不同神经示踪剂组合对大鼠脊髓运动神经元的逆行标记效率比较

Comparison on the efficacy of different retrograde tracers for the spinal motor neurons in rat

目的：探讨神经示踪剂荧光金（FG）、真蓝（TB）和荧光红（FR）两两组合对脊髓运动神经元的标记效率差异，为再生神经重支配准确性研究奠定基础。方法：采用大鼠胫神经示踪模型，采取神经内注射与神经横断后近侧断端浸泡（20min）2种方式，分别对FG、TB和FR的两两组合进行示踪试验。示踪术后5d，取脊髓腰膨大段冷冻纵切，共聚焦显微镜进行显微成像和计数。结果：FG联合TB示踪标记的运动神经元数量最多，其次为FG联合FR，而FR联合TB组标记细胞数最少，双标比例也最小。神经断端浸泡方式使用示踪剂时标记效率仅为神经内注射的2／3左右。结论：FG联合TB以及FG联合FR示踪对脊髓运动神经元的标记效果较好，且神经内注射使用示踪剂效果优子持续20min的神经断端浸泡。

Objective： To test the double-labeling efficacy of different combinations of the neural tracers fluoro-gold （FG）, true blue （TB） and fluoro-ruby （FR）. Methods： The left tibia[ nerve of adult Sprague-Dawley rats was exposed to different tracer combinations, i.e. FG-FR, FG-TB or FR-TB, either by intra-fascicular neural injection or by nerve transection and immediate immersion of the proximal nerve stump into tracer mixture for 20 min. After survival for 5 days, the lumbar enlargement of the spinal cord was dissected out and longitudinally cryo-sectioned under a laser confocal microscope and labeled motor neurons counted. Results： The tracing with a combination of FG and TB gave the largest number of labeled motor neurons, among which 80%-85% was doubly labeled. The combined use of FG and FR also exhibited the second largest number of labeled motor neurons with over 90% double labeling. However, the FR-TB combination group showed poor labeling efficacy in terms of total labeled neurons and double labeling ratio. Exposure to tracers via nerve stump-immersion （20 min） was observed to be significantly less efficient, i.e. 2/3 labeling, than via intra-faseieular neural injeetion. Conclusion： The combination of either FG with TB or FG with FR seemed suitable for sequential retrograde tracing in assessing nerve regeneration aeeuraey, with better labeling efficacy for intra-fascicular neural injection.