匹罗卡品诱发成年大鼠普遍癫痫与难治性癫痫海马神经发生的变化

背景：神经发生包括细胞增殖、迁移、分化和存活等，是人和多数哺乳动物部分脑区产生新生神经元的过程，主要分布在侧脑室下区和海马齿状回。癫痫可导致海马齿状回的神经发生改变，BrdU是目前公认最理想检测未成熟细胞增殖的标记物之一。 目的：观察匹罗卡品诱发成年大鼠癫痫后神经发生的特点，以及普通癫痫与难治性癫痫神经发生的差异。 设计、时间及地点：随机分组，细胞分子生物学实验，于2006-08/2007-06在华中科技大学同济医学院中心实验室及附属协和医院中心实验室完成。 材料：选用健康Sprague-Dawley雄性大鼠100只，随机分为2组，对照组8只，实验组92只，分为普通癫痫组，自发发作组，难治性癫痫组和非耐药组。匹罗卡品为武汉晶美公司产品，兔抗鼠BrdU抗体为美国sigma公司产品,辣根过氧化物酶标记的羊抗兔IgG为武汉博士德公司产品。 方法：对照组大鼠腹腔注射生理盐水；实验组腹腔注射匹罗卡品15mg/kg，最多注射4次，出现癫痫持续状态的大鼠注射水合氯醛终止发作。致癫大鼠癫痫发作终止后6h腹腔注射BrdU，观察自发发作情况。以脑电图、自发发作频率持续时间，无自发发作为普通癫痫组，有自发发作为自发发作组。行卡马西平灌胃2周并记录发作频率，对卡马西平治疗无效，发作频率减少<50%的为难治性癫痫组，治疗有效即发作频率减少>50%为非耐药组。普通癫痫组分别于注射后第1，2，3，7，14，21和28d取鼠脑海马部做冠状连续切片。非耐药组和难治性癫痫组的大鼠，再次行腹腔注射BrdU，每次为50mg/kg，连续注射4次，每次间隔2h，48h后取脑海马部制作石蜡切片。 主要观察指标：脑海马部切片行免疫组化染色，镜下观察不同时间点BrdU阳性细胞的分布、形态和数量及注射匹罗卡品后大鼠癫痫发作状况。 结果：匹罗卡品第1次注射后所有大鼠无癫痫发作，第2次注射发作16只，第3次注射发作42只，第4次注射后发作11只，14只大鼠4次注射仍无癫痫发作，9只大鼠癫痫持续状态后死亡，77只进入结果分析。神经发生主要位于海马颗粒细胞层和齿状回。与对照组比较，普通癫痫组BrdU阳性细胞明显增多（P<0.01），难治性癫痫组新生细胞较普通癫痫组明显减少（P<0.01），神经发生减少。癫痫后第2天BrdU 阳性细胞数目开始增加，14-15d达到高峰，1个月后回到初始水平。 结论：与普通癫痫相比，难治性癫痫可导致神经发生减少。

Proliferation changes in hippocampal neurons following pilocarpine-induced intractable epilepsy in adult rats

Epilepsy can lead to the changes in neurons residing in the dentate gyrus. The present study aimed to observe the cell dividing features following epilepsy in adult rat hippocampi, and to study difference in cell proliferation between adult rats with common epilepsy and intractable epilepsy. Adult, male, Sprague Dawley rats were randomly divided into control (n = 8, treatment with normal saline) and three experimental groups: common epilepsy (n = 33), intractable epilepsy (n = 11), and drug-responsive (n = 25). Pilocarpine (15 mg/kg) was intraperitoneally administered to establish epilepsy in the three experimental groups. Rats that developed epilepsy were treated with chloral hydrate. Rats that did not exhibit spontaneous seizures were enrolled in the common epilepsy group, and rats with spontaneous seizure were included in the spontaneous seizure group. At 6 hours after epileptic attack termination, rats were intraperitoneally injected with bromodeoxyuridine (BrdU; 50 mg/kg), an optimal marker for labeling cell proliferation in vivo, four times. Immunohistochemistry results at 48 hours after BrdU injection indicated that the number of BrdU-positive cells was the highest in the common epilepsy group, followed by the control group, and lastly the intractable group (P < 0.01). In addition, the number of BrdU-positive cells in the common epilepsy group was similar to the drug-responsive group. The present findings demonstrated that intractable epilepsy led to decreased hippocampal neurons in adult rats when compared to common epilepsy.