伪狂犬病毒法示踪大鼠排便反射高级中枢调控的研究

A study on central neural pathway of defecation regulation in rats

关键词:  排便  神经通路  伪狂犬病病毒  钙成像  大鼠

Key words:  defecation  neural pathway  pseudorabies virus  calcium imaging  rat

摘要:

目的通过伪狂犬病毒(pseudorabies virus, PRV)示踪技术结合钙成像技术，研究排便反射的高级中枢定位及相关神经通路。 方法研究将成年SD大鼠分为实验组和对照组，实验组大鼠在直肠壁内注射编码荧光蛋白的PRV病毒，对照组大鼠则注射空载体病毒。注射5d后，处死大鼠并取出直肠和大脑组织进行荧光定量分析。使用基因编码的钙离子指示剂（如GCaMP）结合活体钙成像技术，观察排便活动相关神经元的钙信号动力学变化。 结果实验组大鼠的下丘脑可观察到逐渐增强的荧光信号，通过图形分析可明确椎体神经元是直肠的上游突触连接神经元，通过活体钙成像技术可进一步明确排便活动时下丘脑区域的神经元钙信号明显上升。 结论本研究初步明确了大鼠排便反射的定位中枢，并通过伪狂犬病毒示踪技术和钙成像技术，动态观测到排便活动时相关中枢神经元的显著电活动信号。

Abstract:

ObjectiveTo investigate higher central neural pathways of defecation reflex with pseudo rabies virus(PRV) tracing and calcium imaging techniques. MethodsAdult male SD rats were divided into an experimental group and a control group. The rats in experimental group were injected with PRV virus encoding fluorescent protein into the rectal wall, while the control group rats were injected with empty vector virus. After 5 days of injection, the rats were euthanized and rectal and brain tissues were harvested for fluorescence quantitative analysis. At the same time, using gene encoded calcium ion indicators(GCaMP) and in vivo calcium imaging technology, the dynamic changes of calcium signals in defecation activity related neurons were observed. ResultsThe hypothalamus of the rats in experimental group showed gradually increasing fluorescence signals. Through graphic analysis, it was confirmed that the vertebral neurons were upstream synaptic connecting neurons in the rectum. Through in vivo calcium imaging technique, it was further confirmed that the calcium signal of neurons in the hypothalamus area increased significantly during defecation activity. ConclusionThis study preliminarily identified the localization of centers related to defecation reflex in rats, which provides an experimental basis for further study on the neural regulatory mechanisms of defecation reflex.