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目的基于图像分析探讨用于鉴别周围神经功能束的乙酰胆碱酯酶(AChE)染色Karnovsky-Roots法24小时内的染色规律,为基于乙酰胆碱酯酶染色的周围神经功能束三维重建确定标准化的孵育条件。方法新鲜人体截肢腓总神经标本,连续横断冰冻切片,片厚10μm,共切取56张,随机分为7组,每组8张切片,采用Karnovsky-Roots法进行乙酰胆碱酯酶孵育,其中1～6组分别为1、2、4、8、12和24 h,第7组切片为阴性对照组。采用Image-Pro Plus 6.0图像分析软件对不同时间染色组图像进行图像分析,指标为:(1)AChE反应阳性区域的面积;(2)AChE反应阳性区域的平均光密度;(3)AChE反应阳性区域的累积光密度。组间结果采用SPSS13.0进行t-test统计检验,以P<0.05为有统计学差异。结果 8h组的平均光密度值与12 h组有显著性差异,而12 h与24 h的平均光密度值无显著性差异;8 h、12 h和24 h间的染色面积、累计光密度值均存在显著性差异,但镜下可观察到24 h组存在过度染色。结论孵育12～24 h间的染色效果趋于一致,染色结果稳定可靠,神经束功能性质鉴别容易,神经束膜与染色的神经纤维间有清晰的分界,利于轮廓获取,可作为基于乙酰胆碱酯酶染色的周围神经功能束三维重建的标准化的育条件。
Objective To investigate the staining pattern of Karnovsky-Roots staining for acetylcholinesterase (AChE) staining in peripheral nerves to determine the normalized incubation conditions based on the three-dimensional reconstruction of peripheral nerve function with acetylcholinesterase staining. Methods Fresh human amputated common peroneal nerve specimens were continuously transected frozen sections and the thickness was 10μm. A total of 56 slices were taken and randomly divided into 7 groups with 8 slices in each group. Karnovsky-Roots method was used for acetylcholinesterase incubation. Among them, 1 to 6 Groups were 1, 2, 4, 8, 12 and 24 h, respectively. The seventh group was negative control group. Image analysis was performed with Image-Pro Plus 6.0 image analysis software on the images of stained groups at different times with the following indexes: (1) the area of AChE positive reaction area; (2) the average optical density of AChE positive reaction area; (3) the AChE reaction The cumulative optical density of the positive area. The results between groups using SPSS13.0 t-test statistical test to P <0.05 as a statistically significant difference. Results The average optical density value of 8h group was significantly different from that of 12h group, while the average optical density value of 12 h and 24 h had no significant difference. The staining area, cumulative optical density value between 8 h, 12 h and 24 h There was a significant difference, but the microscope can be observed 24 h group over-staining. CONCLUSION: The staining results of 12-24 h incubation tend to be the same, the staining results are stable and reliable, the functional properties of nerve bundles are easy to distinguish, and there is a clear boundary between the nerve membrane and the stained nerve fibers, which is conducive to contour acquisition and can be used as the basis of acetylcholinesterase Dyeing of peripheral nerve bundles by three-dimensional reconstruction of normalized fertility conditions.