癫癎发作对幼年大鼠海马齿状回神经发生影响的研究

Neurogenesis of dentate granule cells following kainic acid induced seizures in immature rats

目的 探讨癫癎发作对幼鼠齿状回颗粒细胞神经发生的影响。方法 生后第15天Wistar大鼠80只,随机分为癫癎组50只.对照组30只。癫癎组以海藻酸致癫,对照组幼鼠以相同方法注射生理盐水,2组于注射后第5天经腹腔注射5溴脱氧尿苷(BrdU)。采用BrdU标记新生细胞,再以β微管蛋白Ⅲ(βⅢtubulin,TuJ1)、钙调蛋白D28k(CaBP)和胶质原纤维酸性蛋白(GFAP)双标免疫组织化学方法分别标记早期神经元、成熟颗粒细胞和胶质细胞,观察致癫幼鼠齿状回颗粒细胞的神经发生。另外,采用Timm染色对P15致惊幼鼠于致惊后1及2个月的苔藓纤维发芽情况进行观察。结果 BrdU注射后第7天和第21天,癫癎组幼鼠齿状回BrdU阳性细胞数明显高于各自对照组(第7天:244±15与190±10;第21天:218±19与133±12,P均<0.05);BrdU注射后第7天,癫癎组和对照组幼鼠分别约80.2％和78.7％的BrdU阳性细胞同时表达TuJ1(p>0.05);:BrdU注射后第21天,癫癎组和对照组幼鼠分别约60.2％和58.2％的BrdU阳性细胞同时表达CaBP(P>0.05):BrdU注射后第7天和第21天,两组幼鼠均约3％-5％的BrdU阳性细胞同时表达GFAP。P15致惊幼鼠在致惊后1及2个月均未发现内分子层及CA3区苔藓纤维的发芽现象。结论 癫癎发作可增加幼鼠齿状回颗粒细胞的神经发生。

Objective Data accumulated over the past years have led to widespread recognition that neurogenesis, the emergence of new neurons, persists in the hipppocampal dentate gyrus of the adult mammalian brain, and can be increased by seizures in multiple models. Also, aberrant reorganization of dentate granule cell axons, the mossy fiber sprouting, occurs in human temporal lobe epilepsy and rodent epilepsy models. However a number of studies suggest that the immature brain is less vulnerable to the morphologic alteration of hippocampus after seizures. The goal of this study was to determine whether the seizures can induce dentate granule cell neurogenesis and mossy fiber sprouting in the immaure rat Methods Seizures was elicited by unilateral microinfusion of kainic acid(KA,l μg) into the amygdula at postnatal day 15 ( P15 ) . Rat pups were given bromodeoxyuridine ( BrdU) intraperitonealy on day 5 after KA administration and killed 7 d or 21 d later. The brains were processed for BrdU mitotic labeling combined with double-label immunohistochemistry using neuron-specific, early differentiation marker TuJl ( (β Ⅲ tubulin) or granule-specific marker CaBP (calcium-binding protein calbindin D28k) as well as glia-specific marker GFAP(glial fibrillary acidic protein). Mossy fiber sprouting in intermolecular layer and CA3 subfield was assessed in Timm-stained sections both 1 month and 3 months after KA administration by using a rating scale and density measurement. Results The dentate BrdU-immunoreactive cells of the KA-treated rats increased significantly compared with those of control rats on day 7 and 21 after BrdU administration (7d: 244 ± 15 vs. 190 ± 10; 21d:218 ± 19 vs. 133 ± 12,P <0. 05). Approximately 80. 2% and 78. 7% of BrdU-labeled cells coexpressed TuJl in KA-treated rats and control rats on day 7 after BrdU respectively (P > 0.05). On 21d after BrdU, 60.2% and 58.2% of dentate BrdU-labeled cells coexpressd GaBP in KA-treated rats and control rats respectively (P>0. 05). GFAP colocalized with 3%-5% dentate BrdU-labeled cells in the rats of both groups on day 7 and 21 after BrdU. It was also demenstrated that status epilepticus at P15 did not result in any detectable mosssy fiber sprouting within the hippocampus both 1 month and 3 months after KA administration. Conclusions KA induced seizures ran increase granule cell neurogenesis in the immature rat. Most of newly appeared cells migrate from suhgranular proliferation zone (SGZ) into granule cell layer, the hilus as well as the molecular layer, and there they can differentiate into granule tieurons. These observations also indicate that there is an early developmental resistence to seizure-induced mossy fiber sprouting in the immature brain.