神经干细胞移植于慢性癫痫鼠海马CA3区对其癫痫发作影响的研究

The Influence on the Spontaneous Seizures of Adult Chronic Epileptic Rat After Transplanting Neural Stem Cells to its Hippocampal CA3

目的研究神经干细胞(neural stem cells,NSCs)移植到慢性海人酸(kainic acid,KA)癫痫鼠海马CA3区后对大鼠癫痫发作的影响。方法用KA脑室注射制作慢性癫痫模型。将原代培养的、EGFP标记的NSCs移植到慢性癫痫鼠的海马CA3区。分别在移植后第2周、第4周、第8周和第12周连续进行7天观察大鼠癫痫发作频率和程度,在移植后第10周进行发作间期右侧海马深部脑电监测。然后取脑冰冻切片,在倒置荧光显微镜下直接观察移植细胞的存活和迁移,用免疫荧光染色观察移植细胞分化情况,Ti mm's染色观察海马齿状回异常苔状纤维发芽。结果移植后12周仍有大量移植细胞存活(65,045.00±881.72)。NSCs在移植区以胶质细胞分化为主,在齿状回和海马各区以神经元分化为主,γ-氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比率较高。NSCs移植后第4周移植组癫痫鼠的发作次数与对照组相比开始减少,Ti mm's染色计分和发作程度也有明显改善,两组发作间期脑电图尖、棘波在每个观察期的发放次数分别是3.83±4.96和27.16±21.08。结论将NSCs移植到慢性KA癫痫鼠的海马CA3区,移植细胞不仅能够长期存活、迁移到海马齿状回的各区,而且能够分化为神经元和神经胶质细胞,特别是GABA能神经元,同时还能够抑制齿状回颗粒细胞的苔状纤维发芽,从而减少癫痫发作次数,减轻癫痫发作程度。

Objective To investigate the influence on the spontaneous seizures of adult chronic epileptic rats after transplanting neural stem cells （NSCs） into its hippocampal CA3. Methyls The model of adult chronic epileptic rats were created by intracerebroventicular injection of kainic acid（KA）. Neural stem cells were primarily cultured and labeled with the enhanced green fluorescent protein （EGFP） and transplanted in hippocampal CA3 of adult chronic epileptic rats of KA--treated. During the 2,4,8 and 12 weeks after being grafted, spontaneous recurrent seizures were observed with Racine＇s seizure severity scale over 7 days respectively. The electroencephalogram of right hippocampal was detected with deep electrode at 10th week after being grafted. Survival and migration of the NSCs were detected directly under a phase contrast fluorescent microscope and the differentiation of the NSCs was determined by tmmunocytocnemlcat fluorescent staining. The abnormal mossy fiber sprouting （MFS） of hippocampus and denate gyrus were evaluate by Timm＇s staining. Results Large numbers of the grafted cells were existent at week 12 after being grafted（65045.00±881.72）. The NSCs differentiated mainly into astrocyte in the grafted area, while into neuron in hippocampus and dentate gyrus. GABAergic neurons differentiated from the grafted cells were detected in hilar and hippocampal CA3. At 4th week after transplantation of NSCs, the spontaneous recurrent seizure degree of grafted group was decreasing compare with the control group. Both the Racine＇s seizure severity scale and the grade of Timm＇s staining in grafted group were ameliorated. The frequency of discharge of epileptiform spikes of twe groups were 3.83±4. 96 and 27. 16±21.08 at 10th week after NSCs grafted respectively. Conclusion The NSCs after transplantation in hippocampal CA3 of adult chronic epileptic rats not only can migrate to dentate gyrus and all area of hippocampus and survive well for long time, but also can differentiate into neuron and astrocyte, especially GABA-immunoreactive interneurons and reduce MFS of hippocampus and denate gyrus. These findings suggest that NSCs could suppress spontaneous recurrent seizure in adult KA-treated chronic epileptic rats.