脊髓后角神经降压肽和P物质的免疫细胞化学双标法研究——光镜和电镜观察

A DOUBLE LABELLING IMMUNOCYTOCHEMICAL STUDY OF NEUROTENSIN AND SUBSTANCE P IN DORSAL HORN OF SPINAL CORD IN RAT——A LIGHT AND ELECTRON MICROSCOPIC CORRELATIVE STUDY

本文用PAP法在同一切片上作双重免疫染色以二氨基联苯胺DAB和二盐酸联苯胺BDHC作显色剂,对经秋水仙素处理的大鼠脊髓后角内神经降压肽(NT)和P物质(SP)的免疫反应性进行光镜和电镜研究。实验中发现,DAB与BDHC的反应产物具有明显的区别,光镜下,前者为棕色,后者为蓝色;在电镜下,DAB反应产物呈均匀,弥散的电子致密体,而BDHC反应产物呈斑块状电子致密体。这种新的而又简单的方法能在同一张超薄切片上同时显示两种抗原物质,这将有利于进一步研究两种不同的神经元之间的结构和功能关系。本研究发现,NT样免疫阳性反应胞体主要位于第Ⅱ层和Ⅲ层外部,终末位于Ⅰ—Ⅲ层。电镜下,NT阳性反应轴突终末可与未标记轴突或树突构成突触。SP样阳性反应胞体主要位于Ⅱ层,在Ⅰ、Ⅱ层有较高密度的终末。电镜下观察到存在轴(SP)—轴(SP)和轴(SP)—体(SP)突触。在双重NT染色切片上,电镜下发现阳性轴突终末能与SP轴突终末形成触或接触。本研究结果提示:脊髓后角内含NT和含SP的神经元可能参与一级感觉信息的调制过程。

Immunoreactivity of neurotensin(NT) and substance P (SP) were studied by means of PAP method with simultaneous immunocytochemical double staining on the same sections using diaminobenzidine (DAB) for SP and benzidinc dihydrochloride(BDHC) for NT as the chromogens for light and electron microscopy in dorsal horn of spinal cord of rat treated with colchicinc. In our hands the reaction products of DAB and BDHC is quite discernible, under LM the former is brown and later is blue; under EM DAB reaction product is homogeneous and diffuse electron opaque while BDHC reaction product is patch electron opaque, it is a new but simpler approach to demonstrate two antigens simultaneously in the same ultrathin section. It would be conformable further to the studies of morphological and functional relationships between different kinds of neurons. The results showed that NT-like immunoreactive (NT-LI) perikarya were mainly located in lamina Ⅱi and out layer of lamina Ⅲ and NT-LI terminals were mainly located in laminae Ⅰ-Ⅲ. Under EM the NT-LI axon terminals may synapse with the unlabeled axon or unlabeled dendrites. SP-like immunoreactive (SP-LI) perikarya were mainly located in lamina Ⅱ. The density of SP-LI terminals were higher in laminae Ⅰ-Ⅱ, while axo (SP)-axonic (SP), axo (SP)-somatic (SP) synapses were identified with EM. In double labelling sections, furthermore under EM, it was found that NT-LI axon terminal (BDHC patch like electron opaque reaction products)can synapse or contact with SP-LI axon (DAB diffuse electron opaque reaction products). Our results suggested that NT-and SP-containing neurons and terminals in dorsal horn might participate in regulating process of primary sensory transmission.