同时显示大鼠中枢神经系统神经元和神经纤维技术对其定性、半定量、定位研究的意义

背景在神经损伤与修复的研究工作中,常常会涉及损伤后以及修复过程中神经元和神经纤维的定性、定位和半定量的问题.目前神经生物学常规实验技术还很难解决这样的问题.目的探索一种简便易行并能同时显示大鼠中枢神经系统(centralnervous system,CNS)神经元和神经纤维的全程连续切片的块染方法.设计非随机非对照的实验研究.地点和材料第三军医大学组胚教研室(组织学常规技术实验室),实验动物中心.动物健康成年Wistar无菌级大鼠2只(第三军医大学实验动物中心提供),体质量200～300g,雌雄不拘.干预取大鼠嗅脑至脊髓骶段的完整CNS组织,将CNS按顺序以冠状切面切成厚2em的块待切,氨乙醇固定组织吸干后直接入30 g/L硝酸银水溶液,于22℃恒温箱中浸银染色1周.常规脱水、透明、石蜡包埋,全程连续切片的制作.一个大鼠的CNS全程标本可做成3套相近的连续切片.主要观察指标神经元及突起和神经纤维的形态结构.结果大鼠全程CNS连续切片各断面上神经元呈深棕黄色,核膜及核仁呈黑色;神经纤维呈黑色;背景淡黄色.结论运用灌流冲洗后固定、Cajal氏镀银块染、连续石蜡切片的方法,可制作同时显示CNS神经元和神经纤维的全程连续切片.将此技术方法运用到神经损伤与修复的实验研究中,对于神经元和神经纤维的定性、半定量和定位的研究都具有较高的实用价值.

Significance of the technique of simultaneous demonstration of neurons and nerve fibres in central nervous system of rat in the research on its nature determination, semi-quantitation and localization

BACKGROUND: In the research of the neural injury and rehabilitation, the problems as nature determination, localization and semi-quantitation of nerve fibres and neurons after injury and during rehabilitation were often involved. However, recent routine laboratory techniques in neurobiology had difficulties in solving such problems.OBJECTIVE: To explore an easy and practical way of mass staining in,which was able to demonstrate the serial sectioning process in neurons and nerve central nervous system(CNS) in rat.DESIGN: A non-randomized and non-controlled trial study.SETTINGS and MATERIALS: Settings were the Department of Histology and Embryology(Routine Technique Laboratory of Histology) of the Third Military Medical University and the Centre of Experimental Animal. Animals were 2 healthy aseptic Wistar rats obtained from the experimental animal centre of the Third Military Medical University with a body mass from 200 g to 300 g of either gender.INTERVENTIONS: Whole CNS tissues from rhinencephalon to spinal sacral segment of the rat were taken. CNS was sectioned into 2 cm thick coronal slices following sequence and fixed with colamine. After blotting up,the tissues were directly put into 30 g/L of silver nitrate water solution for 1 weeks of silver staining in a constant temperature box with a temperature of 22 ℃. Routine dehydration, clarification, paraffin embed and then serial sectioning were operated. A whole CNS specimen could be prepared into 3sets of similar serial slices.MAIN OUTCOME MEASURES: The morphological structures of neurons and umbos, and nerve fibres as well.RESULTS: Neurons on each section of the whole CNS serial sectioning in rat were in dark nankeen, which the nuclear membrane and nucleolus were in black; nerve fibre was black; the backgTound was canary.CONCLUSION: It can prepare whole serial sectioning, which simultaneously demonstrates neurons and nerve fibres in CNS by introducing the method of fixation after irrigation, Cajal silver staining, and serial paraffin sectioning, which has preferably practical merits in the researches on the nature determination, semi-quantitation and localization of neuron and nerve fibre if this technique is applied in the laboratorial researches of neural injury and rehabilitation.