目的:探讨在细胞水平建立可靠、简便易行的神经元机械性损伤模型及神经系统损伤的分子病理学.方法:将大鼠脑皮层神经元体外培养7d,通过光镜、电镜和免疫组织化学法鉴定培养的神经元.以微量移液器塑料枪头划割细胞,造成神经元机械性损伤.研究神经元机械性损伤后细胞形态学变化.检测细胞存活率和培养液乳酸脱氢酶活性变化,从病理生理学角度阐明神经元机械性损伤的效果.结果:神经元体外培养7d,光镜及电镜检测符合神经元特点.NF200免疫组织化学染色细胞阳性率达90％；机械性划割后可见神经元立即坏死和伤后6h的细胞凋亡,以及伤后72 h神经元损伤修复现象.神经元机械性损伤后细胞存活率下降,伤后30 min起,各损伤组较对照组细胞存活率均明显下降；伤后1～24 h,中、重型损伤组细胞存活率较轻型损伤组明显下降(P＜0.05).伤后30 min起,各损伤组较对照组乳酸脱氢酶活性明显升高；伤后12～24 h重型损伤组乳酸脱氢酶活性高于轻型损伤组(P＜0.05).结论:胎鼠脑皮层体外原代培养生成的神经元纯化率较高；划割损伤对神经元有确切的致伤作用,依据划割范围不同,可造成不同程度的神经元损伤.%Objective: Traumatically injured model of neurons in vitro can establish a foundation for molecular pathological mechanism research of central nervous system injury. In this study, we aim to form a novel, reliable and easy-conducted traumatically injured model of cortical neurons of rat in vitro. Methods: Cortical neurons were identified with microscope, electric microscope and immunohistochemistry after 7 days in vitro cultivation. Mechanical injury was manipulated with a plastic tip crossing onto the neuronal culture, which resulted in three kinds of injury according to the degree of insults. After that, morphological changes were observed. In addition, cellular viability and lactate dehydrogenase (LDH) concentration were detected to elucidate the mechanical injury effects from the pathphysiological aspect. Results: The neuronal nucleus was large and clear with round or elliptical outline. The nucleolus and the membrane of nucleus were easily found. The figures of neurons were various, and the cytoplasm was bright with many dendrites and axons striking out from the cellular body, which connected each other to form a complicated reticular formation. The chromatin was mainly euchromatin. Plenty of organelle including mitochondria and Golgi complex, and neuronal specific structure neurofilament and dendrite were also found. The percent of neu-rofilament 200 (NF200) positive neurons was more than 90%. Phenomena of necrosis and apoptosis were discovered 6 hours after mechanical injury and regeneration of injured neurons 72 hours after injury. The cellular viability in in-jured groups decreased significantly compared with that in the control group since 30 minutes after injury. The LDH concentration of injured groups increased significantly compared with that in the control group since 30 minutes after mechanical injury of neurons in vitro. Conclusion: It is confirmed that tip-caused injury can actually produce neuronal insults, and the degree of damage depended on the extension of tip-caused injury. This simple and effective traumati-cally injured model of neurons can simulate the pathological mechanism of traumatic brain injury, and is convenient for the study of morphology, molecular pathology and therapeutics after traumatic brain injury.
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