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目的观察惊厥持续状态(SC)幼年大鼠海马前凋亡因子CHOP动态表达及神经细胞凋亡的变化,探讨依达拉奉(ED)对二者的影响。方法将195只SD幼年雄性大鼠随机分为9 g.L-1盐水对照组(NS组)、SC组和ED组,每组65只,各组均按SC后处死时间点分为2 h、12 h、24 h、48 h、72 h 5个亚组,每组13只。应用氯化锂-毛果芸香碱建立大鼠SC模型,用半定量反转录-PCR(RT-PCR)动态观察SC后大鼠海马CHOP mRNA的表达,采用免疫组织化学SABC法检测海马CA1区CHOP蛋白表达;并用HE染色观察海马CA1区病理改变,原位细胞凋亡检测法(TUNEL)观察海马CA1区神经元凋亡细胞数。结果 1.RT-PCR法:SC组幼年大鼠海马CHOP mRNA表达于2 h开始增加,于12 h达高峰,之后开始下降;与NS组各时间点比较差异均有统计学意义(Pa<0.01);与SC组比较,ED组(2 h,12 h,48 h)CHOP mRNA表达均明显下降(Pa<0.05,0.01)。2.免疫组织化学结果:SC组幼年大鼠海马CHOP蛋白表达于2 h开始增加,12 h明显增加,24 h达高峰;与NS组各时间点比较差异均有统计学意义(Pa<0.01);ED组各时间点均较SC组明显降低(Pa<0.01,0.05)。3.TUNEL结果:SC组海马CA1区TUNEL阳性细胞数于惊厥后24 h迅速增加,48 h达高峰(Pa<0.05,0.01);与NS组比较,12～72 h时间点组均明显高于NS组(Pa<0.01);与SC组比较,ED组12～72 h CA1区TUNEL阳性细胞数明显减少(P<0.05,0.01)。4.HE染色:SC后出现神经元变性及丢失,48 h组病理改变最显著,与TUNEL表达一致;而ED组病理改变减轻。结论 SC后早期幼年大鼠可能触发了内质网应激中CHOP介导的凋亡信号途径,从而引起了脑损伤;而ED可能通过下调其表达,从而缓解惊厥后脑损伤。
Objective To observe the changes of dynamic expression of CHOP and apoptosis in hippocampus of juvenile rats with convulsion persistent condition (SC), and to explore the effect of edaravone (ED) on both. Methods 195 male SD rats were randomly divided into 9 gL-1 saline control group (NS group), SC group and ED group, with 65 rats in each group. The rats were divided into 2 h, 12 h, 24 h, 48 h, 72 h 5 subgroups, 13 in each group. SCLC model was established by lithium chloride-pilocarpine. The expression of CHOP mRNA in hippocampus of rats after SC was observed by semi-quantitative reverse transcriptase-PCR (RT-PCR). The expression of CHOP protein in hippocampal CA1 region was detected by immunohistochemical SABC The pathological changes of hippocampal CA1 area were observed by HE staining. The number of apoptotic neurons in hippocampal CA1 area was observed by TUNEL. The expression of CHOP mRNA in hippocampus of young rats in SC group began to increase at 2 h and reached a peak at 12 h after RT-PCR, and then began to decrease. Compared with NS group, the difference was significant ). Compared with SC group, the expression of CHOP mRNA in ED group (2 h, 12 h, 48 h) decreased significantly (Pa <0.05, 0.01). Immunohistochemical results: CHOP protein expression in hippocampus of young rats in SC group increased at 2 h, increased significantly at 12 h and reached the peak at 24 h, compared with those in NS group at each time point (P <0.01) ; ED group was significantly lower than SC group at each time point (Pa <0.01,0.05). The results of TUNEL showed that the number of TUNEL positive cells in hippocampal CA1 area of SC group increased rapidly 24 h after SCI and peaked at 48 h (P <0.05, 0.01). Compared with NS group, the TUNEL positive cells in CA1 area of SC group were significantly higher than that of NS group NS group (P <0.01). Compared with SC group, the number of TUNEL positive cells in CA1 area of ED group decreased significantly at 12-72 h (P <0.05, 0.01). 4.HE staining: neuronal degeneration and loss after SC, 48 h group the most significant pathological changes, consistent with the expression of TUNEL; and ED group pathological changes reduced. Conclusion Early childhood SC rats may trigger CHOP-mediated apoptotic signal pathway in endoplasmic reticulum stress and cause brain injury. However, ED may reduce brain injury after seizures by down-regulating its expression.