基于图像分析的周围神经Karnovsky-Roots法染色规律的效果观察

Karnovsky-Roots' stain of peripheral nerve based on image analysis

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 explore the feasibility of image analysis on the result ofacetylcholinesterase stain （Karnovsky-Roots＇method） of peripheral nerve, and provide the reference for the standard incubational conditions of the three-dimensional reconstruction of functional fascicles groups of peripheral nerves. Met.botts The samples came from fresh adult common peroneal nerve, which were serially horizontally sliced into 56 slices with the thickness of 10~m. All slices were randomly divided into seven groups with 8 slices in each group, including 1h, 2h, 4h, 8h, 12h, 24h and negative control group. After AChE histochemical staining, the stain characteristics of different nerve fiber in various stain phrases were observed under optic-microscope. The average area, density and integrated optical density （IOD） of AChE positive region of various stain period were analyzed by an image analytical software （Image-Pro Plus 6.0）. T-test was adopted to distinguish the group differences through SPSS13.0. Results For the mean optical density value, there was notable diversity between 8h and 12h groups, but not for the 12h and 24h groups. The significant differences also appeared among 8h, 12h, and 24h groups for stain area and IOD. Furthermore, overstain was observed on slices of 24h group. Conelt^ioas The staining results from 12h to 24h were stable and reliable, moreover, it is possible to identify different functional fascicles surrounding by perineurium. For three-dimensional reconstruction of functional fascicles groups of peripheral nerves, it can be taken as the standard stain incubational condition.