碳化青和固蓝逆行标记新生大鼠面神经核的比较研究

A comparison of carbocyanine diI and fast blue in retrograde labeling of neonatal rat's facial nucleus

目的 探讨碳化青(diI) 和固蓝(Fast Blue ,FB) 用作鉴定分离培养新生大鼠面神经运动神经元细胞来源的逆行追踪剂的可行性。方法 采用外周注射逆行标记法, 比较不同浓度下碳化青(diI5 % 和10 % - DMSO 液)和固蓝(FB,5 % 和3 % ) 两种荧光染料逆行标记新生大鼠面神经核区运动神经元数量及其分布特点, 观察并比较标记荧光持续时间。结果 固蓝逆行标记面神经核区运动神经元胞浆呈蓝绿色荧光, 荧光淬灭较快; 平均每张切片标记神经元数多于碳化青(t= 3 .187 和t= 3 .880 , P< 0 .01) 。碳化青标记细胞为橙红色荧光, 且持续96 小时以上;10 % 碳化青作追踪剂时, 标记细胞数较5 % 碳化青多( t = 2 .163 , P < 0 .05) 。固蓝和碳化青标记的面神经核区运动神经元胞体分布相似。结论 碳化青有荧光保持时间长、不易向胞体外扩散等优点, 可选作鉴定和识别所分离培养的新生大鼠面神经运动元细胞较理想逆行追踪剂, 而固蓝尽管标记率高,

Objective To investigate the feasibility of Carbocyanine diI(diI) and Fast Blue (FB) as retrograde tracing fluorescent dye for identification of neuronal cultures from the origin of the facial nucleus of neonatal rats. Methods DiI(5% and 10% in DMSO) and FB (5% and 3% in saline) were respectively injected into neonatal rat's whisked pad snouts. The average counts of labeled facial motorneurons from every third section in serial sections of the facial nucleus were compared and distribution of the labeled neurons was analyzed. Fluorescence duration of two fluorescent dyes in the labeled neurons were observed. Results The number of motoneurons labeled with FB was more than diI as tracer (t=3.187 and t=3.880 respectively, P<0.01) in each section and larger counts of labeled neurons were obtained with 10% diI than with 5% diI (t=2.163, P<0.05). Flocculate blue green fluorescence was observed in cytoplasms in FB labeled neurons. while orange fluorescence in diI labeled neurons was mainly on neuronal membrane . In compare with early fading of FB, the fluorescence of diI labeled neurons maintained bright at more than 96 hours. The distribution of labeled neurons with both of the dyes similarly located in lateral, intermediate, and ventral subnucleus of facial nucleus. Conclusion DiI would be a better tracer for labeling of motoneuron cultures of facial nucleus origin due to its long lasting fluorescence, non cytotoxity and realtively slow diffusion. FB would not be used as a tracer for cultured neurons though it has a higher labeling rate.