周围神经虚拟三维重建中神经束功能及形态定位的组织化学染色方法研究

目的探索周围神经虚拟三维重建中不同性质神经纤维与功能束的组织学水平的形态学定位方法。方法取自愿捐献新鲜成人尸体右侧正中神经作为样本进行连续冰冻切片,取形态完整标本切片30个,首先采用单纯Karnovsky-Roots法对神经切片染色(A组,n=30),再依次行甲苯胺蓝染色(B组,n=28)以及丽春红2R染色(C组,n=21)。每种染色完成后即于光学显微镜下分区显微摄影(×100)并拼接成全景图像,比较不同染色方法下全景图像的纹理特征、乙酰胆碱酯酶活性部位数量及平均灰度;并对比计算机自动获取神经束轮廓结果,以确立获取理想图像的染色方案。结果 A、B、C组乙酰胆碱酯酶活性位点数量分别为(21.63±4.06)×102、(20.64±3.51)×102、(20.54±5.71)×102个;平均灰度分别为(1.41±0.06)×102、(1.10±0.05)×102、(1.14±0.07)×102。各组间乙酰胆碱酯酶活性位点数量差异无统计学意义(F=0.64,P=0.54);与A组比较,B、C组平均灰度均较低,差异有统计学意义(P<0.001)。A组仅乙酰胆碱酯酶活性部位着色;B组髓鞘显示不满意;C组可同时显示神经纤维轴突及髓鞘染色,神经束和不同性质神经纤维纹理特征最显著,神经束边界轮廓最清晰,计算机处理时假阳性容易去除,图像分割最精确。结论 Karnovsky-Roots-甲苯胺蓝-丽春红2R三重复染方法不影响神经纤维乙酰胆碱酯酶阳性位点的表达,图像纹理清晰,较符合周围神经虚拟三维重建时在组织学水平分辨及获取神经束功能状态二维图像的相关要求,有望解决周围神经组织形态学表达方式这一技术难题。

Research of histochemical staining for identifying the function and morphology of fascicles in three-dimensional reconstruction of peripheral nerves

Objective To explore the histochemical staining for distinguishing and localizing nerve fibers and fascicles at histological level in three-dimensional reconstruction of peripheral nerves.Methods The right median nerve was harvested from one fresh cadaver and embedded in OCT compound.The sample was serially horizontally sliced with 6 μm thickness.All sections were stained with Karnovsky-Roots method(group A,n=30) firstly and then stained with toluidine blue(group B,n=28) and Ponceau 2R(group C,n=21) in proper sequence.The results of each step were taken photos(× 100).After successfully stitching,the two-dimensional panorama images were compared,including texture feature,the number and aver gray level of area showing acetylcholinesterase(AchE) activity,and result of auto microscopic medical image segmentation.Results In groups A,B,and C,the number of AchE-positive area was(21.63 ± 4.06)× 102,(20.64 ± 3.51)× 102,and(20.54 ± 5.71)× 102,respectively,showing no significant difference among 3 groups(F=0.64,P=0.54);the mean gray level was(1.41 ± 0.06)× 102,(1.10 ± 0.05)× 102,and(1.14 ± 0.07)× 102,respectively,showing significant differences between group A and groups B and C(P < 0.001).In the image of group A,only AchE-positive area was stained;in the image of group B,myelin sheath was obscure;and in the image of group C,axons and myelin sheath could be indentified,the character of nerve fibers could be distinguished clearly and accurately,and the image segmentation of fascicles could be achieved easier than other 2 images. Conclusion The image of Karnovsky-Roots-toluidine blue-Ponceau 2R staining has no effect on the AchE-positive area in the image of Karnovsky-Roots staining and shows better texture feature.This improved histochemical process may provide ideal image for the three-dimensional reconstruction of peripheral nerves.