低频率电刺激海马下托对难治性癫痫的作用及其机制研究

Low frequency stimulation（LFS） is emerging as a promising treatment of epilepsy. Our previous studies demonstrated that LFS at several brain area, such as the cerebellar fastigial nuclear and the entorhinal cortex, can control seizures. We have also reported that LFS at the subiculum(LFS-SUB) significantly inhibit temporal lobe epilepsy with wider therapeutic time window in amygdaloid-kindling and pilocarpine-induced recurrent epilepsy model. The results suggested that the SUB may be a potential and more suitable target for clinical LFS treatment. More interestingly, the latest results of our preliminary studies have indicated that LFS-SUB can effectively inhibit phenytoin(PHT)-resistant refractory epilepsy in rats (universal model of refractory epilepsy). Moreover, the response to PHT showed an improvement in PHT-resistant animals after long-term LFS treatment, which suggested that LFS-SUB may reverse the resistance to PHT in animals with refractory epilepsy. Further research with optogenetics technology has found that these effects may involve with the micro neural circuit of excitatory neurons in the hippocampal Cornu Ammonis 1 (CA1)-SUB region. Thereby, with techniques such as microelectrode recording, whole-cell patch, optogenetics and micro-PET as well as pharmacology method, this study expects to determine the therapeutic effect and charcteristic of LFS-SUB in two refractory epilepsy models in rats and mice and further elucidate whether the therapeutic effect of LFS is mainly through the underlying micro neural circuit of excitatory neurons and relative with molecular target like NMDA receptors et al. in the CA1-SUB area. The study may provide new therapeutic strategies and potential drug targets for the clinical treatment of refractory epilepsy.

低频率电刺激（low frequency stimulation，LFS）是较有应用前景的癫痫治疗新手段。课题组前期研究已发现LFS小脑顶核、内嗅皮层等靶点能有效抑制颞叶癫痫发作；我们还发现LFS海马下托（LFS-SUB）在杏仁核电点燃和匹鲁卡品癫痫模型中发挥明显抗癫痫作用，且具有治疗时间窗较宽的优点，提示SUB可能是一个潜在的临床应用LFS靶点。课题组初步的预实验发现LFS-SUB能有效控制大鼠耐苯妥英钠难治性癫痫发作，且长期使用能逆转其对苯妥英钠的耐药。利用光遗传学技术发现该作用可能与CA1-SUB兴奋性神经微环路有关。因此，本课题将通过多通道微电极记录、离体电生理、光遗传学、影像学、药理学等方法，进一步明确LFS-SUB抗难治性癫痫的作用及其作用特点；着重阐明LFS是否通过CA1-SUB兴奋性微环路起效，及其与NMDA受体的关系。本研究将为难治性癫痫的临床治疗提供新的潜在药物靶点。

癫痫的临床治疗目前仍然以药物控制发作为主，但有大约30%的病人对多种抗癫痫药物耐药，从而发展为难治性癫痫，其中颞叶癫痫病人发展为难治性癫痫的比例甚至更高。除手术外，难治性癫痫尚缺乏有效治疗手段，这已成为一个迫切需要解决的问题。低频率电刺激（low frequency stimulation, LFS）是较有应用前景的癫痫治疗新手段。课题组前期研究已发现LFS小脑顶核、内嗅皮层等靶点能有效抑制颞叶癫痫发作；我们还发现LFS海马下托（subiculum, SUB）在杏仁核电点燃和匹鲁卡品癫痫模型中发挥明显抗癫痫作用，且具有治疗时间窗较宽的优点，提示SUB可能是一个临床应用LFS的潜在靶点。因此，课题组利用大鼠耐苯妥英钠难治性癫痫模型和小鼠耐拉莫三嗪海马快速点燃模型主要在整体水平研究了LFS-SUB对难治性癫痫的作用，发现LFS-SUB能减少难治性癫痫动物的大发作几率、平均发作等级和大发作持续时长，延长大发作潜时，甚至还能逆转难治性癫痫动物的耐药。进一步结合脑电分析、光遗传学和微电极记录等技术，采用行为学、免疫组化、电生理等多种方法，初步明确了该作用与SUB区域锥体神经元之间的关系。结果表明CA1-SUB微环路在难治性癫痫发生过程中有重要作用，SUB兴奋性神经元的发放对苯妥英钠失去响应可能是难治性癫痫动物耐药的原因。通过LFS或光调控选择性抑制此类神经元发放则能明显逆转难治性癫痫动物的耐药，而蓝光特异性激活此类神经元则能促进非难治性癫痫动物向难治性癫痫转变。因此，本研究首次表明LFS-SUB对难治性癫痫可能有较好的直接抑制作用，并可能同时有逆转耐药的作用，这一作用的可能机制是抑制了SUB区域兴奋性神经元的发放。这可能更新了以往对SUB在难治性癫痫发生中所起作用的认识，为未来难治性癫痫的干预提供了潜在靶点和新的治疗策略。