母孕期慢性应激与NMDA诱导的幼鼠痉挛模型的关系

摘要

目的 探讨母孕期慢性应激与子代N-甲基-D-天门冬氨酸受体(NMDA)诱导的痉挛发作的关系.方法 孕鼠分为母孕期慢性应激组(PS组n=6只)和对照组(PC组n=5只),应用母孕期强迫游泳,水温4℃,5 min,建立母孕期慢性应激模型;生后13 d幼鼠,腹腔注射NMDA 7 mg/kg建立幼鼠痉挛模型,观察幼鼠痉挛样发作次数的变化;应用HE染色方法和扫描电镜观察神经细胞大体形态和细胞线粒体超微结构的改变,应用免疫组织化学方法检测幼鼠脑内海马结构、大脑皮层、下丘脑和脑干部脊髓核团神经元NMDA受体表达,比较PS组和PC组幼鼠NMDA受体阳性细胞的差异.结果 PS组幼鼠NMDA致(癎)评分高于PC组(7.84±1.01比5.90±1.12,P=0.000).PS组幼鼠与PC组幼鼠脑切片HE染色均有细胞水肿.电镜下,PS组幼鼠较PC组幼鼠海马和脑干处神经细胞线粒体破坏增多,线粒体直径PS组为(692.9±119.0)μm,而PC组海马和脑于处神经细胞线粒体直径为(817.1±210)μm.免疫组织化学染色显示:NR1、NR2A、NR2B抗体均主要表达在细胞膜,细胞质和细胞核也有表达.PS组幼鼠NR1、NR2B阳性细胞在海马、皮层、下丘脑及脑干较PC组表达量均高,NR1两组比较P均<0.05;NR2B两组比较P均<0.01.PS组幼鼠NR2A阳性细胞数在海马处较PC组增高(P=0.006),在皮层、下丘脑室旁核和脑干脊核处两组差异无统计学意义(P均>0.05).结论 母孕期慢性应激可损伤子代幼鼠脑内神经细胞的线粒体,从而改变细胞的能景代谢,同时增加神经细胞NR1、NR2A、NR2B的表达,增加子代幼鼠痉挛的发生率.%Objective To explore the relationship between the stress during pregnancy and the NMDA-induced spasm. Methods The pregnant rats were divided into two groups: pregnant control group(PC group)and pregnatal stress group(PS group). The rats in PS group were forced to swim in cold water of 4℃ for five minutes. Pups of the two groups were injected NMDA 7mg/kg intraperitoneally on postnatal day 13 to induced infantile spasm-like attacks. Then the clinical behavior of pups of the two groups were observed and recorded. The ultrastructure of mitochondria was detected with scanning electron and appearance of neurons was observed with HE staining. NMDA receptor and its subuniforms (NR2A, NR2B) in hippocampus, cerebral cortex, hypothalamus and raphe nucleus of brainstem were estimated by immunohistochemistry. Results The average score of pups in PS group is higher than in PC group(7.84 ±1.01 vs 5.90 ± 1.12). There were swelling neurons and no cytonecrosis in hippocampuss of both two groups. The uhrastructure of mitochondria in hippocampuss and raphe nucleus of brainstem showed great changes in pups of PS group as membrane in mitochondria of pups in PS group. NR1, NR2A and NR2B distributed mainly on the membrane and also in cytoplasm and cellular nucleus. The NR1 expressions in hippocampus, cerebral cortex, hypothalamus and raphe nucleus in pups of PS group were elevated than those of PC group and the P value is 0.001, 0.009, 0.015, 0.001 respectively. The NR2B expressions in hippocampus, cerebral cortex hypothalamus and raphe nucleus in pups of PS group were elevated than those of PC group and the P value is 0.004, 0.002, 0.003, 0.002 separately. NR2A expressed more in hippocampus in pups of PS group than that in PC group,but there was no difference in cerebral cortex, hypothalamus and raphe nucleus between two groups and the P value is separately 0.006, 0.067, 0.7, 0.5. Conclusions The mitochondria of neurons in rat pup were hurt by maternal severe stress daring pregnancy, which made the energy metabolism of neurons changed. Maternal stress can also elevate the expression of NR1, NR2A and NR2B in cerebral cortex, brainstem and hippocampus and increase the morbidity of infantile spasm-like attacks.