亚低温预处理对谷氨酸诱导原代大鼠皮质神经细胞损伤的保护作用

The protective effect of mild hypothermia pretreatment against injury to primary cultured cortical neurons induced of rat by glutamate

目的 观察亚低温预处理对体外环境下培养大鼠大脑皮质细胞发生类缺血/再灌注（I/R）损伤的保护作用;比较亚低温单用或联合药物预处理的保护作用.方法 选择出生24 h内SD大鼠的大脑皮质细胞,体外培养至第3天,用阿糖胞苷（2.5 mg/L）培养基换液1次;培养6d后随机分为空白对照组、谷氨酸损伤组（加入含200 μmol/L谷氨酸的无血清培养基作用0.5 h）、亚低温预处理组（谷氨酸损伤前1d于33.5℃低温下培养24 h）、亚低温联合依达拉奉预处理组和亚低温联合丙泊酚预处理组（谷氨酸损伤前1d分别更换为100 μmol/L依达拉奉培养基和3 mg/L丙泊酚培养基后,再低温培养24 h）.各组更换正常培养基24 h后检测细胞存活率[四甲基偶氮唑盐（MTT）比色法]、细胞凋亡率、c-fos蛋白表达,苏木素-伊红（HE）染色后显微镜下观察细胞形态改变,铀-铅染色后透射电镜下观察细胞超微结构改变.结果 谷氨酸损伤组细胞存活率明显低于空白对照组[（0.20±0.02）％比（0.97±0.03）％,P＜0.01],细胞凋亡率和c-fos蛋白表达均明显高于空白对照组[细胞凋亡率：（9.85±0.76）％比（0.94±0.20）％,c-fos（ng/L）：6.96±0.75比1.65±0.59,均P＜0.01].亚低温预处理可明显逆转谷氨酸损伤细胞的存活率、凋亡率及c-fos水平,且以亚低温联合丙泊酚预处理作用最为明显[存活率：（0.80±0.04）％比（0.20±0.02）％,凋亡率：（2.26±0.54）％比（9.85±0.76）％,c-fos（ng/L）：2.98±0.46比6.96±0.75,均P＜0.01].显微镜下观察细胞形态发现,空白对照组大部分细胞形态大致正常;谷氨酸损伤组较多细胞质呈深红色,核呈蓝黑色,同缩状态,轴突萎缩明显,个别细胞突起断裂,细胞数量锐减;各预处理组细胞数量减少不明显,可见少量凋亡细胞.透射电镜下观察细胞超微结构发现,空白对照组神经细胞超微结构无明显改变;谷氨酸损伤组细胞质内细胞器数量明显减少,线粒体严重肿胀、嵴消失,呈空泡化或固缩,粗面内质网严重扩张,核膜局部出现囊样改变、甚至溶解断裂,染色质浓缩、凝集在核膜周边;各预处理组病理改变较谷氨酸损伤组明显减轻.结论 亚低温预处理能降低体外培养谷氨酸对大鼠大脑皮质神经细胞的损伤,且亚低温联合丙泊酚预处理组的保护作用更好.

Objective To investigate the effect of mild hypothermia preconditioning against ischemia/ reperfusion （I/R） injury of cultured primary cortical rats neurons,and to compare the protective effect of mild hypothermia only and with its combination with drugs.Methods Cortical neurons of neonatal Sprague-Dawley （SD） rat within 24 hours after birth were harvested and cultured in vitro.On the 3rd day,the cells were cultured in a medium containing 2.5 mg/L cytosine arabinoside to inhibit the growth of glial cells and fibroblast.Having cultured for 6 days they were randomly divided into blank control group,glutamate damaged group （cultured with 200 μmol/L glutamate for 0.5 hour after washing）,mild hypothermia preconditioning group （cultured under 33.5 ℃ for 24 hours before injury induced by glutamate）,mild hypothermia combining with edaravone preconditioning group,and the hypothermia combining with propofol preconditioning group （medium containing 100 μmol/L edaravone and 3 mg/L propofol）.They were cultured under 33.5 ℃ for 24 hours before injury induced by glutamate.After 24 hours of culturing in various medium,apoptosis ratio was observed by flow cytometry.Cell surviving rate was determined with methylthiazolete trazolium （MTT）,c-fos protein expression was assayed,and morphologic change of cells with hematoxylin-eosin （HE） staining under the microscope,and uhrastructure changes were observed after uranyl acetate and lead citrate staining under transmission electron microscope.Results The apoptosis ratio and c-fos protein in glutamate damaged group were significantly higher than those in blank control group [apoptosis ratio：（9.85 ± 0.76）％ vs.（0.94 ± 0.20）％,c-fos （ng/L）：6.96 ± 0.75 vs.1.65 ± 0.59,both P＜0.01],the cell surviving rate was significantly lower than that in blank control group [（0.20 ± 0.02）％ vs.（0.97 ± 0.03）％,P＜0.01].Mild hypothermia preconditioning reversed surviving rate,apoptosis ratio and c-fos protein,and the effect of hypothermia combining with propofol preconditioning was obviously better [cell surviving rate：（0.80 ± 0.04）％ vs.（0.20 ± 0.02）％,apoptosis ratio：（2.26 ± 0.54）％ vs.（9.85 ± 0.76）％,c-fos （ng/L）：2.98 ± 0.46 vs.6.96 ± 0.75,all P＜0.01].The morphology of cortical neurons in blank control group was normal.Most of the cells in glutamate damaged group showed bluish black cytoplasm with pyknic nuclei,with crimpled axons and of them were fractured,and cell number was obviously decreased.In each pre-conditional groups,decrease in cell number was unconspicuous,and only a few cells showed apoptosis.Under transmission electron microscope,it was found that cell membrane,mitochondria and rough endoplasmic reticulum were intact in blank control group,but with reduction in organelles,severely swollen mitochondria,even with formation of vacuole or pyknosis,serious dilation of rough endoplasmic reticulum,with loss of cristac with loss of vacuoles or pyknosis,and marked dilatation of intemal reticular endoplasm,and loss of cristac with vacuolation and chromatin were observed under electron microscope in glutamate damaged group.Compared with the glutamate damaged group,these pathologic changes were markedly alleviated in protected groups.Conclusions Mild hypothermia preconditioning can inhibit glutamate-induced injury to cortical neurons.The protective effect of mild hypothermia combined with propofol is better.