【作者】 吴浩；

【导师】 马正良；

【作者基本信息】 南京大学 ， 麻醉学， 2019， 硕士

【摘要】 工作目的:术前焦虑加重术后疼痛是临床常见现象,其机制尚未明确。心理应激会激活下丘脑-垂体-肾上腺(HPA)轴引起GCs合成释放增多。已有研究表明,GCs可导致神经可塑性发生改变。GABA能系统作为参与形成中枢抑制的最主要部分,GABA能系统活性异常可导致多种神经性病变,其中就包括疼痛。之前的研究发现,一种神经元PAS结构域蛋白(Npas4)可以促进GABA能突触的发生,并在维持平衡的神经网络活动中起着重要作用。本研究将通过建立大鼠术前焦虑模型,研究GCs信号在术前焦虑诱发术后痛觉过敏发展过程中的作用及相关机制,为临床上术前焦虑诱发的术后痛觉过敏的预防和治疗提供新的思路。研究方法:本实验使用单次延长应激法(SPS)构建大鼠术前焦虑模型,机械缩足阈值(PWMT)来评估大鼠跖底切口后的疼痛阈值,从而判断该模型是否建立成功。Elisa法检测大鼠血清CORT变化,Real-time荧光定量PCR和/或Western blotting法检测大鼠L3～L5段脊髓Npas4和GABA能标志物(GAD65、GAD67、GABAAα1和GABAAγ2)(提示GABA能系统活性)的表达,免疫荧光双标法观察Npas4在大鼠脊髓背角处的细胞定位。分别在大鼠SPS前30min时单次腹腔注射糖皮质激素受体(GR)拮抗剂RU486(30mg/kg)和盐皮质激素受体(MR)拮抗剂螺内酯(Spironolactone)(30mg/kg),痛行为学评估其镇痛效应。Real-time荧光定量PCR法检测大鼠L3～L5段脊髓Npas4和GABA能标志物(GAD65、GAD67、GABAAα1和GABAAγ2)的表达。分别在SPS前7d鞘内注射rAAV-Npas4/rAAV-Npas4 RNAi(滴度:4×1010 TU/ml,10μl)过表达/干扰 Npas4,痛行为学评估其镇痛效应,Real-time荧光定量 PCR法检测大鼠L3～L5段脊髓GABA能标志物(GAD65、GAD67、GABAAα1和GABAAγ2)的表达。提取大鼠原代脊髓神经元在体外培养,用外源性皮质酮(CORT)直接刺激,Real-time荧光定量PCR法检测大鼠L3～L5段脊髓Npas4和GABA能标志物(GAD65、GAD67、GABAAα1 和 GABAAγ2)的表达。结果:1.痛行为学检测发现SPS大鼠在跖底切口后有明显的PWMT减少,说明造模成功;2.Elisa检测发现造模后第1～14天血清CORT含量增加;3.real-time 荧光定量 PCR 检测发现造模后第 7 天和第 14 天两个时间 点大鼠 L3～L5 段脊髓Npas4和GABA能标志物减少(提示GABA能系统受损);4.Western blotting检测发现造模后第7～14天Npas4减少;5.SPS前30min时单次腹腔注射RU486或螺内酯,痛行为学检测发现RU486可明显减轻SPS大鼠的术后疼痛,real-time荧光定量PCR提示RU486改善GABA能标志物和Npas4的降低;6.过表达Npas4缓解了 SPS大鼠的术后痛觉过敏行为,以及恢复SPS引起的GABA能标志物的降低,而干扰Npas4则加重SPS大鼠的术后痛觉过敏行为,导致Npas4和GABA能标志物进一步降低;7.大鼠原代脊髓神经元在10μM的CORT刺激24h后,Npas4和GABA能标志物也同样是降低的,且可被RU486逆转。结论:术前焦虑导致脊髓GABA能系统受损,从而引起痛觉过敏,这是由于糖皮质激素诱导的Npas4下调所致。更多还原

【Abstract】 Background:Preoperative anxiety aggravating postoperative pain is a common clinical phenomenon,and its mechanism is not yet clear.Psychological stress can activate the hypothalamic-pituitary-adrenal(HPA)axis and cause increased synthesis of GCs,which has been shown to lead to changes in neural plasticity.Neuronal inhibition is primarily GABAergic mediated,and impaired GABAergic system lead to neurological disorders including pain.Previous studies have shown that a neuronal PAS domain protein(Npas4)promotes GABAergic synapses development and plays an important role in maintaining the balance of neural network activity.This study will establish a rat preoperative anxiety model to study the role of glucocorticoid signaling in development of postoperative hyperalgesia induced by postoperative anxiety,and provide new ideas for the prevention and treatment of postoperative analgesia induced by preoperative anxiety.Methods:In this study,a single prolonged stress(SPS)was used to construct a preoperative anxiety model in rats,and paw withdrawal mechanical threshold(PWMT)was used to evaluate the pain threshold after plantar incision in rats,so as to judge whether the model was successfully established.CORT changes in rat serum were detected by Elisa,both Npas4 and GABAergic markers(GAD65,GAD67,GABAA α1 and GABAAγ2)were detected by real-time quantitative PCR and/or Western blotting.The cellular localization of Npas4 in the dorsal horn of rat spinal cord was observed by immunofluorescence.Rats were intraperitoneally injected with either glucocorticoid receptor(GR)antagonist RU486(30mg/kg)or mineralocorticoid receptor(MR)antagonist Spironolactone(30mg/kg)at 30min before SPS,respectively,and the pain behavior was evaluated.Then Npas4 and GABAergic markers(GAD65,GAD67,GABAAal and GABAAγ2)in L3-L5 spinal cord of rats were detected by real-time quantitative PCR.Intrathecal injection of rAAV-Npas4/rAAV-Npas4 RNAi(Titer:4×1010 TU/mL,10μL)was performed 7 days before SPS,the pain behavior was evaluated and the expression of spinal GABAergic markers(GAD65,GAD67,GABAAal and GABAAγ2)was detected.Primary spinal cord neurons were directly stimulated with CORT in vitro,and Npas4 and GABAergic markers(GAD65,GAD67,GABAAal and GABAAγ2)were detected in L3-L5 spinal cord by real-time quantitative PCR.Results:(1)The pain behavior of SPS rats showed decreased PWMT after plantar incision,indicating that the modeling was successful.(2)The Elisa assay showed that serum CORT was significantly up-regulated for at least 14 days after modeling.(3)Real-time quantitative PCR results showed that Npas4 and GABAergic markers were decreased in L3-L5 spinal cord of rats at 2 time points of day 7 and day 14 after modeling,indicating an impairment of GABAergic system.(4)Western blotting showed that Npas4 decreased on the 7th to 14th day after modeling.(5)Intraperitoneal injection of RU486 30 minutes before SPS could significantly reduce postoperative pain and improve GABAergic markers and Npas4 in SPS rats.(6)Over-expressing Npas4 alleviated the postoperative pain of SPS rats,increased Npas4 and restored decreased GABAergic markers induced by SPS,while interfering with Npas4 aggravated postoperative pain of SPS rats,resulting in further decreased Npas4 and GABAergic markers.(7)After stimulation of rat primary spinal cord neurons with exogenous CORT in vitro,Npas4 and GABAergic markers were also down-regulated,and RU486 reversed that.Conclusions:These findings demonstrated that preoperative anxiety led to GABAergic system impairment in spinal cord and thus caused hyperalgesia due to glucocorticoidinduced down-regulation of Npas4.更多还原