大鼠急性局灶性脑挫裂伤后神经细胞线粒体膜电位变化及其意义

Changes in mitochondrial membrane potential of rat neurons following acute brain contusion and laceration injury

目的研究大鼠急性局灶性脑挫裂伤后神经细胞线粒体膜电位变化及其意义。方法70只SD大鼠采用随机数字表法分为假手术组（n=10）和脑损伤组，损伤组按伤后1、6、24、48、72和168h观察时间点分为6个亚组（n=10）。用Feeney氏自由落体撞击法制作重型颅脑损伤模型。利用激光扫描共聚焦显微镜观察损伤后不同时间荧光探剂JC-1标记的活体脑片细胞线粒体膜电位的动态变化，应用Hochest3342荧光染色和Tunel法观察神经细胞凋亡程度，透射电镜观察神经细胞超微结构。结果（1）与假手术组相比，损伤组脑皮质神经细胞线粒体膜电位在伤后1h即开始显著降低，24h降至最低，之后一直无恢复（P〈0．01）。（2）Hochest33342荧光染色发现神经细胞呈现凋亡的特征。（3）Tunel染色显示损伤组在1h可见少量阳性细胞，6h阳性细胞数明显增加，48h达峰值，与假手术组比较差异有统计学意义（P〈0．05），与线粒体膜电位下降时间比较延迟24h。（4）电镜发现脑损伤后6h神经细胞核发生染色质散在，核仁边集，细胞核核膜增宽等凋亡特征；24h出现核皱缩，核内染色质溶解。结论创伤性脑损伤早期（〈1h）神经细胞线粒体膜电位即下降，并引起神经细胞凋亡。

Objective To investigate the changes in mitochondrial membrane potential of the rat neurons after acute brain contusion and laceration injuries. Methods Seventy Sprague-Dawley （SD） rats were randomized into the sham injury group （n=10） and brain injury group. The brain injury group was divided into 6 subgroups （n=10） for observation at 1, 6, and 24 h and 2, 3, and 7 days after acute brain contusion and laceration injury, which was induced by impact of the brain with a free-falling object. In the brain slices labeled with fluorescent JC-1 staining, the changes in the mitochondrial membrane potential of the neurons were observed under confocal laser scanning microscopy at the indicated time points. The neuronal apoptosis was detected using Hochest33342 staining and TUNEL assay, and the number of apoptotic neurons was determined under optical microscope. The ultrastructural alterations of the neurons were observed with transmission electron microscopy. Results Compared with that of the sham injury group, the mitochondrial membrane potential of the neurons decreased significantly 1 h after the brain injury （P〈0.01）, reaching the lowest level at 24 h, and maintained the low level afterwards. Apoptotic neurons were identified in the brain slices with Hochest33342 staining. One hour after the brain injury, TUNEL assay revealed a small number of positive neurons, which increased significantly at 6 h （P〈0.01） and reached the highest level at 48 h. The alteration pattern of the number of the positive neurons showed a 24-h delay in comparison with that of the mitochondrial membrane potential after the brain trauma. Under electron microscope, the mitochondria of the neurons exhibited only slight swelling at 1 h, and obvious morphological changes of the mitochondria occurred 6 h after the brain trauma, accompanied by chromatin fragmentation, presence of marginal nuclei and broadened nuclear membrane; nuclear condensation and chromatolysis were observed in the neurons 24 h after thebrain trauma. Conclusion In rats with acute brain contusion and laceration injuries, the mitochondrial membrane potential of the neurons decreases early （〈1h） after the brain injury, which induces apoptosis of the neurons.