【作者】 肖智；

【导师】 阮怀珍；

【作者基本信息】 第三军医大学 ， 神经生物学， 2010， 博士

【摘要】 疼痛对人类健康危害极大,因此对疼痛机制的探索一直是临床医学和神经科学研究的热点和难点。慢性神经痛继发于外周或中枢神经损伤,表现有自发痛(spontaneous pain),痛觉过敏(hyperalgesia)以及触诱发痛(allodynia)等。细胞外的三磷酸腺苷(adenosine 5’-triphosphate,ATP)通过与细胞膜上嘌呤受体(purinoceptors)结合,促进疼痛在外周和中枢的传递,其中P2X3受体亚型是嘌呤受体中一个主要传递疼痛信息的受体。中脑导水管周围灰质(midbrain periaqueductal gray,PAG)在疼痛信息传递中处于承上启下的关键部位,是机体内源性疼痛调制系统(endogenous pain modulatory system)的一个重要组成部分,高位中枢神经系统对疼痛信息的调制往往都通过对PAG功能的调节而实现。在东方国家,电针(electroacupuncture, EA)广泛运用于慢性疼痛的临床治疗已有2000多年历史,疗效可靠,但是一直以来对电针镇痛机理尚不明确,在一定程度上阻碍了电针治疗的临床运用和推广。目的:(1)观察Sprague-Dawley(SD)大鼠在外周神经慢性结扎性损伤(chronic constriction injury,CCI)手术所引发慢性神经痛前后热痛阈、机械痛阈的变化和中脑导水管周围灰质外侧部(lateral midbrain periaqueductal gray,lPAG)中P2X3受体表达变化。(2)通过在慢性神经痛大鼠(以下称:CCI大鼠) lPAG微量注射P2X3受体的激动剂和特异性阻断剂,观察CCI大鼠热痛阈和机械痛阈的改变。(3)对CCI大鼠进行多次穴位电针治疗,观察电针治疗前后,CCI大鼠热痛阈和机械痛阈的改变以及lPAG中P2X3受体表达变化,探讨lPAG中P2X3受体表达与电针镇痛的关系,为临床电针治疗慢性神经痛提供新的理论依据。方法: (1)建立SD大鼠慢性右侧坐骨神经结扎性损伤模型,通过辐射热实验(radiant heat test)和von Frey纤维丝实验(von Frey filaments test)检测大鼠在神经损伤前后,建模侧后肢热痛阈(thermal withdrawal latency,TWL)和机械痛阈(mechanical withdrawal threshold,MWT)的变化,通过免疫组织化学技术观察lPAG中P2X3受体表达的变化以及通过免疫印迹技术观察lPAG中P2X3蛋白水平变化; (2)通过对CCI大鼠中脑lPAG微量注射P2X3受体的特异性阻断剂A-317491阻断lPAG中P2X3受体功能后,在lPAG微量注射ATP的类似物α,β-亚甲基ATP (α,β-methylene-ATP,α,β-meATP),观察CCI大鼠热痛阈和机械痛阈变化规律;运用脑片全细胞膜片钳技术,观察乳鼠lPAG神经元的α,β-meATP诱发放电和A-317491对α,β-meATP诱发放电的影响; (3)在CCI大鼠建模侧“足三里”和“三阴交”穴给予多次电针刺激,观察电针治疗前后大鼠热痛阈和机械痛阈以及lPAG中P2X3受体表达变化; (4)用针对P2X3基因的反义寡聚核苷酸(oligodeoxynucleotide,ODN)脑内微量注射降低lPAG中P2X3 mRNA表达后,观察对“足三里”穴位电针治疗镇痛作用的影响,以探讨电针治疗和lPAG中P2X3受体表达的关系。结果:(1)通过对SD大鼠疏松结扎右侧坐骨神经后成功复制出慢性神经痛动物模型,建模成功大鼠的热痛阈和机械痛阈均下降,CCI术后第14天热痛阈和机械痛阈下降至最低值,14天后热痛阈和机械痛阈均有小幅缓慢上升但与正常大鼠热痛阈和机械痛阈比较差异仍有统计学意义。(2)CCI大鼠中脑PAG中P2X3受体阳性表达与正常大鼠以及假手术大鼠比较有轻度上调,主要阳性表达部位在lPAG,同时lPAG中P2X3受体蛋白水平与正常大鼠和假手术大鼠比较也有轻度升高。(3)在lPAG微量注射ATP类似物α,β-meATP后,与注射前比较,CCI大鼠热痛阈和机械痛阈值有明显升高;当用P2X3受体的特异性阻断剂A-317491在lPAG微量注射进行预处理后,α,β-meATP的痛阈升高效果明显减弱,热痛阈和机械痛阈值升高幅度减少,有统计学意义。(4)α,β-meATP可使乳鼠lPAG神经元放电频率增加, P2X3受体特异性阻断剂A-317491可抑制此作用;(5)在CCI大鼠“足三里”和“三阴交”穴位给予多次电针刺激后,伴随P2X3受体在CCI大鼠lPAG表达上调,CCI大鼠痛阈明显升高;相反,对CCI大鼠进行“非穴位”电针,P2X3受体在lPAG表达无明显变化,CCI大鼠痛阈改变无统计学意义;(6)当用针对P2X3基因的反义寡聚核苷酸lPAG微量注射降低P2X3受体在lPAG中表达后,“足三里”穴位电针对CCI大鼠的镇痛效果明显下降。结论:(1)外周神经(坐骨神经)损伤可导致SD大鼠热痛阈和机械痛阈值下降, CCI大鼠lPAG的P2X3阳性表达上升;(2)lPAG微量注射α,β-meATP可引起CCI大鼠热痛阈和机械痛阈值的显著性升高,此作用可因微量注射P2X3受体的特异性阻断剂A-317491预处理而减弱;同时A-317491对lPAG神经元α,β-meATP诱发电流有抑制作用。(3)对CCI大鼠“足三里”穴位给予7次电针刺激,可引起CCI大鼠热痛阈和机械痛阈值的升高以及P2X3受体在lPAG表达的升高;对CCI大鼠“非穴位”进行7次电针,不能引起痛阈升高和P2X3受体在lPAG表达变化;(4)在lPAG多次微量注射针对P2X3基因的反义寡聚核苷酸后明显减弱“足三里”穴位电针对CCI大鼠的镇痛作用。综上所述,lPAG中的P2X3受体在脊髓上疼痛调节中起抑制性作用;“足三里”穴位电针刺激通过上调lPAG中P2X3受体表达加强机体内源性镇痛系统作用,缓解CCI大鼠神经痛症状。lPAG中P2X3受体介入了电针镇痛的脊髓上调节机制。更多还原

【Abstract】 The study on pain mechanisms is always a hot topic in clinic and neuroscience research fields. Neuropathic pain is known as a notorious devastating consequence of central or peripheral nerve injure which is characterized by a combination of spontaneous pain, hyperalgesia and allodynia. Increasing evidence has revealed that the extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain for thousands years in Eastern countries but the mechanisms underlying EA analgesia are still uncertain.Objective to⑴study the function of P2X3 receptors in the lateral midbrain periaqueductal gray (lPAG); the relationship between P2X3 receptor expression and neuropathic pain induced by a chronic constriction injure of the right sciatic nerve on Sprague-Dawley(SD) rats. By immunohistochemistry and Western blotting, we observe the P2X3 receptor protein level and expression location in the lPAG and further explore the effect of pretreatment with A-317491, a novel potent and selective antagonist of P2X3 and P2X2/3 receptors, on the analgesic effect of lPAG microinjection of alpha,beta-methylene ATP(α,β-meATP).⑵investigate whether repetitive administration of electroacupuncture (EA) on‘zusanli’and‘sanyinjiao’acupoints could reduce thermal and mechanical hyperalgesia in a rat neuropathic pain model and to explore the relationship between the antinociceptive effect of EA treatment and the expression of P2X3 receptor in the lPAG.Methods: The experiment was composed by four series:Series 1. Changes of pain thresholds of SD rats after establishment of rat CCI (chronic constriction injury) model. Study the expression changes of P2X3 receptor in the lPAG by immunohistochemical and immunoblotting analysis. Series 2. Observation of effect of pretreatment with A-317491 on the analgesic effect of lPAG microinjection ofα,β-meATP and the effect of A-317491 onα,β-meATP-induced current of the neonatal rat lPAG neurons. Series 3. Influence of EA treatments on the pain thresholds and investigation of change of P2X3 receptor expression in the lPAG posterior to EA or sham-EA treatment and explore the acupoint specificity of EA treatment on CCI rat either. Series 4. Analgesic effect changes of EA treatments in response to lPAG microinjection of P2X3 antisense ODN.Results:Part one⑴The inclined-plane test was used in all experimental rats before algesimetry test to study whether CCI operation, drugs or ODNs microinjection and EA treatment exerted certain adverse effects on rats motor function and proprioception, there was no significant difference on the average maximum angle among all experimental groups.⑵The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured before CCI operation on the right sciatic nerve and at days 2,4,7,10,12,14,18 and 21 respectively after CCI operation. In CCI group, the hind-paw TWL and MWT were decreased gradually and down to the lowest point on day 14 after CCI operation. In the normal and sham-CCI groups, from day 4 to day 21 after CCI operation, the values of TWL and MWT were quite stable.⑶Immunoblots from lPAG homogenates showed an immunopositive band of P2X3 receptors with molecular weight around 55 kDa both in normal, sham-CCI and CCI groups. There was no significant difference between normal and sham-CCI groups. In CCI group, sciatic nerve ligation caused an increase of P2X3 protein level in the lPAG.⑷In CCI groups, P2X3 receptor-specific immunoreactivity was mildly higher and a little more extensive compared with normal group or sham-CCI group at day 14 after CCI operation.Part two⑴Rats were randomly divided into two groups, the rats received intra-lPAG injection with sterile saline (0.3μl) or A-317491 (1.5nmol/0.3μl) once daily for 5 consecutive days. After pre-treatment, TWL and MWT showed no significant difference between above two groups. Microinjection of A-317491 alone cause no long-term effects on pain thresholds of CCI rats .⑵The saline and A-317491 group were then divided into two subgroups before microinjection with PBS orα,β-meATP. lPAG microinjection with vehicle (0.3μl PBS) induced no change of the thermal or mechanical nociceptive threshold. In saline group, lPAG microinjection ofα,β-meATP (30 nmol/0.3μl) significantly elevated nociceptive thresholds compared with beforeα,β-meATP microinjection. The antinociceptive effect ofα,β-meATP was attenuated significantly by A-317491 pretreatment.⑶Whole cell voltage-clamp recording was performed on neonatal rat brain slices to obtain the currents of the lPAG neurons. Our data show that A-317491 attenuated the frequency of theα,β-meATP-induced current in lPAG neurons.Part three⑴Multiple EA treatments had no significant effect on TWL and MWT values of rats in normal plus EA group. On the contrary, multiple EA treatments significantly increased TWL and MWT values of rats in CCI plus EA group.⑵The P2X3 receptor protein level in lPAG was detected after multiple EA treatments, which reached a statistically significant high level in CCI plus EA group but not in normal plus EA group.⑶The pain thresholds of CCI rats were elevated after multiple sham-EA treatments but still remained lower than rats in CCI plus EA group.⑷NA(non-acupoint)-EA treatments had no significant effect on pain thresholds and the P2X3 receptor protein level in the lPAG of CCI plus NA-EA group rats was not changed after NA-EA treatments.Part four⑴TWL and MWT were both increased after 7 times of EA treatment in the antisense ODN or mismatch ODN group rats compared with CCI rats without EA treatment.⑵The antinociceptive effect of EA treatment was attenuated significantly by P2X3 antisense ODN intra-lPAG injection.Conclusion: The expression change of P2X3 receptor in the lPAG and the elevated pain thresholds after intra-lPAG injection ofα,β-meATP indicate that P2X3 receptors in the lPAG play an inhibitory role in the pain modulatory and thus may be a central target for analgesics. EA produces a long lasting analgesic effect on neuropathic pain and increases expression of P2X3 receptors in the lPAG. The present study for the first time addressed the involvement of purinergic signaling system in EA analgesia on neuropathic pain in rat CCI model, as could deepen our understanding of the mechanism of EA analgesia and provide a rational basis for enhancing EA analgesic effect by potentiating the function of purinergic signaling system at supraspinal level.更多还原