低频率电刺激海马下托对癫痫灶点转移的作用及与NKCC1和KCC2的相关研究

Secondary epileptogenesis, i.e, the positive transfer of epileptic mirror focus is closely related to refractory epilepsy. The early effective intervention of positive transfer is of great significance for the prevention and treatment of refractory epilepsy. Our previous studies revealed that 1Hz low frequency stimulation (LFS) of certain brain areas can inhibit epileptogenesis with a narrow therapeutic window (several to tens of seconds) in mode- and timing- dependent manners. However, the stimulation at the subiculum showed a wider therapeutic window (< 2h) in inhibiting epileptogenesis and showed a potential to inhibit the formation of mirror focus in the contralateral amygdala in amygdala kindling model. These data indicated that the subiculum might be an ideal stimulating target for preventing positive transfer. The latest pilot study in our lab found that 1Hz LFS may inhibit the upregulation of sodium-potassium-chloride co-transporter (NKCC1). This implies that it is likely that 1Hz LFS of the subiculum can enhance the inhibitory effect of GABA through modulating the expression of chloride co-transporters. Therefore, with the help of epileptic focus transfer models both in vivo and in vitro as well as techniques such as biochemistry, behavioral observation, siRNA interference, microPET and electrophysiology, this study aims to investigate effects of LFS at the subiculum on positive transfer of epileptic focus when applied with various parameters and modes and to recommend the most optimal approach. Moreover, this study also aims to observe roles of NKCC1 and KCC2 as well as the function of GABAa receptors in the process of positive transfer and the relation of LFS at the subculum with their modulation. Our study will elucidate the effects of LFS at the subculum on positive transfer of epileptic focus and its potential mechanisms, providing experimental data for understanding the occurrence of and developing preventive drugs for refractory epilepsy.

癫痫灶点转移与难治性癫痫的发生密切相关，早期有效抑制灶点转移具有重要意义。我们的前期研究发现1Hz电刺激特定脑区可抑制癫痫发生，但时间窗效应明显（几秒到几十秒），而刺激海马下托有较宽的时间窗（< 2h），并对杏仁核点燃引起的镜灶形成有抑制作用，提示海马下托是一个潜在的抑制灶点转移的刺激靶点。此外，课题组预实验还发现1Hz电刺激海马下托可抑制氯离子转运体NKCC1上调，提示其可能是通过调节氯离子转运体功能而增强GABA的抑制性作用。因此，本课题拟在在体和离体癫痫灶点转移模型，采用siRNA干扰、microPET、电生理学等技术，研究不同刺激参数和模式电刺激海马下托对灶点转移的作用和最优的刺激方式，并进一步观察LFS海马下托与NKCC1和KCC2及GABAA受体功能调控的关系。本研究可阐明低频率电刺激海马下托对癫痫灶点转移的作用及可能机制，为深入认识难治性癫痫的发生和开发防治药物提供实验依据。

难治性癫痫是约占癫痫的三分之一，是临床上最为棘手的疾病之一。癫痫灶点的发生和镜灶转移是与难治性癫痫密切相关的两个重要病理现象，对此进行早期有效干预对于防治难治性癫痫具有重要意义。本研究采用多种癫痫模型，系统研究了低频率电刺激（LFS）海马下托对癫痫灶点形成和镜灶转移的作用和可能机制。结果发现，LFS而不是高频率电刺激海马下托既可抑制杏仁核电点燃诱发的癫痫灶点形成，也可抑制癫痫大发作，并对灶点转移至对侧有抑制作用。在匹鲁卡品诱导的具有对称性灶点的难治性癫痫模型，LFS单侧海马下托可减少自发性癫痫的发作，提示一侧海马下托的LFS处理可抑制镜灶内神经元异常兴奋。对于耐苯妥英钠的难治性癫痫和耐拉莫三嗪的难治性癫痫，LFS海马下托也能显著抑制癫痫大发作，提示LFS海马下托具有治疗难治性癫痫的潜力。此外，我们还发现，在杏仁核完全点燃已经发生镜灶转移的动物，海马神经元KCC2的表达量和上膜量均减少，LFS海马下托可抑制这种减少，提示LFS海马下托可能通过调节氯离子转运体在神经元上的表达和分布而增强GABA的抑制性作用。为进一步寻找调节癫痫灶点发生和转移的可能靶点，我们通过在体电生理和病毒学追踪技术，发现中脑黑质网状部与癫痫灶点的形成和镜灶转移密切相关。利用光遗传学和化学遗传学方法，发现特异性激活或抑制该部位的GABA能神经元对癫痫放电的扩散和镜灶形成具有调控作用。我们的结果表明海马下托在难治性癫痫的灶点形成和和镜灶转移中发挥关键作用，LFS海马下托对癫痫灶点的形成和镜灶转移可能具有治疗作用。在此基础上，我们还发现了中脑黑质网状部对海马癫痫灶点的远距离调控作用。本研究发现海马下托和黑质网状部是潜在的抑制灶点转移的刺激靶点，也为深入认识难治性癫痫的发生和开发防治药物提供实验依据。