体外培养的乳鼠皮层神经元氧糖剥夺后轴突损伤的实验观察

Experimental observation of OGD-induced axonal injury in neonatal rat models with primary cultured cortical neurons

目的：建立乳鼠皮层神经元体外培养模型，观察氧糖剥夺处理对神经元轴突损伤的影响。方法取出生24 h 内的 SD 大鼠大脑皮层神经元，采用逐渐降低培养液中血清浓度和添加阿糖胞苷的方法培养、纯化神经元，观察其形态并鉴定。建立氧糖剥夺模型，随机分为正常对照组和氧糖剥夺处理组（2、4、6、8 h 各1组）。采用噻唑蓝比色法测定神经元活性，βⅢ-tubulin 免疫荧光染色观察轴突形态学变化。结果神经元培养3～5 d 后，胞体变得饱满并分出轴突和树突，突起之间逐渐交织成密集网络，神经元微管相关蛋白-2（MAP-2）标记阳性率＞95％。与正常对照组相比，随着氧糖剥夺时间的延长，轴突网络逐渐损伤、崩解，同时神经元活性呈梯度下降（P ＜0.01）。结论该研究成功建立了神经元体外原代培养及氧糖剥夺模型，并观察到在氧糖剥夺处理过程中神经元的轴突损伤及同时出现的神经元活性下降。

Objective The neonate rat models with primary cultured cortical neurons were established. The impact of oxygen and glucose deprivation （ OGD） treatment on neuronal axonal injury was observed. Methods Cerebral cortical neurons of neonatal Sprague-Dawley （SD） rats aged 〈 24 h were obtained. The neurons were cultured and purified by gradual reduction of serum concentration and supplement of cytarabine （Ara-c） into the culture medium. The neuronal morphology and identification were carried out.OGD models were established. Cultured cortical neurons were randomly divided into control and OGD treatment groups （2 ,4 ,6,8 h group）. Neuronal activity was determined by methylthiazolyldiphenyl-tetrazolium-bromide （ MTT） assay. Morphological changes of the axon were observed with p IH -tubulin immunofluorescence staining. Results After 3-5 d culture, the neuron cell body became plump presenting with axons and dendrites ,which gradually constructed a dense network. The positive rate of microtubule associated protein 2 （MAP-2） was 〉95%. Compared with the control group, the axonal network was gradually damaged and disrupted along with the prolonged OGD treatment. Meantime, the neuronal activity was decreased in a gradient manner （P 〈0. 01）. Conclusion In this study, OGD-treated neonate rat models with primary cultured cortical neurons were established. Neuronal axonal injury accompanied with decreased neuronal activity was observed after OGD treatment.