)脊束核尾側(cè)亞核TRPA1、TRPV1、TRPV2和TRPM8的表達(dá)顯著升高,且有一動(dòng)態(tài)變化過(guò)程。A型肉毒毒素治療三叉神經(jīng)痛的機(jī)制可能是抑制三叉神經(jīng)脊束核尾側(cè)亞核中TRPA1、TRPV1和TRPV2的高表達(dá),降低中樞敏化,從而發(fā)揮抗傷害感受作用。
[Abstract]:Trigeminal neuralgia is one of the most painful and common neuropathic pain in the clinic. Clinical manifestations are unilateral transient electric shock or knife cut pain limited to the trigeminal distribution area. Its first choice is oral medication, and surgical treatment can be considered in the case of ineffective oral medication. However, there are many patients. After a variety of treatments, the effect of botulinum toxin type.A is a kind of exotoxin released by Gram positive anaerobic bacillus botulinum. It is widely used in the field of dystonia and Cosmetology. In recent years, more and more studies have been made on the treatment of pain. In 2012, we changed the technology of botulinum toxin type A to treat trigeminal neuralgia. Route, for the first time, a randomized, double blind, placebo-controlled trial for the first time confirmed that botulinum A can effectively alleviate the pain and slight adverse reactions of trigeminal neuralgia for a long time. Several subsequent clinical studies have drawn similar conclusions. However, the mechanism of A botulinum toxin for the treatment of trigeminal neuralgia is still unclear. We established a rat model of trigeminal neuralgia with chronic suborbital coarctation of the suborbital nerve to explore the therapeutic effect, location and mechanism of botulinum toxin A on trigeminal neuralgia. It provides the basis for the treatment of trigeminal neuralgia and other painful diseases by botulinum toxin type A in the first part of the rats. The behavior and histology of the trigeminal neuralgia model aim to establish a rat model of trigeminal neuralgia with chronic constrictive ring of the suborbital nerve, to simulate the classical trigeminal neuralgia, to observe the pain behavior and the histological changes of the suborbital nerve in the rats after the operation. Von Frey filaments was used to evaluate the pain behavior of the two groups of rats before and after the operation. HE staining and MBP immunohistochemical staining were used to evaluate the histological changes of the suborbital nerve 14 days before and after the operation. The results showed an obvious refractory period in the experimental group fourth days after operation. The threshold of pain in the same side was increased significantly, the sixth day pain threshold reached a peak of 24.6 + 8.6g, which was significantly different from that before the operation (P0.05). Then the threshold of pain decreased sharply and reached 5.5 + 2.3g (P0.05) fourteenth days after the operation. This trend was at least 34 days after the operation. The pain threshold of the group before and after the operation was not statistically significant. The pain threshold of the experimental group and the control group was 13.5 + 3.6g and 12.6 + 5.5G, respectively. There was no statistical difference between the two groups (P0.05). The pain threshold between the two groups was 5.5 + 3.2g and 13.6 + 4.8g after the operation, and there was a statistically significant difference between the groups (P0.05). The difference lasted to at least thirty-fourth days after the operation,.HE and MBP immunity. Histochemical staining showed that there were obvious demyelination changes in the suborbital nerve in the experimental group fourteenth days after operation, but there was no significant change in the control group. Conclusion the model of trigeminal neuralgia for the chronic suborbital constriction ring operation of the rat's suborbital nerve conforms to the clinical characteristics and histological changes of the classical trigeminal neuralgia. This model studies the pathogenesis of trigeminal neuralgia and studies new treatment methods. Second part of the anti nociceptive effect of botulinum toxin type A on the trigeminal neuralgia model in rats. Aim to intervene the trigeminal neuralgia model with A botulinum toxin and observe the change of pain behavior in rats. Method the chronic constriction ring of the suborbital nerve in rats. Fourteenth days after the operation of the trigeminal neuralgia model, the experimental group was subcutaneously injected with botulinum toxin A (3U/Kg and 10U/Kg) and the control group was injected with equal amount of physiological saline in the same part. Von Frey filaments was used to evaluate the pain and pain behavior of each group before and after the operation. Results the treatment of A botulinum toxin was found. Fourth days after treatment (eighteenth days after operation), the pain threshold of both group 3U and group 10U was significantly higher than that of the control group (P0.05). This difference lasted for at least 20 days after the treatment (34 days after the operation) and the pain threshold of the.10U group was higher than that of the 3U group, but there was no statistical difference between the two groups (P0.05). Conclusion A botulinum toxin must be found. Subcutaneous injection can significantly increase the pain threshold of the rat trigeminal neuralgia model and improve its anti nociceptive ability. Third Research on the role of botulinum toxin type A in the treatment of trigeminal neuralgia in rats, based on the study of the first two parts and the detection of C SNAP-25, a specific marker for botulinum toxin type A, study A The role of botulinum toxin in the treatment of trigeminal neuralgia and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A. Methods 13.5 days after the operation of the chronic constrictive ring of the suborbital nerve in rats, the experimental group injected the axon transport inhibitor colchicine in the trigeminal ganglion and injected the same amount of normal saline to the group for 14 days after operation. A botulinum toxin (10U/Kg) or saline solution was injected subcutaneously on the same side of the facial facial pad. The changes of pain stimulation in each group of rats at different time points were observed and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A was investigated. C SNAP-25, a specific marker for botulinum toxin type A, was examined by Western blot and immunofluorescence. Test, observe the role of botulinum toxin type A in the rat model of trigeminal neuralgia. Results the experimental group used colchicine blocking axonal transportation to reduce the anti nociceptive effect of type A botulinum toxin. The pain threshold of the experimental botulinum toxin type A was significantly lower than that of the control group at fourth days after the treatment, and the difference was statistically significant (P0.05). The difference continued to be sustained. 7 days after the treatment of botulinum toxin type.A, the level of C SNAP-25 in the nucleus caudal subnucleus of the trigeminal nucleus of the rat was significantly increased by Western-blot and immunofluorescence. After blocking the axon transport by injection of colchicine, the trigeminal ganglion could block the.Western blot Biao Mingsan ganglion injection of colchicine to block axonal transportation The C SNAP-25 in the posterior trigeminal nucleus caudal subnucleus was significantly lower than that in the control group (P0.05). Conclusion colchicine inhibited the anti nociceptive effect of A type botulinum toxin by colchicine blocking axon transport and the effect of.A type botulinum toxin on the nucleus caudal subnucleus of the trigeminal nucleus through axon. Fourth part TRPs was observed. Study on the mechanism of A botulinum toxin in the treatment of trigeminal neuralgia in rats. Objective to explore the mechanism of pain related TRPs (TRPA1, TRPV1, TRPV2 and TRPM8) in the anti nociceptive effect of botulinum toxin type A on rat trigeminal neuralgia model. Methods TRPA1, TRPV1, TRPV2 and TRPM8 are the ion channels associated with pain in the TRPs family, for the purpose of The mechanism of the anti nociceptive sensitivity of botulinum toxin A to trigeminal neuralgia was further clarified, and the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of trigeminal neuralgia model was detected by Western blot, and the dynamic changes of these proteins in the trigeminal neuralgia model were observed. At the same time, Western blot was used. The effects of subcutaneous injection (3U/Kg and 10U/Kg) on the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of the trigeminal neuralgia model of botulinum toxin type A botulinum toxin (3U/Kg and 10U/Kg). Results Western blot showed that the expression level of TRPA1 and TRPV1 in the caudal nucleus of the trigeminal nucleus of the spinal trigeminal tract was performed by chronic constrictive ring of the orbital nerve After fourteenth days (P0.05), the expression increased significantly (P0.05) (P0.05) at the seventh day after operation (P0.05), and continued to increase on the twenty-eighth day after operation (P0.05).TRPM8 began to increase on the seventh day after the operation, and reached the peak at the fourteenth day after the operation, and the twenty-eighth day after the operation was still statistically different (P0.05).A type botulinum toxin compared to the control group (P0.05). On the fourteenth day after treatment, Western blot showed that the expression of TRPA1 and TRPV1 in the nucleus caudal nucleus of the trigeminal nucleus of the rat was significantly lower than that of the control group (P0.05). The expression of TRPA1 and TRPV1 of the A type botulinum toxin was dose-dependent. The expression of TRPA1 and TRPV1 in the 10U group was statistically different from that of 3U. The expression of TRPV2 was significantly reduced in the last fourteenth days (P0.05), and the expression level of TRPV2 in group 3U was not significantly different from that in the control group (P0.05). Compared with the control group, A type botulinum toxin (3U/Kg and 10U/Kg) did not affect the expression of TRPM8 (P0.05). Conclusion the caudal nucleus of the trigeminal nucleus of the trigeminal nucleus after the operation of the suborbital nerve of the rat is TRPA1, TRPV1, and The mechanism of botulinum toxin type.A in the treatment of trigeminal neuralgia may be to inhibit the high expression of TRPA1, TRPV1 and TRPV2 in the caudal nucleus of the trigeminal nucleus, and reduce the central sensitization, thus exerting anti nociceptive effect.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R745.11
，

本文編號(hào)：1914210