清醒大鼠神经递质和脑电联合检测方法在镇静药物机制研究中的应用

【作者】 卢英俊；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2004， 博士

【摘要】 中枢神经系统的兴奋与抑制是一个多层次，多途径，多种物质相互作用的动态变化过程。在正常的条件下，中枢神经系统的兴奋与抑制保持着动态平衡，以维持机体正常的生理功能。如果受到外界过度的刺激，这种动态平衡将被打破，中枢神经系统的兴奋与抑制之间将失去平衡，从而产生分子、细胞、组织、及整体行为等不同层次的异常症状。其深层机制，与脑内神经递质水平失调和神经电生理的变化密切相关。目前研究兴奋—抑制的主要方法有行为学方法，电生理方法和神经生化方法。酸枣仁皂甙A(JujubosideA，JuA)是镇静催眠中药酸枣仁的一种主要的皂甙类成分。行为学实验和海马离体脑片灌流实验证明酸枣仁皂甙A有显著的中枢抑制作用，但是进一步的机制尚待阐述。 本课题的主要目的就是：综合利用神经生化和神经电生理技术，并建立清醒大鼠神经递质和脑电同时检测的方法，应用于JuA对青霉素诱导的中枢神经系统兴奋抑制作用及机制的研究。 本文结合高效液相色谱(HPLC)双泵梯度洗脱法、微透析在体取样及侧脑室微量给药技术，建立了清醒大鼠海马脑区谷氨酸在体检测方法；并在此基础上检测了青霉素及JuA作用后中枢海马区域谷氨酸水平的变化。谷氨酸是使神经组织过度兴奋的主要的兴奋性神经递质之一。青霉素直接刺激神经组织可以一定程度地打破神经系统的兴奋与抑制之间的平衡，是兴奋与抑制研究中一种常用的广谱兴奋剂。低剂量青霉素(1000KIU／L)侧脑室给药能够诱导海马谷氨酸水平的提高，青霉素剂量过高时则引发实验动物癫痫样发作症状。0.1g／L JuA与青霉素同时给药能显著抑制1000KIU／L青霉素的作用；安定及苯巴比妥与青霉素同时侧脑室给药也能显著抑制青霉素的作用。说明青霉素主要通过升高神经组织内的谷氨酸水平破坏神经系统的兴奋和抑制的平衡，而JuA则可以逆转青霉素引起的谷氨酸的升高，在递质的水平起到神经抑制和保护作用。 本文在神经生化与神经电生理技术基础上，建立了麻醉大鼠纹状体神经递质和皮层脑电同时检测的方法，初步研究了大鼠局灶性脑缺血条件下纹状体谷氨酸，单胺类神经递质与皮层脑电的变化情况；为进一步系统研究脑缺血作用的神经生化和神经电生理机制及其相关性打下基础。 进一步建立了清醒大鼠海马谷氨酸与皮层脑电同时检测的新方法，并应用脑电功率谱分析技术，研究了皮层脑电信号与海马谷氨酸水平在侧脑室青霉素给药后的变化、以及电信号与生化信号之间的相关性。结果显示，给药后大鼠皮层脑电重心频率显著升高，62 and 61频谱段功率显著下降，海马谷氨酸升高与脑电兴奋性存在一定的相关性。在JuA与青霉素同时给药后，皮层脑电信号重心频率显著下降，低频故and剐段功率显著上升，脑电兴奋性得到抑制;而安定虽然同样降低重心频率，升高低频62 and 61段功率，却显著地增加了高频瞬成分。这一结果表明，安定和JuA有不同的脑电抑制机制，安定的p增强作用可能与其副作用有关。说明J叭对青霉素诱导的中枢兴奋性神经递质和皮层脑电兴奋均具有显著的抑制作用，且无西药安定所引起的p增强副作用。 本研究对中枢神经兴奋与抑制平衡在神经递质和脑电生理水平上进行了研究，并在此基础上研究了J叭对中枢神经系统的抑制作用。结果发现J叭能够在递质与脑电的水平上参与了中枢神经系统兴奋与抑制平衡这一过程，两者之间存在一定的联系。建立的神经递质与脑电联合检测实验方法及脑电功率谱分析应用方法，对其他中西药的药理和作用机制研究比较具有一定的指导意义。更多还原

【Abstract】 Neuroelectrophysiological and neurochemical signals are of great significance to various physiological and pathological phenomena. Researchers had studied these two different systems separately for many years. The development of microdialysis technique paved the way for freely continuous neurochemical sampling from the brains of waking animals. Acquisition of cortical EEG and deep-seated EEG signals by electrodes implantation offered the means of extracting spontaneous EEG and evoked potential from waking animals. Automatic analysis(including time-domain analysis and frequency-domain analysis) of mass data by computer technique greatly relieved us from the burdensome task of manual analysis.The balance of excitatory and inhibitory signal pathways in CNS (central nervous system) is a dynamic interacting procedure of different substances, cells, structures etc. In normal status, this procedure keeps running smoothly to maintain many functions of CNS. However, when a body is over stimulated, this balance will corrupt, which will bring abnormal changes in the body including molecules, cells, structures and even behavior. As a result, the system might lose its balance, and the body is very likely to be affected by all kinds of diseases. The mechanisms of excitation and inhibition in CNS closely correlated with the changes of neuroelectrophysiological and neurochemical signals.Most of the pharmacological methods that have been used to study excitation and inhibition phenomena mainly fall into three categories: behavior experiment methods, neurophysiological experiment methods and neurochemical experiment methods. Jujuboside A ( JuA) is a main component of Jujubogenin extracted from the seed of Ziziphus (Ziziphus jujuba Mill Var spinosa ( Bunge ) Hu ex H F Chou ) , a popular Chinese herb medicine, which has long been known as a sedative-hypnotic on the CNS. The behavioral experimental studies had revealed that, the extraction of the seed of Ziziphus could inhibit the spontaneous activity and facilitate the hypnotic action of pentobarbital, but the detailed mechanism of its action is still not fully understood.The main purpose of this paper was to set up new pharmacological study methods by using cross-disciplinary modern detection techniques, and establish a method of simultaneous in vivo cortical EEG and neurotransmitters detection in rats to monitor neuroelectrophysiological and neurochemical signals at the same time. Then with the indexes of behavior change, CNS neuroelectrophysiological and neurochemical change obtained, we can investigate the effects of drugs on CNS synthetically, analyze and reveal the action mechanisms of sedative drug JuA. The correlation between neuroelectrophysiological and neurochemical signals can also be investigated.The method of glutamate detection in hippocampal area with the HPLC system and the microdialysis/microinjection technique was set up. It was reported that penicillin could break the balance of excitatory and inhibitory signal pathways in CNS when applied to the neural tissue directly and is always used as an agitator of CNS. The change of the glutamate level in hippocampal area induced by penicillin was detected with HPLC associated with the microdialysis and in vivo sampling technique. Penicillin was injected into lateral ventricle through microinjection probe and the microdialysis was performed to detect the level of hippocampal glutamate. The glutamate concentration increased greatly with the application of penicillin 1000KIU/L and higher dose brought the behavioral seizure symptoms. When JuA was co-injected into the lateral ventricle with penicillin, the increase of glutamate induced by penicillin decreased with the dose of JuA. From this, it can be deduced that JuA reversed the effects of penicillin on the level of hippocampal glutamate.A new method of simultaneous in vivo cortical EEG and hippocampal glutamate(Glu) detection in rats was established in order to monitor neuroelectrophysiological and neurochemical signals at the same time. EEG signals were recor更多还原