【作者】 张乐；

【导师】 张孟元；

【作者基本信息】 山东大学 ， 麻醉学， 2017， 硕士

【摘要】 研究背景神经病理性痛是最常见的慢性痛之一,常表现为不依赖于外周伤害性刺激的自发性痛,而神经病理性痛相关厌恶情绪反应如焦虑、恐惧等对患者的危害往往比痛本身更大,因此,我们应当更加重视痛相关厌恶情绪的临床治疗。前扣带皮层吻侧部(rACC)是介导痛情绪形成的重要脑区,NMDA受体NR2B亚基是痛情绪产生过程中的关键因子。神经损伤能够显著上调NR2B的表达,但是,当脑rACC局部给予NR2B特异性受体拮抗剂,其表达的上调能够被完全阻断,同时痛情绪反应也有所减轻。BDNF,一种活动依赖性的神经营养因子,广泛分布于中枢神经系统,在LTP、突触可塑性改变、学习记忆以及慢性痛的产生和维持等过程中起着关键性作用。最新研究发现,大鼠足底注射CFA,可以诱导ACC内BDNF的表达上调和神经兴奋性增强,并产生持续性痛觉过敏。而局部注射TrkB受体拮抗剂,阻断BDNF信号通路,能够显著降低神经兴奋性,同时减轻冷刺激诱发的痛觉过敏和被动回避行为。此外,有证据表明,BDNF/TrkB-NR2B信号通路是脊髓中枢敏化及痛觉过敏形成的重要机制之一。但是,脑rACC中的BDNF相关信号通路是否参与神经病理性痛相关厌恶情绪的形成以及参与该过程的确切分子机制还尚不清楚。因此,本研究采用外周神经损伤引起的神经病理性痛大鼠模型,并在该基础上探讨rACC神经元BDNF参与痛情绪形成的分子机制。研究目的本研究拟通过建立大鼠保留性坐骨神经损伤(SNI)引起的神经病理性痛模型,探讨脑rACC神经元BDNF在痛相关厌恶情绪形成过程中的作用和机制。研究方法与结果1.内源性BDNF参与神经病理性痛相关厌恶情绪的形成方法将大鼠随机分组。手术组(保留性坐骨神经损伤,即SNI模型)、假手术组(行假手术)、空白对照组(无任何处理),分别于术前1天,术后第1、3、7、10和14天检测各组大鼠术侧后爪的机械痛阈,用后肢缩爪阈值(PWT)来表示。其中行SNI手术的大鼠再分为CTX-B组(脑rACC注射CTX-B)和CTX-B-P组(脑PFC注射CTX-B),术后第7天,行可乐定诱导的条件位置偏爱(CPP)训练,检测痛情绪。处死大鼠取rACC脑组织,采用免疫组化技术检测BDNF蛋白的表达,采用Western blot技术检测其高度特异性受体TrkB的表达。结果与对照组相比,手术组大鼠PWT显著降低,同时产生了明显的位置偏爱。而CPP训练前手术组大鼠rACC(而非PFC)脑区局部注射TrkB受体拮抗剂CTX-B,能够阻断CPP的形成。此外,免疫组化和Western blot结果显示,手术组大鼠脑rACC神经元BDNF蛋白及其高度特异性受体TrkB的表达较对照组显著增高。2.外源性BDNF通过调控NR2B受体参与痛厌恶情绪的形成方法将大鼠随机分组。Naive组(无处理)、BDNF组(脑rACC注射BDNF)、DMSO 组(脑 rACC 注射 DMSO)、CTX-B 组(脑 rACC 注射 BDNF 和 CTX-B)、Ifenprodil 组(脑 rACC 注射 BDNF 和 Ifenprodil)、BDNF-P(脑 PFC 注射 BDNF)组。各组大鼠行条件位置回避(CPA)训练,检测痛情绪。行为学训练完成后,处死大鼠取rACC脑组织,采用Western blot技术检测NR2B受体的表达,采用免疫荧光技术检测两者在脑rACC神经元的分布以及是否存在共表达。结果与DMSO组相比,正常大鼠rACC(而非PFC)脑区注射外源性BDNF能够产生明显的位置回避现象,而CPA训练前脑rACC局部给予TrkB或NR2B受体拮抗剂均能够阻断BDNF诱导的CPA的形成。此外,BDNF组大鼠脑rACC中NR2B的表达较对照组明显增加,而CTX-B能够减弱NR2B表达的上调。同时,免疫荧光结果显示,TrkB和NR2B受体在rACC神经元存在共表达。结论rACC神经元BDNF/TrkB信号通路通过激活NR2B受体参与神经病理性痛相关厌恶情绪的形成。本研究在动物实验中证明了抑制脑rACC神经元BDNF及其相关受体能够显著缓解神经病理性痛相关厌恶情绪反应,该研究可能为临床慢性痛患者的全方位治疗以及痛情绪相关的研究提供新的理论指导。更多还原

【Abstract】 BackgroundNeuropathic pain,as one of the most common types of chronic pain,always behaves in a spontaneous manner(a continuous or paroxysmal pain that is not related to an external noxious stimulus).There has been considerable evidence suggesting that patients with chronic pain suffer from much more affective disturbances than pain itself,such as anxiety,fear and inferiority.Thus,increased attention should be paid to the clinical treatment of pain affection.The rostral anterior cingulate cortex(rACC)is an important brain area involved in the formation of emotional pain.NMDA receptor NR2B subunits in the rACC play a key role during the process of pain-related aversion.Nerve injury significantly increases the expression of NR2B,but the administration of NR2B antagonist into the rACC completely blocks the upregulation and relieves the pain affect.Brain-derived neurotrophic factor(BDNF)is commonly regarded to be an activity-dependent neuronal modulator in central nerve system that plays an important role in synaptic plasticity,learning-memory and chronic pain.lately,a new study found increased BDNF expression in the ACC of rats with inflammation,while local injection of a TrkB receptor antagonist mostly alleviated neuronal hyperexcitability,cold stimulation-induced hyperalgesia and prevented passive avoidance behavior.Moreover,it was reported that the BDNF/TrkB-mediated signaling pathway in the spinal cord promoted the development of central sensitization and hyperalgesia,and that this process was dependent upon the activation of dorsal horn NR2B receptors.However,whether and how BDNF in the rACC contributes to neuropathic pain-related aversion is still unclear.Thus,the present study was aim to explore the mechanisms of BDNF in pain-related aversion formation in the rat model of neuropathic pain.ObjectiveTo establish the rat model of partial sciatic nerve injury and investigate the function and mechanisms of BDNF on neuropathic pain-related aversion.Methods and Results1.Endogenous BDNF is involved in neuropathic pain-related aversionMethods Rats were randomly divided into groups.Operated group(spared nerve injury model,SNI),Sham group(sham operation),Naive(no treatment).The paw withdraw threshold(PWT)of all rats,was evaluated at 1 day before and also on 1,3,7,10 and 14 days after surgery.The SNI rats were randomly divided into CTX-B group(CTX-B+rACC)and CTX-B-P group(CTX-B+PFC).On day 7 after surgery,clonidine-induced conditioned place preference(CPP)was performed to evaluate pain affect.After the behavioral tests,rats were decapitated and the rACC was quickly removed for the determination of BDNF and TrkB expression by western blot and immunohistochemistry.Results Compared with control group,the PWT was significantly decreased in surgical rats,and CPP was only acquired in SNI rats.However,when TrkB receptor antagonist CTX-B was pre-injected into rACC(not PFC)before CPP test,the preference phenomenon disappeared.Moreover,the expression of BDNF and its high affinity receptor TrkB were significantly upregulated in the rACC of SNI rats.2.Exogenous BDNF contributes to pain affection via regulating NR2B receptorsMethods Rats were randomly divided into groups.Naive group(no treatment),BDNF group(BDNF+rACC),DMSO group(DMSO+rACC),CTX-B group(BDNF+CTX-B),Ifenprodil group(BDNF+Ifenprodil),BDNF-P group(BDNF+PFC).After surgery,conditioned place avoidance(CPA)test was conducted to evaluate pain-related aversion.After behavioral tests,rats were decapitated and the rACC was quickly removed for the determination of expression of TrkB and NR2B by western blot and immunofluorescence.Results Compared with DMSO group,administration of exogenous BNDF into the rACC(not PFC)of normal rats was sufficient to produce CPA.However,when the TrkB or NR2B receptor antagonist was pre-injected into rACC before test,the BDNF-CPA phenomenon was completely disappeared.Moreover,exogenous BDNF significantly increased the NR2B expression when compared with control group,but this upregulation was mostly alleviated by CTX-B.The immunofluorescence results showed that TrkB was co-localized with NR2B receptors in rACC neurons.ConclusionBDNF/TrkB signaling pathway in the rACC contributes to neuropathic pain-related aversion via activation of NR2B receptors.The results of this study are demonstrated in animal experiments,which confers a clinical significance that a new strategy targeting BDNF and relative receptors in the rACC might be useful for the prevention of pain-related emotional disturbance due to peripheral nerve injury.更多还原