基于下丘脑-小脑神经环路的针刺预处理对心肌缺血再灌注损伤保护效应的调控机制研究

Our previous studies showed that acupuncture has explicit effects of protecting myocardial ischemia and reperfusion injury, the mechanism is closely related to the central regions of hypothalamus and hippocampus, and cerebellum may play a role in the mechanism of acupuncture effect. Neuroscience research found that there is a direct neural circuit between hypothalamus and cerebellum, and which provided the basis for the study of the acupuncture mechanism of the cerebellum through the hypothalamus. Therefore, we hypothesized that the cerebellum is involved in the regulation effects of acupuncture on cerebral ischemia reperfusion injury, and dependent on the hypothalamic cerebellar neuronal circuits. Accordingly, this study was designed to observe the effects of acupuncture on neuronal activity, neurotransmitter content and expression of c-fos in the cerebellum and hypothalamus of rats with acute myocardial ischemia reperfusion injury, detected the conduction pathway of acupuncture signal in the brain by tracing technique of nerve tract. To investigate the main nucleus and material base of cerebellum and hypothalamus of acupuncture effect on myocardial ischemia reperfusion injury. To further clarify the main pathway of the hypothalamic cerebellar nerve pathway involved in the effects of acupuncture on myocardial ischemia reperfusion injury through nuclear damage, nerve fiber signal blocking or strengthening experiment. The aim of this study was to provide evidence for further study of the mechanism of the involvement of the hypothalamus and cerebellum in the acupuncture effect, and to lay the foundation for revealing the central integration mechanism of acupuncture effects.

项目组前期研究表明，针刺预处理可有效减轻心肌缺血再灌注损伤，其作用机制与下丘脑、海马等部位的调控密切相关，小脑可能参与了针刺效应机制的发挥。神经解剖学研究发现下丘脑和小脑之间存在直接的神经环路，为研究小脑经下丘脑途径参与针刺效应机制提供了依据。因此，我们推测小脑参与了针刺抗心肌缺血再灌注损伤效应的脑调控机制，并依赖于下丘脑-小脑神经环路而发挥作用。据此，本项目拟观察针刺对急性心肌缺血再灌注损伤大鼠小脑与下丘脑不同核团神经元活动、神经递质含量、c-fos蛋白及基因表达的影响，利用神经束路示踪技术观察针刺信号在脑内的传导通路，初步探明小脑、下丘脑参与针刺抗心肌缺血再灌注损伤效应的主导核团和物质基础；并通过核团损毁、神经纤维信号阻断或强化实验，进一步明确下丘脑-小脑神经环路参与针刺抗心肌缺血再灌注损伤效应的主导通路及其神经纤维性质，为进一步探明下丘脑-小脑神经环路参与针刺效应的作用机制提供依据。

长期以来，针灸效应的神经调控机制一直是学者们关注的重点研究领域之一。项目组前期研究表明，针刺预处理可有效减轻心肌缺血再灌注损伤（MIRI），其作用机制与下丘脑、海马等部位的调控密切相关，小脑可能参与了针刺效应机制的发挥。神经解剖学研究发现下丘脑和小脑之间存在直接的神经环路，为研究小脑经下丘脑途径参与针刺效应机制提供了依据。因此，我们推测小脑参与了针刺抗MIRI效应的脑调控机制，并依赖于下丘脑-小脑神经环路而发挥作用。本项目采用免疫组织化学法和免疫荧光法观察针刺预处理对MIRI大鼠小脑、下丘脑c-fos蛋白表达的影响；采用微透析结合高效液相-电化学检测法观察针刺预处理对MIRI大鼠小脑、下丘脑不同核团细胞间液多巴胺（DA）、5-羟色胺（5-HT）、谷氨酸（Glu）、γ-氨基丁酸（GABA）含量的影响；采用Plexon系统记录小脑和下丘脑神经元放电和场电位，利用Offline Sorter软件进行神经元信号聚类分析；采用Neuro Explorer软件对神经元放电波形进行自相关与互相关、放电频率和实时频谱分析，观察电针预处理对MIRI大鼠下丘脑/小脑神经元电活动的影响；观察损毁小脑/下丘脑不同核团对针刺抗MIRI效应及下丘脑/小脑神经元活动、神经递质含量和c-fos蛋白表达的影响。通过本项目的研究，进一步明确了小脑在针刺调节心脏功能效应中的作用，初步探明小脑顶核（FN）、下丘脑室旁核、下丘脑外侧区（LHA）参与了针刺减轻MIRI的效应，FN、LHA中DA、5-HT、Glu、GABA可能是其参与针刺调控效应的重要物质基础；初步明确小脑通过下丘脑-小脑投射参与了针刺改善心脏功能的作用，FN-LHA是下丘脑-小脑神经环路参与针刺抗MIRI效应的主导通路，初步探明针刺对锥体神经元的调控是其调节心脏功能效应的重要机制，为进一步明确下丘脑-小脑神经环路参与针刺效应的调控机制提供了实验依据。