锂-匹鲁卡品致痫后大鼠齿状回颗粒细胞下层神经前体细胞增殖活性的变化

Changes of the proliferative activities of neural precursors in the lower layer of dentate gyrus granular cells in rats after seizures induced by lithium-pilocarpine

目的:观察锂-匹鲁卡品致痫后大鼠齿状回颗粒细胞下层神经前体细胞增殖活性的变化,探讨自体神经前体细胞在颞叶癫痫病理进程中的潜在作用。方法:实验于2004-08/12在华中科技大学同济医学院附属同济医院神经内科实验室进行。取健康成年SD雄性大鼠64只,随机分为致痫组和对照组2组,每组32只。①给药:致痫组采用锂(3mmol/kg)-匹鲁卡品(50mg/kg)腹腔注射以诱导大鼠急性癫痫模型,持续抽搐达30min后,给予阿托品(1mg/kg)腹腔注射,30min后或大鼠抽搐濒危时再给予地西泮(10mg/kg),水合氯醛(5mL/kg)联合腹腔注射;对照组以生理盐水(4mL/kg,4mL/kg)取代氯化锂和匹鲁卡品,其余用药步骤及方法同致痫组。每组又分给药后3,7,14和28d4个时间点,每个时间点8只。②标本制备:两组大鼠于相应时间点前1d以增殖性细胞的标记物5-溴脱氧尿苷50mg/kg腹腔注射给药3次,间隔4h1次。24h后麻醉状态下处死取材。③观察指标:利用Nissle染色观察海马区域神经元的丢失情况,免疫组化方法观察齿状回颗粒细胞下层5-溴脱氧尿苷阳性细胞数目的变化,评估自体神经前体细胞增殖情况。结果:经补充后64只大鼠进入结果分析。①各致痫组大鼠齿状回门区、CA1和CA3区可见不同程度的内氏小体减少或者消失。②齿状回颗粒细胞下层5-溴脱氧尿苷阳性细胞数:致痫组致痫后即逐渐上升,第14天达到高峰,第28天已开始下降(F=655.61,P<0.0001)。致痫组致痫后7,14和28d高于对照组[(31±5),(95±5),(18±3)个/mm2;(10±2),(11±1),(11±1)个/mm2,P<0.0001]。结论:急性痫性发作可造成脑海马神经元脱失,并能促进大鼠海马齿状回颗粒细胞下层神经前体细胞的明显增殖,后者在颞叶癫痫的病理进程中具有意义。

AIM： To study the change of the proliferation activities of the neural precursor in lower layer of dentate gyrus granular cells after seizures induced by lithium-pilocarpine in rats and discuss the potential role of the self-neural precursor in the pathological course of the temporal lobe epileptics. METHODS： The experiment was taken in the Laboratory of Department of Neurology, Tongji Affiliated Hospital, Tongji Medical College, Huazhong University of Science ＆ Technology from August to December 2004. Sixtyfour healthy adult SD rats were selected and assigned randomly into 2 groups, seizures group and control group with 32 rats in each group.① Administration： The rats in seizures group were given lithium （3 mmol/kg） and pilocarpine （50 mg/kg） to induce acute epilepsy models injected intoabdominal cavity. After lasting tic for 30 minutes, atropine （1 mg/kg） was given injected into abdominal cavity and after 30 minutes or when the tic was dangerous diazepam （10 mg/kg） the rats was associated injected with chloral hydrate （5 mL/kg） through abdominal cavity; The rats in control group were given saline （4 ml/kg, 4 ml/kg） instead of lithium chloride and pilocarpine, and the procedure and method of the other drugs was the same as that in seizures group and randomized into 4 time points in every group 3, 7, 14 and 28 days after administration with 8 rats in each group. ② The preparation of sample： The rats in the two groups were treated with 5 bromium deoxyuridine 50 mg/kg, the marker of proliferation cells, through abdominal cavity for administration 3 times and once every 5 hours one day before the corresponding time point. After 24 hours under drugged state the rats were killed to gain materials. ③ Observation index： The losing condition of neuron in hippocampus area was observed with Nissle staining. The change of number of positive cells of 5-bromium deoxyuridine in dentate gyrus gramular cell lower layer was observed with immunohistochemistry technique to evaluate the proliferation condition of self-neural precursor. RESULTS： After compensation 64 rats were involved in the result analysis. ① Negri bodies of dentate gyrus area, CAI and CA3 area in different degree decreased of disappeared in rats of seizures group.② The number of positive ceils of 5-bromium deoxyuridine in dentate gyrus gramular cell lower layer： It was rising gradually after seizures in seizures group, reached the peak at the 144 day and decreased at the 28^th day （F =655.61, P〈 0.000 1 ）. It was higher in seizures group after seizures at 7, 14 and 28 day than that in control group [（31±5）, （95±5）, （18±3） per mm^2; （10±2）, （11±1）, （11±1） pertain2, P〈0.000 1]. CONCLUSION： Acute seizures can induce hippocampus neuron of brain to doff, and accelerate the significant proliferation of neural precursor in dentate gyrus gramular ceil lower layer in hippocampus in-rats. The latter has significance in the pathological course of temporal lobe epilepsy.