显微外科技术构建家兔股动脉局部去交感神经模型

A study on constructing： a rabbit model of local sympathetic denervation of femoral artery by microsurgery method

目的研究显微外科技术在体内去除兔股动脉交感神经的可行性。排除血管外膜交感神经的影响，为进一步研究组织工程骨神经化机制提供可靠的动物实验模型。方法2014年7月至2015年7月，将21只新西兰大白兔随机分4组：对照组（n=3）、4周组（n=6）、8周组（n=6）、12周组（n=6）。分离21只新西兰大白兔双侧股动脉，实验组于股动脉中段剥除血管外膜，对照组仅做分离。于术后4周、8周、12周分别取材行苏木精-伊红（HE）染色观察血管壁结构、乙醛酸荧光染色观察交感神经分布及其荧光强度、Westemblot检测交感神经特异性蛋白酪氨酸羟化酶（TH）的含量。结果HE染色的结果：对照组动脉血管各层结构完整，3个实验组动脉外膜层缺失或明显变薄。乙醛酸染色结果：对照组、4周组、8周组、12周组的荧光强度A值分别为0．8626±0．03519、0．3631±0．03019、0．3964±0．02239、0．3487±0．02356，各实验组相比对照组荧光分布范围明显缩小，荧光强度显著减弱，差异有统计学意义（P〈0．05），各实验组之间的荧光强度差异无统计学意义（P〉0．05）。Westernblot结果：对照组、4周组、8周组、12周组WB条带半定量分析值分别为0．08124±0．00260、0．02920±0．00206、0．02661±0．00233、0．03094±0．00211，各实验组与对照组相比，TH的含量均明显减少，结果具有统计学意义（P〈0．05）；而3个实验组间差异无统计学意义（P〉0．05）。结论显微外科手术剥除法可有效去除动脉血管外膜的交感神经。该方法作用持久，交感神经去除彻底且在4周、8周、12周3个观察点内无论从神经递质方面还是特异性蛋白方面都没有观察到交感神经的明显再生，故该方法可为研究单纯血管因素对组织工程骨的影响提供持久、有效、可靠的动物模型。

Objective To study the feasibility of microsurgical technique to denervate sympathetic of femoral artery in rabbit, providing a reliable animal experimental model for further study of the mechanism of neuralization in bone tissue engineering. Methods From July, 2014 to July, 2015, 21 New Zealand white rabbits were divided in- to 4 groups randomly： the control group （n = 3）, the 4 weeks group （n = 6）, the 8 weeks group （n = 6） and the 12 weeks group （n = 6）. Bilateral femoral arteries of the 21 rabbits were exposed. Adventitia of femoral arteries in 3 test groups were removed for about 2cm by microsurgical technique, whereas adventitia of the control group remained in- tact without any treatment. The arteries samples were collected at 4 weeks, 8 weeks and 12 weeks after treatment. The structure of vascular were indicated by hematoxylin-eosin （HE） staining, and the distribution and volume of the sympathetic fibers were evidenced by glyoxylic acid staining and the expression of tyrosine hydroxylase （TH）, the marked protein of sympathetic. Results The adventitia of 3 test groups were invisible or lost most of it while the control group remained intact shown by HE staining. For glyoxylic acid staining, the fluorescence intensity value of the control group, 4 weeks group, 8 weeks and 12 weeks were 0.08124 ± 0.00260, 0.02920 ± 0.00206, 0.02661 ± 0.00233, 0.03094 ± 0.00211, respectively （n = 6）. The distribution and fluorescence intensity of sympathetic nerve were both significantly reduced in test groups compared to the control group （P 〈 0.05）. And there was no statistical difference among the 3 test groups （P 〉 0.05）. Semi-quantitative analysis of the expression of TH was 0.8626 ± 0.03519, 0.3631 ±0.03019, 0.3964 ± 0.02239, 0.3487 ± 0.02356 respectively, which showed the same tendency as glyoxylic acid staining test. Conclusion Microsurgical technique is promising as an ideal method for the local denervation of sympathetic nerve from artery system as it can significantly reduce sympathetic fibers on adventitia without regeneration during the experimental period.