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用荧光素逆行双标和免疫组化结合三标技术,研究脊神经节含CCK-8的感觉神经元的皮肤-内脏分支投射。将fast blue(FB)和nuclear yellow(NY)分別注入大鼠左侧腹腔神经节和左侧第9～11肋间神经皮支内。灌注固定,取左侧胸9～11脊神经节,恒冷箱切片。在Nikon荧光显微镜下观察,激发光波长为365nm。镜下可见三种不同标记的神经元:NY标记神经元,胞核发黄色荧光;FB标记神经元,胞浆发蓝色荧光;NY+FB双标神经元(具有分支投射的神经元),胞浆发蓝色荧光和胞核发黄色荧光。双标神经元约占所有荧光标记神经元的2.8％。含有双标神经元的切片,用CCK-8抗血清孵育,用改良的PAP法显色。镜下可见CCK免疫反应阳性神经元。在CCK免疫反应阳性神经元中,可以区分:1.被CCK标记的NY神经元(NY+CCK)。2.被CCK标记的FB神经元(FB+CCK)。3.被CCK标记的NY+FB双标神经元(NY+FB+CCK)。4.单纯CCK免疫反应阳性神经元。NY+FB+CCK三标神经元约占NY+FB双标神经元的11.5％,仅占CCK阳性神经元的0.4％。以上结果表明:脊神经节中有些感觉神经元的周围突具有分支,分别投射到内脏和皮肤;这类神经元中,有些神经元含有CCK-8。从而提示:内脏牵涉性痛的会聚可在脊神经节进行;CCK可能参与内脏和皮肤感觉会聚的调节。因此,本文为牵涉性痛的发生机制和躯体-内脏反射提供了重要的化学神经解剖学资料。
Using retrograde double-labeled fluorescein and immunohistochemistry combined with triple labeling technique, the skin-visceral branch projection of sensory neurons containing CCK-8 in the spinal ganglia was studied. Fast blue (FB) and nuclear yellow (NY) were injected into the left celiac ganglion and left intercalary neurons of the 9th to 11th branches, respectively. Perfusion fixed, take the left chest 9 ~ 11 spinal ganglia, constant cold box section. Observed under a Nikon fluorescence microscope, the excitation light wavelength was 365 nm. There were three kinds of neurons with different marks in the microscope: NY-labeled neurons with yellow fluorescence in nuclei; FB-labeled neurons with blue fluorescence in cytoplasm; NY + FB double-labeled neurons with branches projecting neurons; Pulp blue fluorescence and nuclear hair yellow fluorescence. Double-labeled neurons make up about 2.8% of all fluorescent-labeled neurons. Sheets containing double-labeled neurons were incubated with CCK-8 antiserum and developed with a modified PAP method. CCK immunoreactive positive neurons can be seen under the microscope. Among CCK immunoreactive neurons, one can distinguish between: 1. NY neurons that are CCK-labeled (NY + CCK). 2. FB neurons labeled with CCK (FB + CCK). 3. NY + FB double labeled neurons (NY + FB + CCK) labeled with CCK. 4. Simple CCK immunoreactive neurons. NY + FB + CCK three-labeled neurons accounted for about 11.5% of NY + FB double-labeled neurons, accounting for only 0.4% of CCK-positive neurons. The above results show that: Some of the sensory neurons in the dorsal root ganglion have branches that project to the viscera and skin, respectively; some of these neurons contain CCK-8. Suggesting that the involvement of visceral pain may occur in the dorsal root ganglia; CCK may be involved in the regulation of visceral and skin sensory convergence. Therefore, this article provides important chemical neuroanatomy data for the mechanisms involved in pain and somatic-visceral reflexes.