鼠面神经修复后轴突非同步生长与靶肌多支配终板形成的关系

To date, it is believed that functional improvements appear to be consistently associated with reduced polyinnervated motor end-plates after facial nerve repair. However, the reason for the formation of polyinnervated motor end-plates is unclear. We hypothesize that this is because early reaching terminal axons become sprouting along the surface of muscle fibers and establishing polyinnervated motor end-plates, induced by denervated muscle fibers. To test this we plan to evaluate the effects of asynchronously growing axons after facial nerve transection and immediate end to end suture in the rat, combined structural and functional analyses. The vibrissal motor performance is monitored periodically using a video-based motion analysis in order to determine the time of motor recovery (include the vibrissae erect again, restoration of rhythmical whisking, protraction and retraction movement, and finished recovery of function). At 3 selective time (i.e. time of the vibrissae erect again, the vibrissal movement and finished recovery of function), neuron counts and accuracy of target reinnervation are studied using preoperative/postoperative labeling in the vibrissal pad muscles and double retrograde labeling (Fluoro Gold, FG and Fast Blue, FB) of facial motoneurons respectively, the proportion of mono-, poly-,and noninnervated motor endplates in the levator labii superioris muscle is determined by means of immunocytochemistry for β-tubulin and histochemistry with α-bungarotoxin, the expressive levels of neural cell adhesion molecule (NCAM) and brain-derived neurotrophic factor (BDNF) in the tartget muscle is observed by immunohistochemistry and real time polymerase chain reaction (RT-PCR). By botulinum toxin injection in vibrissal pad muscles as a paralysis model, the time of functional recovery is considered as the time from paralysis induced axonal sprouting to complete new neuromuscular joint. In addition, some rats are treated with antibodies against NCAM and BDNF to inhibit axonal sprouting at early period of functional recovery (i.e. the vibrissae erect again), respectively. The efficacy of 2 treatment paradigms is assessed by both vibrissal motor performance and morphological parameters. Our experiment may be significant in determining the relationship between asynchronously growing axons and formation of polyinnervated motor end-plates in the target muscle.

面神经功能恢复程度与靶肌多支配终板减少密切相关，但仍不清楚多支配终板形成的原因。本研究拟用基于视频运动分析法，确定大鼠面神经干修复后触须恢复直立、运动及功能稳定时间；用双重逆行示踪法评估术前、术后不同时间触须支配神经元数量和分布，明确再支配轴突到达靶肌的时间有无差异；用免疫细胞化学法和组织化学法分析终板支配形式，结合逆行示踪结果，说明失支配肌纤维是否诱导已到达靶肌的轴突发芽、形成多支配终板；用免疫组织化学法和RT-PCR评价靶肌神经细胞粘附分子(NCAM)和脑源性神经营养因子(BDNF)的表达，说明轴突发芽是否受NCAM、BDNF调控；用A型肉毒杆菌毒素致瘫大鼠触须，观察功能恢复时间，从时间方面间接说明失支配肌纤维能否诱导轴突发芽；触须恢复直立后，用抗NCAM、抗BDNF抗体进行干预，观察触须再支配神经元数量和分布、终板支配形式、功能恢复的变化；明确轴突非同步生长与多支配终板形成的关系。

一、研究内容.采用基于视频的大鼠触须运动分析方法，从功能角度反映面神经干钳夹损伤和切断吻合后触须获得神经再支配的时间和程度；在触须运动功能恢复的4个时间节点，采用神经元逆行示踪法记录，比较到达靶肌的运动神经元数量，明确再生轴突到达靶肌的时间是否存在差异，即是否存在再生轴突的非同步生长。.二、重要结果.1、术前所有动物的触须摆动行为均显示双侧同步和对称特点。面神经干钳夹损伤组术后2周，术侧触须平均摆动幅度达到正常侧的77%（37.8°/49.2°）；至12周时摆动幅度恢复至正常侧的96%（47.3°/49.2°）。面神经干切断吻合组术后4周，术侧触须平均摆动幅度达到正常侧的40%（18.7°/50.0°）；至12周时术侧触须摆动幅度恢复至正常侧的77%（37.8°/49.2°）。术后4周起，钳夹损伤和切断吻合组术侧触须摆动频率与正常侧相比，差异均无统计学意义。假手术组动物术后2周即展现了与术前一致的触须摆动行为。面神经干切断不修复组动物在观察期内术侧触须无任何摆动恢复迹象。.2、面神经干钳夹损伤组和切断吻合组动物的正常侧触须标记神经元数量差异无统计学意义。钳夹损伤组术后2周，术侧标记细胞数为正常侧的19.27%；术后4周时，术侧标记细胞数为正常侧的86.15%；术后8、12周，术侧标记细胞数分别达正常侧的126.5%和128.49%。切断吻合组术后2周时，术侧无明显可见的标记细胞；术后4周，术侧标记细胞数为正常侧的58.46%；术后8周、12周，术侧标记细胞数分别达正常侧的116.27%和131.91%。假手术组术后2周时，两侧标记细胞数无明显差异。面神经干切断不修复组术后12周时，术侧标记神经元数为0。.三、科学意义.1、普通视频技术与大鼠身体限制措施、运动分析软件相结合，可以满足面神经损伤或缺损修复后功能恢复评价的需要，为进一步确定逆行示踪再支配神经元的时间点提供了客观、量化的功能恢复依据。.2、通过荧光金逆行示踪再支配触须运动神经元数，初步明确了面神经干损伤后再生轴突存在非同步生长现象，为进一步研究再生轴突非同步生长与靶肌多支配终板形成之间的关系奠定了基础。