星形胶质细胞酸敏感离子通道1a在症状性癫痫发生机制中的作用研究

Symptomatic epilepsy is often resistant to antiepileptic drugs and its underlying mechanisms still remain unknown. Previous studies suggest that brain inflammation and astrocytic activation are the most common pathological charactors involved in the development of symptomatic epilepsy following various brain insults. Recently, we found that expression of ASIC1a was increased in astrocytes in epileptogenic focus of patients with epilepsy. Activation of ASIC1a by the acidosis accompanied by inflammation can lead to influx of Ca2+ in astrocytes, subsequently enhance astrocytic release of glutamate, and finally contribute to the occurrence of epilepsy. Based on these facts, we hypothesize that increased expression of ASIC1a in activated astrocytes following brain insults leads to influx of Ca2+ , then influx of Ca2+ enhances astrocytic release of glutamate which contributes to spontaneous recurrent seizures. To test our hypothesis, we will observe the temporal and spatial patterns of ASIC1a's astrocytic expression in symptomatic epilepsy models in rats and ASIC1a-/- mice. Then, intracellular concentration of Ca2+ in astrocyte and extracellular concentration of glutamate will be investigated in vitro after upregulated or downregulated expession of ASIC1a in astrocytes. Finally, we will assess the effects of upregulated or downregulated expession of ASIC1a on neural network excitability and epileptogenesis. Our study will provide a new approach to treatment and prevention of symptomatic epilepsy.

症状性癫痫临床治疗棘手，发生机制不清。各种脑损伤致症状性癫痫形成过程中脑内共同的病理改变是反应性星形胶质细胞（AST）增生和组织炎症。新近我们在癫痫患者病灶中发现AST异常表达钙通透性的酸敏感离子通道1a（ASIC1a），ASIC1a能被炎症所伴随的组织酸化激活引起钙离子内流，而AST胞内钙浓度升高引起胞外谷氨酸增加已被证实参与了癫痫的发生。因此我们假设：脑损伤后AST上ASIC1a的异常表达和激活升高胞内钙离子浓度，促进AST释放谷氨酸而参与癫痫发生。为此，我们拟应用经典的慢性癫痫模型结合ASIC1a-/-小鼠，首先观察慢性癫痫形成过程中海马AST上ASIC1a的时空表达特征，进一步在离体层次调控AST上ASIC1a的表达，观察其对AST胞内钙浓度以及胞外谷氨酸浓度的影响；最后观察干预AST上ASIC1a通路对神经网络兴奋性及癫痫发生的影响。本项目的完成将为症状性癫痫的防治提供新的靶点。

症状性癫痫患者常发生于各种脑损伤后，其临床疗效和预后都相对较差，发病机制仍不清楚，目前已知各种脑损伤后共同的病理改变为组织炎症反应和反应性星形胶质细胞增生。我们根据前期研究发现癫痫患者病灶中异常增生的星形胶质细胞高表达ASIC1a，提出假设：脑损伤后异常高表达的星形胶质细胞酸敏感离子通道ASIC1a，受到炎症所伴随的组织酸化激活，引起钙离子的内流，进而引发星形胶质细胞谷氨酸的大量释放，参与癫痫的发生。为了验证这一假说，我们首先观察了ASIC1a的时空表达特征；然后在离体水平通过调控ASIC1a表达观察其对星形胶质细胞内钙离子浓度的影响；最后构建针对星形胶质细胞ASIC1a腺相关病毒，同时借助ASIC1a敲除的转基因动物，在体靶向干预海马星形胶质细胞ASIC1a的表达观察对癫痫发生的影响。研究发现：1、颞叶癫痫患者及锂-匹鲁卡品所诱导的癫痫小鼠模型海马星形胶质细胞明显活化，伴ASIC1a表达显著增高；2、脂多糖LPS刺激体外培养的星形胶质细胞，ASIC1a的表达及上膜明显增多，且可以在外周酸化诱导下引起胞内钙离子含量升高；3、降低野生型小鼠星形胶质细胞上ASIC1a表达可以减少癫痫慢性期自发性发作频率，而恢复ASIC1a敲除小鼠星形胶质细胞上ASIC1a的表达可以增加癫痫慢性期自发性发作频率。因此，本项目证实了星形胶质细胞ASIC1a在癫痫发生中的关键作用，为症状性癫痫的防治提供了新的靶点。