药物防护电磁脉冲所致海马神经元损伤的可能性(英文)

Possibility of medicine in preventing and protecting electromagnetic pulse-induced injury of hippocampal neurons

背景:电磁脉冲照射可引起实验大鼠学习记忆能力下降,并可造成离体海马神经元的胞内钙超载,进而发生坏死和凋亡,物理屏蔽可减轻电磁辐射对实验动物的损伤,但缺乏在细胞模型上进行的药物防护研究。目的:观察药物防护电磁脉冲致离体海马神经元损伤的可能性。设计:随机对照动物实验。单位:承德医学院基础部。材料:实验于2004-01/2005-01分别在军事医学科学院和承德医学院完成。实验动物选用Wistar乳鼠若干只。方法:断头取脑分离海马组织,进行海马神经元原代培养与鉴定,原代培养的海马神经元经MK801(N-甲基-D-天冬氨酸受体拮抗剂)和尼非地平(L型钙离子通道阻断剂)预处理后进行电磁脉冲照射。电磁脉冲照射条件为6×104V/m,脉冲上升时间20ns,脉宽30μs,频率2.5脉冲/min,作用2min。将培养在特殊培养皿的细胞分为正常对照组、电磁脉冲照射组、MK80120μmol/L组和MK80120μmol/L+尼非地平1μmol/L组。采用MTT法对细胞活力进行测定;FACS法检测细胞凋亡率;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:各组细胞内钙超载、细胞活力、细胞调亡的情况比较。结果:①电磁脉冲组在照射后即刻的[Ca2+]i荧光强度显著高于正常对照组,差异有显著性意义([107.34±26.14),(54.93±16.08),P<0.05];MK80120μmol/L组比电磁脉冲照射组的[Ca2+]i荧光强度有所下降(81.29±19.96,P<0.05),而MK80120μmol/L+尼非地平1μmol/L组的[Ca2+]i荧光强度呈进一步下降趋势(69.82±25.54,P<0.05)。但两个给药组的[Ca2+]i荧光强度仍高于正常对照(P<0.05)。②MK80120μmol/L组和MK80120μmol/L+尼非地平1μmol/L组反映细胞增殖活力的吸光度值分别为0.25±0.06和0.27±0.07,明显高于电磁脉冲照射组(0.17±0.08,P<0.05),但仍低于正常对照组(0.33±0.08,P<0.05)。③电磁脉冲照射组在照后即刻的细胞凋亡率显著高于正常对照组,差异有显著性意义([68.63±9.04)%,(20.14±4.34)%,P<0.01];MK80120μmol/L组比电磁脉冲照射组的细胞凋亡率有所下降(62.12±11.08)%,MK80120μmol/L+尼非地平1μmol/L组的细胞凋亡率呈进一步下降趋势([53.69±13.60)%,P<0.05)],但两个给药组的细胞凋亡率仍高于正常对照组(P<0.01)。结论:MK801和尼非地平预处理可部分阻断电磁脉冲所致的海马神经元损伤;细胞内钙超载在电磁脉冲损伤机制中发挥重要作用。

BACKGROUND： Electromagnetic pulse （EMP） irradiation can cause the decline of learning and memory abilities of rats, and lead to the intracellular calcium overloading of hippocampal neurons in vitro, and then result in necrosis and apoptosis. Physical shield can alleviate the damage of electromagnetic irradiation on experimental animals, but studies of the medicine prevention and protection on cell models are still in lack. OBJECTIVE： To observe the possibility of medicine in preventing and protecting the EMP-induced injury of hippocampal neurons in vitro. DESIGN： A randomized controlled animal experiment. SETTING： Division of Basic Medical Sciences, Chengde Medical College. MATERIALS： The experiments were carried out in the Academy of Military Medical Sciences and Chengde Medical College from January 2004 to January 2005. Several neonatal Wistar rats were used. METHODS： The neonatal Wistar rats were killed by cutting heads to remove brain, and the hippocampal neurons were primarily cultured and identified. After pretreatment with MK801 [N-methyl-D-aspartate （NMDA） receptor antagonist] and nifedipine （L-type Ca^2＋ channel blocking agent）, the primarily cultured hippocampal neurons were irradiated with EMP. The condition of our experiment was 6×10^4 V/m, pulse rise time was 20 ns, pulse width was 30 ms, and frequency was 2.5 pulse per minute for 2 minutes. The neurons cultured in special petri dish, which could be observed under LSCM high amplified resolution, were divided into EMP irradiation group, MK801 20 μmol/L group, MK801 20 μmol/L＋ nifedipine 1 μmol/L group. The cellular activities were detected with methyl-thiazol-tetrazolium （MTT） colorimetry; The rate of apoptosis was detected with FASC method; The intracellular free Calcium concentration （[Ca^2＋]i） was determined by loading with Fluo-3-AM Ca^2＋ fluorescent probe （Molecular Probes Company） on the laser scanning confocal microscope. MAIN OUTCOME MEASURES： The intracellular calcium overloading, cellular activity and rate of apoptosis were compared. RESULTS： ① The [Ca^2＋]i fluorescent intensity in the EMP irradiation group immediately after irradiation was significantly higher than that in the normal control group （107.34±26.14, 54.93±16.08, P 〈 0.05）; As compared with the EMP irradiation group, the [Ca^2＋]i fluorescent intensity was decreased in the MK801 20 μmol/L group （81.29±19.96, P 〈 0.05）, and further decreased in the MK801 20 μmol/L＋ 1 μmol/L nifedipine group （69.82±25.54, P 〈 0.05）, but both were higher than that in the normal control group （P 〈 0.05）. ② The A values that reflected the activity of cell proliferation MK801 20 μmol/L group and MK801 20 μmol/L＋ 1 μmol/L nifedipine group （0.25±0.06, 0.27±0.07） were obviously higher than that in the EMP irradiation group （0.17±0.08, P 〈 0.05）, but still lower than that in the normal control group （0.33±0.08, P 〈 0.05）. ③ The rate of apoptosis in the EMP irradiation group immediately after irradiation was significantly higher than that in the normal control group [（68.63±9.04）%, （20.14±4.34）%, P 〈0.01]： As compared with the EMP irradiation group, the rate of apoptosis was decreased in the MK801 20 μmol/L group （62.12±11.08）%, and further decreased in the MK801 20 μmol/L＋ 1 μmol/L nifedipine group [（53.69±13.60）%, P 〈 0.05], but both were higher than that in the normal control group （P 〈 0.01）. CONCLUSION： Pretreatment with MK801 and nifedipine can partly block EMP induced damage in hippocampal neurons in vitro. Intracellular Ca^2＋ Overloading may play an important role in the injury of EMP on hippocampal neurons.