【摘要】：疼痛(pain)是由傷害性刺激引起的復(fù)雜生理心理活動(dòng),在正常情況下,是機(jī)體重要的警告信號(hào),當(dāng)傷害性刺激發(fā)生時(shí),機(jī)體通過(guò)全身皮膚和有關(guān)組織中分布的的傷害性感受器將各種形式的刺激轉(zhuǎn)換成神經(jīng)沖動(dòng)的電活動(dòng)信號(hào),沿著傳入神經(jīng),背根神經(jīng)節(jié)到達(dá)脊髓背角或三叉神經(jīng)脊束核中的相關(guān)神經(jīng)元,再由對(duì)側(cè)的腹外側(cè)索傳至丘腦、其他腦區(qū)以及大腦皮質(zhì),產(chǎn)生痛覺(jué),從而對(duì)有害刺激做出回避反應(yīng)。另一方面,過(guò)度的疼痛會(huì)產(chǎn)生負(fù)面影響,長(zhǎng)期的劇烈疼痛會(huì)對(duì)機(jī)體產(chǎn)生難以忍受的精神和軀體的折磨,所以減輕異常疼痛,提高人類(lèi)生活質(zhì)量始終是醫(yī)學(xué)研究的關(guān)注點(diǎn)。介導(dǎo)疼痛的傳導(dǎo)通路及分子作用復(fù)雜,在疼痛機(jī)制的研究長(zhǎng)河中各種學(xué)說(shuō)層出不窮。其中,作為疼痛四大學(xué)說(shuō)之一的閘門(mén)控制學(xué)說(shuō)相對(duì)比較確切的闡述了病理性疼痛的發(fā)生機(jī)制,認(rèn)為在整個(gè)痛覺(jué)的傳導(dǎo)過(guò)程中,脊髓背角膠狀質(zhì)(Substantia gelatinosa,SG)中的某些神經(jīng)細(xì)胞控制著痛覺(jué)信息的傳遞,作用機(jī)理類(lèi)似閘門(mén)的開(kāi)放,這些神經(jīng)元本身就受粗、細(xì)纖維傳入活動(dòng)和高級(jí)中樞下行控制作用的影響,本學(xué)說(shuō)的提出為揭示疼痛的發(fā)生機(jī)制開(kāi)辟了新的方向,基于此理論,我們意識(shí)到,脊髓背角膠狀質(zhì)作為疼痛傳導(dǎo)的中樞起著信息轉(zhuǎn)換的重要作用,必然也是研究開(kāi)發(fā)新型鎮(zhèn)痛藥物的重要靶點(diǎn)。癲癇是因腦部神經(jīng)元異常放電引起的一組臨床綜合征,由已知或未知病因引起,臨床特征表現(xiàn)為反復(fù)、短暫、刻板的神經(jīng)系統(tǒng)功能失常。隨著人類(lèi)醫(yī)學(xué)研究的不斷進(jìn)步,新時(shí)期提出的整合醫(yī)學(xué)認(rèn)為將相關(guān)各領(lǐng)域最先進(jìn)的醫(yī)學(xué)發(fā)現(xiàn)加以整合可以形成更加全面的醫(yī)學(xué)知識(shí)體系。隨著人們對(duì)癲癇和疼痛機(jī)制研究的不斷深入,循著整合醫(yī)學(xué)的方向,查閱資料后我們發(fā)現(xiàn)癲癇和疼痛的的發(fā)病機(jī)制部分類(lèi)似。新型抗癲癇藥物盧非酰胺屬于三唑類(lèi)衍生物,目前臨床作為輔助治療藥物應(yīng)用于4歲以上兒童和成人Lennox-Gastant綜合征(LGS)相關(guān)癲癇發(fā)作。研究發(fā)現(xiàn)它不僅在癲癇的治療上作用顯著,而且具有潛在的鎮(zhèn)痛作用,與此同時(shí),它具有穩(wěn)定情緒的獨(dú)特優(yōu)勢(shì)。近年來(lái),盧非酰胺已經(jīng)成功用于癲癇疾病的治療,但是鎮(zhèn)痛機(jī)制尚不明確,尤其是對(duì)疼痛重要調(diào)節(jié)位點(diǎn)的脊髓背角膠狀質(zhì)神經(jīng)元及其突觸傳遞的作用機(jī)制缺乏有力的研究報(bào)道。本課題主要利用動(dòng)物行為學(xué)測(cè)試技術(shù)、膜片鉗全細(xì)胞記錄探討盧非酰胺對(duì)脊髓背角SG神經(jīng)元的興奮性以及對(duì)傷害性刺激突觸傳遞的影響,為闡明盧非酰胺的脊髓背角鎮(zhèn)痛機(jī)制提供實(shí)驗(yàn)依據(jù)。實(shí)驗(yàn)一:盧非酰胺對(duì)腰5脊神經(jīng)結(jié)扎(Spinal nerve ligation,SNL)神經(jīng)病理性疼痛大鼠模型的鎮(zhèn)痛作用目的:觀察抗癲癇藥物盧非酰胺對(duì)腰5脊神經(jīng)結(jié)扎神經(jīng)病理性疼痛大鼠模型(SNL)的機(jī)械、熱痛覺(jué)超敏的影響。方法:選取180~220g雄性SD大鼠,于SNL模型制作前3d給予連續(xù)的相同時(shí)間同一環(huán)境適應(yīng),前1d測(cè)定大鼠疼痛基礎(chǔ)閾值。選取疼痛閾值在正常范圍內(nèi)的大鼠制備腰5脊神經(jīng)結(jié)扎(SNL)神經(jīng)病理性疼痛動(dòng)物模型。術(shù)后5d同一時(shí)間測(cè)試機(jī)械性縮足反射閾值、熱痛縮足反射潛伏時(shí)間,若疼痛閾值未下降或出現(xiàn)后足偏癱,行為異常視為造模失敗,剔除出組。神經(jīng)病理性疼痛動(dòng)物模型被隨機(jī)分為三組:(1)大劑量實(shí)驗(yàn)組:將50 mg/kg盧非酰胺溶入1%DMSO中,生理鹽水稀釋至1ml,單次腹腔注射。(2)小劑量實(shí)驗(yàn)組:25 mg/kg盧非酰胺溶入1%DMSO中用生理鹽水稀釋至1ml單次腹腔注射。(3)對(duì)照組:與實(shí)驗(yàn)組等體積、等比例的1%DMSO單次腹腔注射。分別于注射后20 min、40 min、60 min、4 h、12 h、24 h進(jìn)行行為學(xué)評(píng)估。觀察盧非酰胺對(duì)大鼠L5神經(jīng)損傷后形成的神經(jīng)病理性疼痛的鎮(zhèn)痛效果。結(jié)果:SNL術(shù)前對(duì)大鼠基礎(chǔ)疼痛閾值進(jìn)行測(cè)定,左、右后足機(jī)械性縮足反射閾值(PWMT)分別為(21.87±5.69)g和(18.33±4.18)g,熱痛縮足反射潛伏時(shí)間(TWL)為(24.43±3.32)s和(22.31±4.28)s,無(wú)統(tǒng)計(jì)學(xué)差異。術(shù)后第5 d,形成穩(wěn)定神經(jīng)病理性疼痛,與術(shù)前相比,左后足的閾值顯著降低至(6.00±2.13)g(P0.001,one-way ANOVA,n=22),熱痛縮足反射潛伏時(shí)間降為(13.45±2.17)s(P0.001,one-way ANOVA,n=22),證實(shí)模型建立成功。當(dāng)腹腔注射不同濃度的盧非酰胺后,測(cè)試機(jī)械、熱痛閾值發(fā)現(xiàn)兩種濃度的盧非酰胺均可以顯著緩解已形成的慢性疼痛,并且鎮(zhèn)痛作用具有明顯的濃度依賴(lài)性,并在給藥后1 h作用達(dá)到高峰,與溶劑組相比可顯著提升大鼠術(shù)側(cè)機(jī)械性縮足反射閾值至(19.99±7.17)g和(17.00±7.32)g(P0.001,one-way ANOVA,n=8),增加熱痛縮足反射潛伏時(shí)間至(16.23±3.14)s和(19.90±2.41)s(P0.001,one-way ANOVA,n=8)這一作用可維持至注藥后12 h。實(shí)驗(yàn)二:盧非酰胺對(duì)脊髓背角膠狀質(zhì)(SG)神經(jīng)元興奮性的影響目的:應(yīng)用膜片鉗全細(xì)胞記錄技術(shù),在脊髓矢狀位切片上觀察盧非酰胺對(duì)SG神經(jīng)元?jiǎng)幼麟娢?Action potential,AP)發(fā)放頻率的影響。方法:取4~5周齡雄性SD大鼠,制備厚度為400-500μm帶后根的脊髓腰骶膨大段矢狀位切片。低倍鏡下確定脊髓背角淺層,高倍鏡下選擇狀態(tài)較好的SG神經(jīng)元做全細(xì)胞記錄,鉗制電流為0 p A,待細(xì)胞狀態(tài)穩(wěn)定5 min后。觀察盧非酰胺對(duì)SG神經(jīng)元?jiǎng)幼麟娢话l(fā)放頻率的影響。結(jié)果:抗癲癇藥物盧非酰胺可顯著減少脊髓背角SG神經(jīng)元?jiǎng)幼麟娢坏陌l(fā)放頻率(P0.01,paired t-test),給予一段時(shí)間洗脫,盧非酰胺的抑制作用被成功逆轉(zhuǎn)(P0.01,paired t-test)。實(shí)驗(yàn)三:盧非酰胺對(duì)傷害性感覺(jué)通路突觸傳遞的選擇性抑制作用目的:全細(xì)胞記錄,電壓鉗模式下給予后根刺激,觀察抗癲癇藥物盧非酰胺對(duì)中等直徑Aδ纖維和小直徑C纖維介導(dǎo)的脊髓背角SG神經(jīng)元的興奮性突觸后電流(e EPSCs)及自發(fā)性興奮性突觸后電流(s EPSCs)的影響。方法:同實(shí)驗(yàn)二,制備SD大鼠脊髓切片,選取SG神經(jīng)元,鉗制電壓為-70 m V,給予從小到大的后根恒壓電刺激,先記錄給藥前誘發(fā)的興奮性突觸后電流(e EPSCs)15條曲線(xiàn),平均后作為對(duì)照,浴槽內(nèi)不間斷灌流200μM盧非酰胺1 min后相同方法再次記錄,根據(jù)文獻(xiàn)報(bào)道不同的纖維傳導(dǎo)具有的電生理特異性,對(duì)記錄的細(xì)胞進(jìn)行分類(lèi)統(tǒng)計(jì)。(1)觀察盧非酰胺對(duì)Aδ纖維介導(dǎo)的e EPSCs的作用。(2)觀察盧非酰胺對(duì)C纖維介導(dǎo)的e EPSCs的作用。(3)觀察盧非酰胺對(duì)脊髓背角SG神經(jīng)元s EPSCs的影響。結(jié)果:(1)單次灌流不同濃度的盧非酰胺對(duì)Aδ纖維介導(dǎo)的e EPSCs幾乎不產(chǎn)生影響(P0.05,paired t-test)。(2)盧非酰胺可顯著抑制C纖維介導(dǎo)的e EPSCs的峰值(P0.01,paired t-test)。(3)盧非酰胺灌流可以明顯抑制SG神經(jīng)元s EPSCs的發(fā)放頻率(P0.001,paired t-test),但對(duì)其幅度沒(méi)有明顯影響(P0.05,paired t-test)。結(jié)論:1.單次腹腔注射不同濃度的盧非酰胺均可以有效緩解已形成的慢性疼痛,鎮(zhèn)痛作用在給藥后1h達(dá)到高峰且具有濃度依賴(lài)性。2.盧非酰胺可以顯著抑制脊髓背角SG神經(jīng)元?jiǎng)幼麟娢坏陌l(fā)放頻率,這一抑制作用可以在給藥后10 min內(nèi)被有效洗脫。3.不同濃度的盧非酰胺幾乎不影響Aδ纖維支配的單突觸SG神經(jīng)元所產(chǎn)生的e EPSCs峰值,與對(duì)照組e EPSCs相比,無(wú)統(tǒng)計(jì)學(xué)意義。盧非酰胺可以顯著抑制C纖維介導(dǎo)e EPSCs的峰值,作用時(shí)間大于10 min。持續(xù)灌流盧非酰胺1 min還可以顯著降低SG神經(jīng)元s EPSCs的頻率,但不影響s EPSCs的幅度。
[Abstract]:Pain is a complex physiological and psychological activity caused by noxious stimuli. Normally, it is an important warning signal of the body. When noxious stimuli occur, the body converts various forms of stimuli into electrical signals of nerve impulses through noxious receptors distributed in the skin and related tissues of the body, along the afferent nerves. The dorsal root ganglion (DRG) reaches the neurons in the dorsal horn of the spinal cord or the nucleus of the spinal trigeminal tract, and then passes from the contralateral ventrolateral cord to the thalamus, other brain regions, and the cerebral cortex, producing a sense of pain, thus avoiding harmful stimuli. On the other hand, excessive pain can have a negative impact, and long-term severe pain can be difficult to produce. It is always the focus of medical research to alleviate abnormal pain and improve the quality of human life because of the mental and physical torture endured. The pathways and molecular functions that mediate pain are complex. Various theories emerge in endlessly in the study of pain mechanism. The mechanism of pathological pain is expounded. It is believed that some neurons in the spinal dorsal horn gelatinosa (SG) control the transmission of pain information during the whole process of pain transmission. The mechanism of action is similar to the opening of gates. These neurons themselves are thick, thin fiber afferent activities and advanced central downward control. Based on this theory, we realize that the gelatinous substance of the spinal dorsal horn, as the center of pain transmission, plays an important role in information conversion and is also an important target for the development of new analgesic drugs. Epilepsy is caused by abnormal release of neurons in the brain. A group of clinical syndromes, caused by known or unknown causes, characterized by repeated, transient, and rigid dysfunction of the nervous system. With the continuous progress of human medical research, integrated medicine proposed in the new era considers that the integration of the most advanced medical discoveries in related fields can lead to a more comprehensive medicine. Knowledge system. With the deepening of research on the mechanism of epilepsy and pain, following the direction of integrated medicine, we found that the pathogenesis of epilepsy and pain is similar in part. Lufenamide, a new antiepileptic drug, belongs to triazole derivatives. It is currently used as an adjuvant therapy in children and adults over 4 years old. Epilepsy related to human Lennox-Gastant syndrome (LGS). Studies have found that lufenamide has not only a significant role in the treatment of epilepsy, but also a potential analgesic effect. At the same time, it has a unique advantage in stabilizing mood. In recent years, lufenamide has been successfully used in the treatment of epilepsy, but the mechanism of analgesia is still unclear, especially for pain. In order to elucidate the effect of lufenamide on the excitability of spinal dorsal horn SG neurons and the synaptic transmission of noxious stimuli, we investigated the effects of lufenamide on the excitability of spinal dorsal horn SG neurons and the synaptic transmission of noxious stimuli by using animal behavioral testing techniques and patch clamp whole cell recording. Objective: To observe the analgesic effect of lufenamide on lumbar 5 spinal nerve ligation (SNL) induced neuropathic pain in rats. METHODS: Male SD rats of 180-220 g were selected and adapted to the same environment at the same time for three days before the establishment of the SNL model. The basic pain threshold was measured at the first day. The neuropathic pain animal model of lumbar 5 spinal ligation (SNL) was established in rats with normal pain threshold. Neuropathic pain animal models were randomly divided into three groups: (1) High-dose experimental group: 50 mg/kg lufenamide was dissolved in 1% DMSO, normal saline was diluted to 1 ml, single intraperitoneal injection. (2) Low-dose experimental group: 25 mg/kg of lufenamide dissolved in 1% DMSO and diluted to 1 ml of normal saline for a single intraperitoneal injection. (3) Control group: 1% DMSO was injected into the abdominal cavity in the same volume and proportion as the experimental group. Behavioral evaluation was carried out 20 minutes, 40 minutes, 60 minutes, 4 hours, 12 hours and 24 hours after injection, respectively. Results: The mechanical shrinkage reflex thresholds (PWMT) of left and right hind feet were (21.87 [5.69] g and (18.33 [4.18] g respectively, and the latency time (TWL) of hot pain shrinkage reflex was (24.43 [3.32] s] and (22.31 [4.28] s, respectively. There was no significant difference between them. Compared with preoperative neuropathic pain, the threshold of left hind foot was significantly reduced to (6.00 (+ 2.13) g (P 0.001, one-way ANOVA, n = 22) and the latent time of foot reflex was reduced to (13.45 (+ 2.17) s (P 0.001, one-way ANOVA, n = 22), which confirmed the success of the model. Lufenamide at different concentrations could significantly alleviate chronic pain, and the analgesic effect was obviously concentration-dependent, and reached its peak at 1 hour after administration. Compared with the solvent group, Lufenamide significantly increased the threshold of mechanical foot contraction reflex to (19.99 (7.17) g and (17.00 (7.32) g (P 0.001, one-way ANOVA, n=8), and increased the fever. The effect of lufenamide on the excitability of spinal dorsal horn gelatinous (SG) neurons was observed on sagittal sections of the spinal cord using patch clamp whole cell recording technique. Methods: Sagittal sections of lumbosacral enlarged spinal cord with posterior roots were prepared from 4 to 5 weeks old male SD rats. The superficial layer of spinal dorsal horn was determined at low magnification, and the better SG neurons were selected at high magnification for whole cell recording. The clamping current was 0 P A and the cells were waiting for cells. Results: Antiepileptic drug lufenamide could significantly reduce the action potential release frequency of SG neurons in spinal dorsal horn (P 0.01, paired t-test). After a period of elution, the inhibitory effect of lufenamide was reversed (P 0.01, paired t-test). Experiment 3: Selective inhibitory effect of lufenamide on synaptic transmission in nociceptive pathways Objective: Whole cell recording, posterior root stimulation under voltage clamp mode, and observation of excitatory postsynaptic currents (e EPSCs) and spontaneous activity of antiepileptic drug lufenamide on SG neurons in spinal dorsal horn mediated by medium-diameter A delta fibers and small-diameter C fibers METHODS: In the same experiment 2, the spinal cord sections of SD rats were prepared, and SG neurons were selected. The clamping voltage was - 70 m V, and constant voltage stimulation was given to the posterior roots from small to large. Fifteen curves of excitatory postsynaptic currents (e EPSCs) were recorded before administration, and then, on average, were used as controls. The same method was recorded 1 minute later, and the recorded cells were classified according to the electrophysiological specificity of different fiber transduction reported in the literature. (1) To observe the effect of lufenamide on E EPSCs mediated by A Delta fiber. (2) To observe the effect of lufenamide on E EPSCs mediated by C fiber. (3) To observe the effect of lufenamide on spinal cord dorsal part. Results: (1) Single perfusion with different concentrations of lufenamide had little effect on Adelta fiber-mediated EPSCs (P 0.05, paired T-T e s t). (2) Lufenamide significantly inhibited the peak of C-fiber-mediated EPSCs (P 0.01, paired T-T e s t). (3) Lufenamide perfusion could significantly inhibit the development of SG neurons EPSCs. Radiation frequency (P 0.001, paired t-test), but no significant effect on its amplitude (P 0.05, paired t-test). Conclusion: 1. Single intraperitoneal injection of different concentrations of lufenamide can effectively alleviate chronic pain, the analgesic effect reached a peak 1 hour after administration and has a concentration-dependent. 2. Lufenamide can significantly inhibit SG nerves in spinal dorsal horn. The inhibitory effect was eluted within 10 minutes after administration. 3. Different concentrations of lufenamide had little effect on the peak value of e-EPSCs produced by single synaptic SG neurons innervated by Adelta fibers, which was not statistically significant compared with the control group. Continuous perfusion of lufenamide for 1 minute also significantly decreased the frequency of s EPSCs in SG neurons, but did not affect the amplitude of s EPSCs.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R614

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