海人藻酸建立内侧颞叶癫痫小鼠模型的研究

Establishment of a mouse model of medial temporal lobe epilepsy using kainic acid

目的 本研究采用立体定向手术在一侧海马注射海人藻酸(kainic acid, KA),建立内侧颞叶癫痫(medial temporal lobe epilepsy, MTLE)慢性模型,通过行为学、电生理学和病理学验证其有效性。方法 取健康的C57BL/6野生型雄鼠22只,随机分成对照组(n=6)和实验组即KA注射组(n=16)。对照组和实验组分别在海马CA3区进行微量注射生理盐水和KA,1周后再次进行立体定向手术,植入脑电记录电极。植入术后1周开始记录小鼠脑电活动,统计癫痫发作次数和持续时间。通过对小鼠的观察与记录,分别从行为学、电生理学和病理学方面验证慢性癫痫模型。结果 本实验对22只C57BL/6野生型雄鼠进行实验,对照组均无癫痫发作,而实验组存活的小鼠均出现癫痫发作。成模的小鼠在行为学上发生了凝视、咀嚼、头面部肌肉抽搐、肢体痉挛等慢性癫痫自发发作行为,有2只小鼠因手术死亡、4只小鼠在急性发作期死亡、10只小鼠模型成功建立;脑电图呈内侧颞叶癫痫样脑电图改变;在病理组织学方面,经免疫荧光染色发现:CA3区神经元丢失,星形胶质细胞大量增生,符合海马硬化特征性的病理改变。结论 通过使用KA单侧、单次颅内注射构建的模型具有耗时、易操作、易成型等多项优势,该模型展现出与人类MTLE相似的脑电图、行为与神经病理改变,有助于研究治疗颞叶癫痫的药物,是癫痫外科手术预后判断的理想动物模型。

Objective To optimize the simulation of the pathological characteristics and seizure behavior of medial temporal lobe epilepsy(MTLE),we aimed to establish a chronic epilepsy model of MTLE by unilateral,single hippocampal injection of kainic acid(KA)via stereotactic surgery,and to validate this epilepsy model in terms of behavior,electrophysiology,and pathology.Methods A total of 22 healthy C57BL/6 wild-type male mice were divided randomly into a control group(n=6)and an experimental group,which received KA injections(n=16).Both groups underwent microinjection of equal doses of saline or KA in the hippocampal CA3 area via stereotactic surgery.One week later,all mice were implanted with electrodes in the hippocampal CA3 area to facilitate electroencephalogram(EEG)recording.Seizure frequency and duration were analyzed statistically.The chronic epilepsy model was assessed in terms of behavior,electrophysiology,and pathology.Results No mice in the control group experienced seizures,while all surviving mice in the experimental group developed seizures.Adult model mice exhibited chronic spontaneous seizure behaviors,such as staring,chewing,head and facial muscle twitching,and limb spasms.Two mice died as a result of the surgery,four mice died during the acute seizure phase,and ten model mice were successfully established.EEG recordings showed epileptiform changes consistent with MTLE.Immunofluorescence staining revealed neuronal loss in the CA3 area and astrocytic changes,consistent with characteristic pathological changes of hippocampal sclerosis.Conclusions The model constructed by single unilateral intrahippocampal KA injection demonstrated several advantages such as being timeefficient,easy to operate,and reproducible.This model exhibited EEG,behavioral,and neuropathological changes similar to human MTLE,making it valuable for studying effective treatments for temporal lobe epilepsy and serving as an ideal animal model for predicting outcomes of epilepsy surgery.