BDNF-TrkB通路对癫痫大鼠海马神经元突触可塑性的影响

Epilepsy is kind of brain functional disorder caused by sudden discharge of brain neurons, with high incidence and mortality. More and more focuses have been put on the relationship between neuronal plasticity and epilepsy mechanisms. Researches have confirmed that there is a close relationship between BDNF and epilepsy development, and BDNF will protect the neuronal function. Therefore, BDNF-TrkB pathway may be a new promising target in the regulation of plasticity of hippocampal neuronal synapse. Our research group has successfully established in vitro system to culture neuron and kainic kindling model and found that the BDNF level is higher in epileptic rat hippocampus compared with normal tissue. We have also found through the microscope that there is a high expression level of BDNF in perikaryon. Based on the fact above, we will block respectively the upstream and downstream pathway of BDNF-TrkB signaling by specific inhibitors, and overexpress BDNF. Then, the technology of behavior test, immunohistochemistry, molecular biology and electron microscope will be utilized to (1) observe morphological structure changes of hippocampal neuron, (2) test hippocampal synaptic plasticity marker and certain proteins changes in the BDNF-TrkB signaling pathway, (3) explore the relationship between BDNF-TrkB signaling pathway and plasticity of hippocampal neuronal synapse in epileptic rat, (4) uncover the effect of BDNF-TrkB pathway on plasticity of hippocampal neuronal synapse in epileptic rat, (5) provide important theoretical evidence for regulating neuronal plasticity and developing new epilepsy theropeutic targets.

癫痫是常见的脑功能障碍性疾病，病因和发病机理不明，至今缺乏有效的治疗手段。目前神经元可塑性与癫痫发病机制关系的研究备受重视。研究证明BDNF与癫痫的发生发展密切相关。因此，BDNF-TrkB通路对神经元突触可塑性的调控有望成为改善癫痫症状的新靶点。项目组在前期研究中已成功建立了体外培养神经元模型和海仁酸点燃癫痫大鼠模型，发现癫痫大鼠海马组织的BDNF表达升高，并观察到神经元内BDNF的高表达。据此，本项目用特异性抑制剂分别阻断BDNF-TrkB上、下游信号通路和BDNF过表达的方法，通过免疫组化、分子生物学、激光共聚焦显微镜以及电镜等技术观察海马神经元的形态结构变化，检测海马突触可塑性标志物和BDNF-TrkB通路相关蛋白水平及mRNA的动态变化，探索BDNF-TrkB通路与癫痫大鼠海马神经元突触可塑性改变之间的关系，为调控癫痫的神经元可塑性和开发新的癫痫治疗靶点提供重要的理论依据。

癫痫是常见的脑功能障碍性疾病，病因和发病机理不明，至今缺乏有效的治疗手段。目前神经元可塑性与癫痫发病机制关系的研究备受重视。研究证明BDNF与癫痫的发生发展密切相关。因此，BDNF-TrkB通路对神经元突触可塑性的调控有望成为改善癫痫症状的新靶点。项目组在前期研究中已成功建立了体外培养神经元模型和海仁酸点燃癫痫大鼠模型，发现癫痫大鼠海马组织的BDNF表达升高，并观察到神经元内BDNF的高表达。据此，本项目用特异性抑制剂阻断BDNF-TrkB信号通路的方法，通过免疫组化、分子生物学、激光共聚焦显微镜以及电镜等技术观察海马神经元的形态结构变化，检测神经元突触可塑性标志物和BDNF-TrkB通路相关蛋白水平及mRNA的动态变化，探索BDNF-TrkB通路与癫痫大鼠海马神经元突触可塑性改变之间的关系。结果显示，癫痫初期TrkB表达量急剧升高，随后BDNF表达也逐渐升高，BDNF和TrkB表达均高于正常对照组。随着病情发展BDNF反复出现先增加后减少的变化，而TrkB逐渐减少到正常水平以下。注射阻断剂之后BDNF量显著减少，海马受损伤后无明显变化，而TrkB受损伤后快速升高后迅速减少，由此说明阻断BDNF-TrkB信号通路，可抑制BDNF的表达，进而调控BDNF对损伤的保护作用。BDNF-TrkB通路被阻断后，PSD-95、MAP-2和SYP 表达量均减少，而MAP-2的表达量减少最为显著，由此说明BDNF与其特异性受体TrkB结合诱导PSD-95、MAP-2和SYP的表达，改变突触可塑性，影响癫痫发作过程。综上所述，BDNF-TrkB信号通路对突触可塑性关键物质PSD-95、MAP-2和SYP表达有重要的诱导作用，而这种作用可被K252a抑制。本实验的结果对癫痫的发生机制有重要意义，并对癫痫的临床治疗提供新的思路，为调控癫痫的神经元可塑性和开发新的癫痫治疗靶点提供了重要的理论依据。