大鼠“京骨”和“大钟”原络配穴的神经解剖学基础——霍乱毒素亚单位B结合荧光素488和594双标法

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 Alexa Fluor 488,594,CTB-Alexa 488,594)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-Alexa 488和CTB-Alexa 594两种示踪剂各5μL分别注入3只雄性SD大鼠后肢"京骨"和"大钟"穴区。72 h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-Alexa 488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L6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-Alexa 488和594荧光双标记示踪技术观察到了与原穴"京骨"和络穴"大钟"相关的感觉和运动神经元在脊神经节和脊髓的分布规律。

Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594

Objective To study the specific correlation between "Jinggu"(BL 64) and "Dazhong"(KI 4) in the nervous system by using a double-labeling of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594(CTB-Alexa 488,594) in rats,so as to investigate its neuroanatomical basis for clinical joint-application of Yuan-Source and Luo acupoints.Methods Three male SD rats were used in the present study.Under anesthesia(10% urethane),0.1% CTB-Alexa 488(5 μL) and CTB-Alexa 594(5 μL) were respectively injected into the border area between the red and white flesh,distal to the tuberosity of the fifth metatarsal bone,and the depression anterior to the medial attachment of the calcaneal tendon,the corresponding sites of the acupoints Jinggu(BL 64) and Dazhong(KI 4) in the human body.After 3 surviving days,the rat's brain,spinal cord and dorsal root ganglia(DRGs) of L3-L6 were dissected following perfusion with 4% paraformaldehyde,cut into sections and observed under fluorescent microscope equipped with a digital camera.The labeled neurons were recorded and counted.Results It was found under fluorescent microscope that the single-labeled neurons and the dual-labeled neurons were ipsilaterally located on the injected side.Among the single-labeled neurons,the labeled sensory neurons related to "Jinggu"(BL 64) and "Dazhong"(KI 4) were found to be in the DRGs of L3-L6,with a higher concentration in the DRGs of L4(27/162,102/332) and L5(130/162,204/332).The dual-labeled 7 neurons were found to be in DRGs of L4 and L5.In addition,the labeled motoneurons related to "Jinggu"(BL 64) and "Dazhong"(KI 4) distributed in the dorsolateral portion of lamina Ⅸ,forming a longitudianal column from L3-L6 with a higher concentration at L4 and L5.Conclusion The labeled sensory and motor neurons innervating Yuan-acupoint "Jinggu"(BL 64) and Luo-acupoint "Dazhong"(KI 4) distribute in DRGs of the same spinal segments and spinal ventral horns from L3-L6.