电磁脉冲对海马神经元的损伤及其对[Ca2+]i的影响

背景:电磁脉冲照射可影响实验大鼠的学习记忆功能,并造成大鼠海马组织损伤和超微结构改变。目的:观察电磁脉冲对离体培养的海马神经元的损伤效应及其对[Ca2 ]i的影响,以深入分析电磁脉冲致脑损伤的可能机制。设计:随机对照动物实验。单位:承德医学院病理教研室。材料:Wistar乳鼠若干只,雌雄各半。电磁脉冲辐射源:高场强电磁脉冲模拟源。方法:实验于2004-03/12分别在军事医学科学院和承德医学院完成。Wistar乳鼠若干只,麻醉下断头取脑,分离海马组织,调整细胞悬液浓度至5×108L-1接种。分组:①培养细胞分为对照组和照射组,于照射后即刻(0h)收集细胞进行形态学观察和胞内游离钙离子浓度测定;②另培养细胞分为对照组和照射后0h组和12h组,进行细胞凋亡和坏死率的测定。(培养细胞用量:每项检查每组1个培养瓶,重复3次。)电磁脉冲辐射条件为6×104V/m,脉冲上升时间为20ns,脉宽为30μs,频率为2.5脉冲/min,作用2min。原代培养的海马神经元经电磁脉冲辐射后,倒置相差显微镜下观察照射前后神经元形态学变化;FACS法检测细胞凋亡和坏死;Fluo-3-AM荧光探针负载、激光共聚焦显微镜扫描测定神经元胞内游离钙离子浓度[Ca2 ]i。主要观察指标:神经元形态学变化;细胞凋亡率和坏死率;胞内游离钙离子浓度[Ca2 ]i。结果:①电磁脉冲辐射后即刻,神经细胞逐渐发生液化,神经元突起回缩、变性。②电磁脉冲辐射后12h凋亡率较辐射后即刻有所恢复,但与对照组相比均显著增加[(59.27±1.27)%,(72.17±6.21)%,(17.45±5.63)%,P<0.05]。③在辐射后即刻和12h坏死率比对照组升高,但差异无统计学意义[(13.71±2.31)%,(11.96±1.04)%,(8.45±0.67)%,P>0.05]。④电磁脉冲辐射后即刻[Ca2 ]i荧光强度显著高于对照组(107.34±26.14,54.93±16.08,P<0.05)。结论:电磁脉冲致海马神经元形态学损伤、坏死与凋亡率增加,神经元内的Ca2 荧光强度明显增加。

Injured effects of electromagnetic pulse on hippocampal neurons and [Ca2+]i

BACKGROUND: Electromagnetic pulse (EMP) radiation can affect the learning and memory function of experimental rats and induce injury of hippocampal issues and change of ultrastructure of rats.OBJECTIVE: To observe the effects of EMP on injury of hippocampal neurons cultured in vitro and [Ca2+]i, and analyze deeply possible mechanism of cerebral injury induced by EMP.DESIGN: Randomized controlled animal experiment.SETTING: Department of Pathology, Chengde Medical College.MATERIALS: Several Wistar neonate rats, of either sex (half and half),were selected. Source of EMP radiation was high intensity EMP dummy source.METHODS: The experiment was performed from March to December 2004 at the Academy of Military Medical Science and Chengde Medical College, respectively. Several Wistar neonate rats were decapitated to take out the brains under narcotization. Hippocampal tissues were isolated. The cell suspension was adjusted to 5×108 L-1 for inoculation. Grouping: ①Cultured cells were assigned into control group and radiation group. Cells were collected immediately after radiation to perform observation of morphology and determination of free calcium ion concentration. ②Other cultured cells were divided into control group, 0-hour radiation group and 12-hour radiation group. Cell apoptosis rate and necrosis rate were determined. (Dosage of cultured cells: one culture flask of each group was checked in each item for 3 times). EMP radiation was in 6×104 V/m, with pulse rise time of 20 ns,pulse width of 30 μs, frequency of 2.5 pulses/min, totally for 2 minutes.EMP radiation was performed in primary cultured hippocampal neurons,and then morphological change of neurons was observed under inverted phase contrast microscope before and after radiation. Cell apoptosis and necrosis were measured with FACS method; Free [Ca2+]i concentration in neurons was measured with Fluo-3-AM fluorescent probe loading and laser confocal microscopy scanning.MAIN OUTCOME MEASURES: Morphological change of neuron, cell apoptosis rate and necrosis rate and free [Ca2+]i concentration.RESULTS: ①Immediately after EMP radiation, the onset of colliquation appeared in nerval cells gradually, and neurite was recovery and degeneration. ②Apoptosis rate after 12-hour EMP radiation recovered as compared with that at hour 0 after radiation, but significantly increased as compared with the control group [(59.27±1.27)%, (72.17±6.21)%, (17.45±5.63)%,P＜0.05]. ③Necrosis rate at hour 0 and hour 12 after radiation increased as compared with the control group, but there was no statistical significant difference [(13.71±2.31)%, ( 11.96±1.04)%, (8.45±0.67)% ,P ＞ 0.05].④[Ca2+]i fluorescence intensity at hour 0 after EMP radiation was higher obviously than that in the control group (107.34±26.14,54.93±16.08,P ＜ 0.05).CONCLUSION: EMP induces morphological injury, necrosis and increase of apoptosis rate in hippocampal neurons, and Ca2+ fluorescence intensity increases markedly in neurons.