【作者】 张伟；

【导师】 陈力学；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 背景:慢性偏头痛(Chronic migraine,CM)是一种致残性疾病,其主要的发病机制是中枢敏化。CM相关的认知和记忆障碍是其致残因素之一,对患者生活工作造成极大影响。多巴胺受体广泛分布于中枢神经系统并调节多种生理或病理性活动,在众多病理性疼痛模型中展现出巨大治疗潜力;在精神分裂或帕金森病中,多巴胺受体可显著改善其相关的认知和精神障碍。因此,本研究旨在探讨CM中多巴胺受体的变化并探究多巴胺受体对CM及其相关的记忆障碍的影响。方法:(1)通过硬脑膜滴注炎性汤建立CM大鼠模型。(2)通过检测大鼠足底和眶周痛阈评价CM模型;通过水迷宫行为学测试评估CM大鼠学习记忆能力。(3)使用Western blot、q-PCR检测TNC和海马部位的DRD1和DRD2表达变化。(4)侧脑室注射DRD2激动剂(quinpirole)和DRD2抑制剂(sulpiride)、Glu A1抑制剂(NASPM)、PI3K抑制剂(LY294002)以及PI3K激动剂(740YP)后通过痛阈检测以评估DRD2、Glu A1和PI3K通路对CM痛敏的影响。(5)通过Western blot检测磷酸化ERK和PSD95蛋白表达以及高尔基染色可视化TNC部位树突棘以评价DRD2、Glu A1和PI3K通路对CM中枢敏化的影响。(6)通过体外培养神经元并使用钙离子探针研究DRD2和PI3K对神经元钙内流的影响。(6)腹腔注射DRD1激动剂(SKF38393)和DRD2激动剂(quinpirole)通过水迷宫实验观察DRD1和DRD2对CM大鼠学习记忆的影响。(7)使用Western blot检测PSD95和p-NR2B蛋白表达,使用Golgi-Cox染色和透射电镜观察树突和突触结构以评价DRD1和DRD2对海马内突触可塑性的影响。结果:(1)炎性汤持续刺激使大鼠产生慢性偏头痛样特征,并损害大鼠学习和记忆能力。(2)CM大鼠TNC内DRD2表达下调;海马内DRD1和DRD2表达下调。(3)DRD2激动作用改善CM大鼠中枢敏化、抑制膜上Glu A1表达以及PI3K通路;DRD2抑制作用加剧CM大鼠中枢敏化、促进膜上Glu A1表达以及PI3K通路。(4)抑制Glu A1和PI3K通路缓解CM大鼠中枢敏化。(5)激活PI3K通路逆转DRD2的激动作用,使大鼠再次出现中枢敏化特征。(6)DRD2激动作用和PI3K抑制作用抑制TNC树突棘异常增高,并在体外减轻神经元钙离子内流。(7)DRD1和DRD2激动剂均改善了CM大鼠学习记忆能力。(8)激动DRD1和DRD2改善了CM大鼠内受损的突触可塑性。结论:在TNC部位DRD2通过PI3K通路调节突触后膜上Glu A1表达进而调节CM大鼠中枢敏化;在海马部位DRD1和DRD2通过挽救受损的突触可塑性改善CM大鼠的学习记忆障碍。更多还原

【Abstract】 Background: Chronic migraine(CM)is a disabling disease whose main pathogenesis is central sensitization,and the cognitive and memory deficits associated with CM are one of the disabling factors that have a significant impact on patients’ life and work.Dopamine receptors are widely distributed in the central nervous system and regulate a variety of physiological or pathological activities,showing great therapeutic potential in numerous models of pathological pain;in schizophrenia or Parkinson’s disease,dopamine receptors significantly improve the cognitive and psychiatric impairments.Therefore,the aim of this study was to investigate the changes of dopamine receptors in CM and to explore the effects of dopamine receptors on CM and its associated memory impairment.Methods:(1)CM rat models were established by dural drip injection of inflammatory soup.(2)The CM model was evaluated by detecting the plantar and periorbital pain thresholds of rats;the learning memory ability of CM rats was assessed by Morris water maze behavioral test.(3)the expression of DRD1 and DRD2 in TNC and hippocampal were detected using Western blot and q-PCR.(4)Lateral ventricular injection of DRD2agonist(quinpirole),DRD2 inhibitor(sulpiride),Glu A1 inhibitor(NASPM),PI3 K inhibitor(LY294002)and PI3 K agonist(740YP)was followed by pain thresholds assay to assess the effects of DRD2,Glu A1 and PI3K pathway on central sensitization in CM rats.(5)Using Western blot to detect the phosphorylated ERK and PSD95 protein expression and using Golgi-cox staining to visualize the dendritic spines in TNC to evaluate the effects of DRD2,Glu A1 and PI3 K pathways on central sensitization in CM.(6)The effects of DRD2 and PI3 K on neuronal calcium inward flow were studied by using calcium probes in neurons cultured in vitro.(6)Intraperitoneal injection of DRD1 agonist(SKF38393)and DRD2 agonist(quinpirole)was used to observe the effects of DRD1 and DRD2 on learning memory in CM rats through the Morris water maze experiments.(7)Using Western blot to detect the PSD95 and p-NR2 B protein expression and using Golgi-cox staining and transmission electron microscopy to observe the synaptic structures to evaluate the effects of DRD1 and DRD2 on synaptic plasticity in the hippocampus in CM rats.Results:(1)Continuous stimulation of inflammatory soup produced chronic migraine-like features in rats and impaired their learning and memory abilities.(2)DRD2 expression was downregulated in TNC in CM rats;DRD1 and DRD2 expression was downregulated in the hippocampus in CM rats.(3)DRD2 agonism improved central sensitization,inhibited synaptic Glu A1 expression and PI3 K pathway in CM rats;DRD2 inhibition exacerbated central sensitization,promoted synaptic Glu A1 expression and PI3 K pathway in CM rats.(4)Inhibition of Glu A1 and PI3 K pathways alleviated central sensitization in CM rats.(5)Activation of the PI3 K pathway reversed the agonistic effect of DRD2,causing the re-emergence of central sensitization in rats.(6)DRD2 agonism and PI3 K inhibition inhibited the abnormal increase of dendritic spines in TNC and attenuated neuronal calcium inward flow in vitro.(7)Both DRD1 and DRD2 agonism improved the learning memory ability of CM rats.(8)DRD1 and DRD2 improved the impaired synaptic plasticity in the hippocampus in CM rats.Conclusion: DRD2 regulated central sensitization by mediating synaptic membrane Glu A1 via the PI3 K pathway in TNC in CM rats;DRD1 and DRD2 improved learning and memory by rescuing the impaired synaptic plasticity in the hippocampus in CM rats.更多还原