【作者】 王娜娜；

【导师】 李治华；

【作者基本信息】 郑州大学 ， 人体解剖与组织胚胎学， 2023， 硕士

【摘要】 背景和目的神经病理性疼痛及其引起的焦虑抑郁一直以来是临床治疗的难题。由于疼痛和焦虑抑郁共患的复杂性,一直缺乏有效的治疗手段。近年来,前扣带皮层(Anterior Cingulate Cortex,ACC)在慢性疼痛和焦虑抑郁中的重要作用成为研究热点,疼痛和焦虑抑郁时ACC发生突触可塑性改变以及神经元兴奋性改变,其中ACC的谷氨酸能神经元在疼痛时被激活,而谷氨酸能神经元的标志物是Ca MKIIα,暗示调节ACC区Ca MKIIα+神经元的兴奋性可缓解疼痛,改善痛情绪,是治疗疼痛的新方向。载脂蛋白E受体2(Apolipoprotein E Receptor 2,ApoER2),在神经系统分布广泛,尤其在锥体神经元中,与成人大脑神经元的损伤修复和突触的形态、功能密切相关。神经系统中ApoER2发挥信号传导功能激活细胞内的磷酸化级联通路,促进谷氨酸N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)磷酸化,参与长时程增强。ACC的ApoER2是否通过改变ACC神经元的兴奋性参与疼痛及焦虑抑郁的调节还未见报道。本研究首先验证了ACC区特异性表达Ca MKIIα的谷氨酸能神经元兴奋导致慢性坐骨神经压迫(CCI)小鼠痛觉过敏和焦虑抑郁样行为。然后探索在神经元突触可塑性中起重要作用的ApoER2是否通能过其胞内结构域(Intracellular domain,AICD)促进N-甲基-D-天冬氨酸受体2B亚基(NR2B)的表达和激活,增强ACC区Ca MKIIα+神经元的兴奋性,导致CCI小鼠疼痛和焦虑抑郁样行为。方法1.利用Western Blot,RT-PCR和IF技术检测CCI后ApoER2的表达变化,利用Western Blot技术检测CCI后AICD、p-CREB、CREB、p-NR2B、NR2B表达量的变化;2.利用双光子钙成像技术检测神经元兴奋性的变化;3.利用si RNA下调ApoER2,检测p-CREB、CREB、p-NR2B、NR2B表达,神经元兴奋性和小鼠行为学的变化;4.利用化学遗传技术激活或抑制Ca MKIIα+神经元,检测小鼠行为学的变化;5.利用腺病毒过表达AICD,检测相关蛋白水平的变化及小鼠行为学的变化。结果1.CCI术后21 d出现痛觉过敏和焦虑抑郁的小鼠ACC的Ca MKIIα+神经元表达c-Fos增多,对同等刺激的钙信号增强,兴奋性升高。化学遗传抑制CCI小鼠ACC区Ca MKIIα+神经元兴奋性,缓解小鼠的痛觉过敏和焦虑抑郁样行为。化学遗传激活正常小鼠ACC区Ca MKIIα+神经元,引发小鼠痛觉过敏和焦虑抑郁样行为。2.CCI后ACC区ApoER2的m RNA和蛋白水平都增加,并与Ca MKIIα大量共标。si RNA降低CCI小鼠ApoER2水平后,Ca MKIIα+神经元对同等刺激钙信号减弱,神经元兴奋性降低,小鼠的痛觉过敏现象和焦虑抑郁样行为得到缓解。3.CCI后ACC区AICD增加,核内的AICD也增加。siRNA和DAPT降低AICD水平后,CCI后升高的p-CREB/CREB、NR2B和p-NR2B水平降低,CCI小鼠的痛觉过敏也有所缓解。在正常小鼠ACC区Ca MKIIα+神经元中选择性过表达AICD时,小鼠出现痛觉过敏和焦虑抑郁样行为,p-CREB/CREB、NR2B和p-NR2B含量都增加。结论ACC区的ApoER2通过其胞内结构域AICD,增强Ca MKIIα+神经元的兴奋性,增加NR2B的表达,导致CCI小鼠痛觉过敏和焦虑抑郁样行为。更多还原

【Abstract】 Background and objectiveNeuropathic pain and anxiety-depression it causes are a long-lasting persistent challenge for clinical treatment,due to the complexity of the coinstantaneous pain and anxiety-depression disorders.In recent years,the anterior cingulate cortex(ACC)has been verified to play an important role in pain and anxiety-depression.There are substantial changes of synaptic plasticity and neuronal excitability in the ACC in the process of pain and anxiety-depression.The excitatory glutamatergic neurons in the ACC are activated in pain and Ca MKIIαis a marker of the excitatory glutamatergic neurons,which suggests that both the sensory and emotion of pain can be modulated by altering the excitability of Ca MKIIαneurons in the ACC region.Apolipoprotein E Receptor 2(ApoER2),which is widely distributed in the nervous system,especially in pyramidal neurons,is closely associated with repair of injury and synaptic morphology and function in adult brain neurons.ApoER2 in the nervous system can function as a signaling pathway,activate intracellular phosphorylation cascade pathways,promote the phosphorylation of NMDAR,and participate in long-term potentiation.However,it remains unclear whether ApoER2 in the ACC is involved in the regulation of pain and anxiety-depression by altering the excitability of ACC neurons.In the present study,we first verified that excitation of glutamatergic neurons which specifically expressed Ca MKIIαin the ACC region led to nociceptive hypersensitivity and anxiety-depression-like behavior in chronic constriction injury(CCI)mice.Then we explored whether ApoER2,which plays an important role in neuronal synaptic plasticity,promotes the expression and activation of NR2B through its intracellular domain(AICD)and enhances the excitability of Ca MKIIαneurons in the ACC region,thus leading to neuropathic pain and anxiety-depression-like behavior in CCI mice.Methods1.The changes in ApoER2 expression after CCI were detected via Western Blot,RT-PCR and IF.Western-blot was used to detect the changes of AICD,p-CREB,CREB,p-NR2B and NR2B after CCI;2.Two-photon calcium imaging technique was used to detect the effect of CCI on neuronal excitability;3.ApoER2 was downregulated with si RNA to detect the alteration of the expression of p-CREB,CREB,p-NR2B,NR2B,neuronal excitability and mouse behavior;4.Chemical genetic techniques were used to activate or inhibit neurons,and then detected the change of mice behavior;5.AICD was overexpressed by viral transfection of ACC to detect changes of p-CREB,CREB,p-NR2B,NR2B protein and mice behavior.Results1.Ca MKIIαneurons in the ACC of mice that developed nociceptive hypersensitivity and anxiety-depression-like behavior in 21 days after CCI showed increased expression of c-Fos,enhanced calcium signaling,and elevated excitability.Chemical genetics inhibited Ca MKIIαneuronal excitability in the ACC region of CCI mice,which alleviated nociceptive hypersensitivity and anxiety-depression-like behavior in mice.Chemical genetics activated Ca MKIIα+neurons in the ACC region of normal mice,which induced nociceptive hypersensitivity and anxiety-depression-like behavior in mice.2.Both m RNA and protein expression levels of ApoER2 in the ACC region were increased after CCI.ApoER2 was extensively colocalized with Ca MKIIα.After reducing ApoER2 levels in CCI mice withsi RNA,Ca MKIIαneurons showed attenuated calcium signaling to equivalent stimuli,decreased neuronal excitability,and nociceptive hypersensitivity and anxiety-depression-like behaviors were alleviated.3.AICD was increased in the ACC region after CCI,and consistent results could also be seen in the nucleus.After the decreased level of AICD by si RNA and DAPT,the elevation of p-CREB/CREB,NR2B and p-NR2B caused by CCI were reversed,hyperalgesia caused by CCI were alleviated.When AICD was selectively overexpressed in Ca MKIIαneurons in the ACC region of normal mice,mice developed nociceptive hypersensitivity and anxiety-depression-like behavior,and the expression of p-CREB/CREB,NR2B and p-NR2B were increasedConclusionApoER2 in the ACCenhances the excitability of Ca MKIIαneurons and increases the expression of NR2B through AICD,leading to hyperalgesia and anxiety-depression-like behavior in CCI mice.更多还原