目的：用霍乱毒素亚单位B结合荧光素488和594（cholera toxin subunit B conjugated with A1exa Fluor488，594，CTB—Alexa488，594）双标记示踪技术揭示“京骨”和“大钟”穴在大鼠神经系统中的特异性联系，探讨原络配穴的神经解剖学基础。方法：用微量注射器将0．1％CTB-Alexa488和CTB-Alexa594两种示踪剂各5μL分别注入3只雄性SD大鼠后肢“京骨”和“大钟”穴区。72h后将动物灌流固定，取出脑、脊髓和脊神经节，并制成组织切片，然后直接用荧光显微镜系统观察和记录被标记的神经元。结果：在相应的激发光下，CTB-Alexa488和594标记的神经元分别呈绿色和红色，双重标记的神经元呈黄色，均位于示踪剂注入侧的相关神经组织中。在所标记到的神经元中，与“京骨”和“大钟”穴相关联的感觉神经元集中在L4～L5脊神经节，少量分布在L3和L5脊神经节，其中双标的感觉神经元主要出现在L4～L5脊神经节；与两穴相关联的运动神经元以L4～L5节段为中心呈柱状分布在L3～L6节段脊髓前角第Ⅸ层的后外侧部。结论：应用CTB-Alexa4gg和594荧光双标记示踪技术观察到了与原穴“京骨”和络穴“犬钟”相关的感觉和运动神经元在脊神经节和脊髓的分布规律。
Objective:To investigate the biochemical characteristic of the neurons associated Zusanli(ST 36)in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B(AF594-CTB)neural tracing and calcitonin gene-related peptide(CGRP)fluorescent immunohistochemical techniques.Methods:Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body.After 3 surviving days,the rat's spinal cord and dorsal root ganglia(DRGs)at lumbar segments were dissected following perfusion with 4%paraformaldehyde,cut into sections,and then stained with CGRPfluorescent immunohistochemical method.Results:AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG,and the labeled motor neurons located in the dorsolateral and intermediate regions of laminaⅨfrom L3-L5 segments with high concentration at L4.Meanwhile,CGRPpositive neural labeling distributed symmetrically on both sides of DRGs,anterior and dorsal homs of spinal cord.In the AF594-CTB labeled neurons,37%sensory neurons and 100%motor neurons expressed CGRPpositive.Conclusion:These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns,and contained CGRP-expression.
