电磁脉冲对大鼠脑海马N-甲基-D-天冬氨酸受体活性的影响

背景电磁脉冲是一种高能非电离辐射,它涵盖的频谱极宽,对电子仪器具有极强的破坏力,并具有一定的生物损伤效应.本课题组前期实验发现,电磁脉冲可造成实验大鼠学习记忆能力的显著下降,并且影响其海马长时程增强效应的形成.目的观察电磁脉冲照射对大鼠海马组织中氨基酸类神经递质含量和N-甲基-D-天冬氨酸受体的影响.设计随机对照的实验,方差分析.单位军事医学科学院放射与辐射医学研究所.材料实验于2004-01/03在军事医学科学院放射与辐射医学研究所完成.实验选用雄性Wistar大鼠32只,随机分为电磁脉冲组26只和对照组6只.方法电磁脉冲组大鼠经电磁脉冲照射(6×104V/m,脉冲上升时间20ns,脉宽30μs,频率2.5脉冲/min,作用2 min)后即刻,3,6,24,48 h麻醉状态下断头取脑,剥离海马;用高效液相色谱法测定氨基酸含量,并以3H标记的谷氨酸为配基进行放射性配基-受体结合实验.对照组麻醉处死前不做任何处理.主要观察指标①各组大鼠海马组织兴奋性氨基酸和抑制性氨基酸含量的变化.②各组大鼠海马N-甲基-D-天冬氨酸受体的最大结合量和平衡解离常数的变化.结果32只大鼠全部进入结果分析.①电磁脉冲照射后即刻、3 h、6 h可见天冬氨酸的含量显著升高峰值(17.25±1.63)μmol/L],明显高于对照组(10.56±1.5)μmol/L,P＜0.05],谷氨酸含量也于上述3个时间点升高峰值(13.67±0.95)μmol/L],显著高于对照组(6.94±1.1)μmol/L,P＜0.05],两者含量均于照射后24 h渐趋恢复,48 h接近正常水平.3抑制性氨基酸(甘氨酸、牛磺酸、γ-氨基丁酸)的含量也于电磁脉冲照射后不同时间点升高,并于48 h恢复.谷氨酸/γ-氨基丁酸比值于照后即刻明显升高(P＜0.05),照后24 h明显下降,48 h恢复至接近对照组.②电磁脉冲照射后即刻大鼠海马中N-甲基-D-天冬氨酸受体的平衡解离常数开始下降,照后3 h下降最显著(P＜0.05),6 h渐有恢复,48 h恢复至正常水平;受照大鼠海马中N-甲基-D-天冬氨酸受体的最大结合量于照后3 h和6 h显著下降(P＜0.05),24 h渐有恢复,照后48 h明显升高,超过正常组水平(P＜0.05).结论电磁脉冲照射导致大鼠海马组织中兴奋性氨基酸含量及谷氨酸/γ-氨基丁酸比值升高;同时N-甲基-D-天冬氨酸受体的亲和力升高及受体密度下降.提示实验动物认知障碍可能与兴奋性氨基酸的过度释放和N-甲基-D-天冬氨酸受体功能改变有关.

Effects of electromagnetic pulse on N-methyl-D-aspartate receptor activity in rat hippocampus

BACKGROUND: Electromagnetic pulse (EMP) is high-energy non-ionizing radiation covering a wide spectrum with strong capability of destruction of electronic devices and inducing injuries in biological organisms. In previous studies we found EMP could cause obvious reduction in the learning and memory ability of rats and affect the formation of long-term potentiation of rat hippocampus.OBJECTIVE: To observe the effect of EMP on the content of amino acid neurotransmitters and N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampus.DESIGN: Randomized controlled experiment and analysis of variance.SETTING: Institute of Radiation Medicine, Academy of Military Medical SciencesMATERIALS: The experiment was conducted in the Institute of Radiation Medicine, Academy of Military Medical Sciences from January to March 2004. Totally 32 male Wistar rats were used and randomized into EMP group (n=26) and control group (n=6).METHODS: Rats in the EMP group were decapitated and the hippocampi were removed immediately 3, 6, 24 and 48 hours after EMP radiation (6×104 V/m, with pulse rise time of 20 ns, pulse width of 30 μs, frequency of 2.5 pulses/minute for 2 minutes). Glutamate contents were detected with high-performance liquid chromatograpy (HPLC). Radioactive ligand-receptor binding test was performed with glutamate as the ligand with 3H labeling. The rats in the control group were not given any treatment before put to death under anesthesia.MAIN OUTCOME MEASURES: ① Changes in excitatory and inhibitory amino acid contents in the hippocampus of the rats. ② Rat hippocampal Bmax of NMDA receptor and changes in equilibrium dissociation constant (Kd).RESULTS: All the 32 rats entered the result analysis. NMDA content was significantly increased in the EMP group with a peak level of (17.25±1.63)μmol/L immediately, 3 and 6 hours after EMP radiation in comparison with that in the control group (10.56±1.5)μmol/L, P ＜ 0.05]. Glutamate content was also increased at the 3 time points with the peak level of (13.67±0.95) μmol/L, which was significantly higher than that in the control group (6.94±1.1) μmol/L, P ＜ 0.05]. NMDA and glutamate contents was both decreased 24 hours after EMP exposure and resumed the normal level after 48 hours. The contents of glycine, taurine and gamma-aminobutyric acid also rose after EMP exposure and recovered 48 hours after the radiation. The ratio of glycine/gamma-aminobutyric increased markedly immediately after EMP exposure (P ＜ 0.05), but decreased obviously 24hours after the exposure and was restored to the level of the control group.The Kd of NMDA receptor began to decrease immediately after EMP exposure, which was the most obvious at 3 hours (P ＜ 0.05), followed then by gradual increase at 6 hours and reaching the normal level at 48 hours. The Bmax of NMDA receptor in rat hippocampus was decreased significantly 3and 6 hours after irradiation (P ＜ 0.05), and increased gradually since 24hours to a level significantly higher than the normal at 48 hours (P ＜ 0.05).CONCLUSION: EMP exposure induces increases in excitatory amino acid contents and the Glu/γ-aminobutyric acid (GABA) ratio in rat hippocampus, increases the affinity and decrease the density of NMDA receptors, suggesting that excessive release of excitatory amino acids and changes in NMDA receptor activity may contribute to cognitive impairment in rats.