抑制颞叶癫痫慢性期白细胞-内皮粘附作用促进海马神经构筑的内源性修复研究

Developing new strategies for prevention of epilepsy is urgently needed, i.e.reconstructing hippocampal neural architecture by endogenous neurogenesis in chronic stage of the disease. By driving inflammation in epileptic brain, leukocyte-endothelial adhesion interaction may play an important role in creating a detrimental environment for shrunken hippocampal neurogenesis in the chronic stage of temporal lobe epilepsy (TLE). Hippocampal neurogenesis in this stage, however, may be useful for hippocampal reconstruction. We hence hypothesize that inhibiting leukocyte-endothelial adhesion interaction in the chronic stage of TLE will be potentially beneficial for augmenting hippocampal neurogenesis, reconstructing neural architecture of hippocampus and hence alleviating seizures. Inhibiting leukocyte-endothelial adhesion molecules （ICAM-1 and VCAM-1） with blocking antibodies in the chronic stage of pilocarpine induced mouse model of epilepsy, the project will: 1) examine the basal proliferation rate, survival, migration, differentiation and functional intergration of neural stem cell in subgranular zone (SGZ) to explore the effect of inhibition of leukocyte-endothelial adhesion interaction on neurogenesis of hippocampus; 2) study functional integration of newly born cells, forming extension to pyramidal cells in the CA3 area of hippocampus, the mossy fiber sprouting and synaptic connection in CA3 /granular layer by morphologic methods, to demonstrate that inhibition of leukocyte-endothelial adhesion interaction could promote reconstructing hippocampal neural architecture in the chronic stage of TLE. 3) clarify the effect of inhibiting leukocyte-endothelial adhesion interaction in the chronic stage of TLE on spontaneous recurrent seizures (SRSs) by EEG + video monitoring. For demonstrating the effect is derived from newly born cells, the dentate neurogenesis will be blocked by Methylazoxymethanol Acetate as control. The project will shed lights on the future treatment of epilepsy, especially on endogenous stem cell-based strategies.

白细胞-内皮粘附作用通过介导脑内炎性反应而减弱颞叶癫痫（TLE）慢性期海马神经再生，而促进海马神经再生在TLE慢性期具有修复海马神经构筑的潜能。我们假设：抑制TLE慢性期白细胞-内皮粘附可以促进海马神经再生、修复海马神经构筑并因此减轻自发性反复癫痫发作（SRSs）。课题采用pilocarpine诱导的癫痫模型，在小鼠TLE慢性期应用细胞间粘附分子（ICAM-1）和血管粘附分子（VCAM-1）抗体而阻断白细胞-内皮粘附反应：①BrdU标记海马齿状回颗粒下层（SGZ）神经干细胞，采用形态学方法研究对SGZ神经干细胞增殖、存活、迁移、分化和功能整合的影响，明确对海马神经再生的作用；②海马CA3区注射神经逆行示踪剂CTB，采用形态学方法研究对新生细胞向CA3区的投射、CA3区突触量、苔藓纤维芽发和海马神经元数量的影响，明确对海马神经构筑修复的作用；③EEG+视频监测方法研究在TLE慢性期抑制白细胞-内皮粘附通过海马神经再生对SRSs的影响。课题的顺利完成将为进一步的相关机制研究奠定理论基础，并将有助于建立内源性神经干细胞治疗TLE的新策略。

颞叶癫痫（Temporal lobe epilepsy, TLE）慢性期海马神经再生活动明显下降，但新生细胞仍可正常迁移到颗粒细胞层，形成顶树突并功能性整合到海马局部神经网路中。脑内炎症反应是阻碍TLE慢性期海马神经再生的重要因素。环氧化酶-2（COX-2）是介导脑内炎症反应的重要路径。本项目主要研究了抑制COX-2能否增强TLE慢性期海马细胞新生活动、修复海马神经构筑并改善疾病转归。为此，课题采用Pilocarpine诱导的小鼠癫痫疾病模型，在TLE慢性期应用BrdU标记海马新生细胞，采用CA3区注射CTB进行逆行神经束路追踪、C-fos激活表达等方法将结构和功能整合的海马新生细胞可视化，研究COX-2抑制剂Aspirin对TLE慢性期海马颗粒细胞下层（subgranular zone, SGZ）细胞增殖、新生细胞存活、向神经元方向的分化、向CA3区的投射以及新生细胞的功能整合；应用视频监测和Morris 水迷宫等行为学实验研究在TLE慢性期应用Aspirin抑制COX-2对小鼠自发反复性癫痫发作（spontaneous recurrent Seizures, SRSs）频次和学习记忆障碍的改善作用。取得的重要研究发现是：1）TLE慢性期应用Aspirin可以有效减少小鼠海马COX-2的表达；2）TLE慢性期应用Aspirin可以有效增加迁移到颗粒细胞层并分化为成熟神经元的新生细胞数量；3）TLE慢性期应用Aspirin可以有效增加向海马CA3锥体细胞形成投射的新生细胞数量；4）TLE慢性期应用Aspirin可以有效增加功能性整合到海马神经网路中的新生细胞数量；5）Aspirin改善TLE慢性期小鼠海马神经再生的作用主要是通过增强SGZ神经祖细胞增值率和新生细胞的存活能力实现的；6）小鼠TLE慢性期应用Aspirin可以有效减轻SRSs频次，并可以有效改善TLE慢性期小鼠空间记忆障碍。