Functional differentiation and visualization of nerve fibers in the human sciatic nerve

doi:10.12307/2022.925

Abstract

Abstract: BACKGROUND: It is the precondition of nerve recovery that nerve tracts are accurately stitched after sciatic nerve injury. However, it is unclear about the trajectory of the distal and proximal fracture ends of nerve tracts in different functional regions, and thus it is unable to accurately fix and repair the fracture end, which is the difficulty of surgical treatment, leading to fault regeneration of different functional nerve tracts. It has a great influence on postoperative neurological recovery.
OBJECTIVE: To study a set of methods for the reconstruction of motor and sensory nerve fibers in peripheral nerves, so as to provide a feasible method for seeking the distribution and trajectory of motion fibers and other fibers in the sciatic nerve and to provide the basis for accurate suture after nerve injury.
METHODS: In patients with upper-middle thigh amputation, a 1 cm sample of the sciatic nerve was taken 20 cm from the upper femoral condyle, marked using hair, fixed with paraffin, and then sliced continuously. Following immunohistochemistry and immunofluorescence staining, the nerve sections were scanned under a microscope to obtain two-dimensional image information. The muscular branch of the sural nerve and the cutaneous branch of the superficial peroneal nerve were taken from the same limb as negative and positive controls. Using CINEMA 4D software developed by Maxon Computer (Germany), the stained sections were used to reconstruct the three-dimensional images of the internal movement of the sciatic nerve and other nerve fibers.
RESULTS AND CONCLUSION: The specific antibody of motor fibers in the sciatic nerve, Anti-HB9/HLXB9 Antibody, was used to stain motor fibers, while other fibers were not dyed. Using CINEMA 4D software, two-dimensional images could be used to form three-dimensional images of nerve fibers in the sciatic nerve, and then motor fibers could be accurately distinguished from the other fibers using immunofluorescent staining. It is suggested that motor nerve fiber bundles and other nerve fiber bundles in the sciatic nerve can be accurately distinguished after immunohistochemical staining. To conclude, advanced three-dimensional reconstruction software can be used to accurately reconstruct the internal structure of the sciatic nerve, to provide an effective method for accurately differentiating motor fibers and other fibers and exploring the trajectory of nerve functional bundles.

Key words: sciatic nerve, nerve fiber, immunohistochemistry, three-dimensional reconstruction, visualization

CLC Number:

Li Xiaojun, Li Jia, Gao Feng, Li Lang, Li Qin, Ouyang Xiangyu. Functional differentiation and visualization of nerve fibers in the human sciatic nerve[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(32): 5148-5154.

Figures/Tables 4

2.1 坐骨神经二维图像横断面的观察结果二维图像横断面显示，人坐骨神经位于大腿肌间隙，呈小圆点状。将人坐骨神经节段连续切片，切片厚度为5 μm，切片间隔45 μm，间距内的石蜡组织切片的二维横断面图像信息只有极小可忽略不计的改变。电子显微镜下逐个观察切片样本的形态结构，判断坐骨神经内部结构的走向。采用参照物校正位置，保持切片的连续性。将取下的人坐骨神经石蜡包裹固定后连续组织切片，免疫组化染色后使用电子显微镜中的照相功能提取其二维图像信息，观察发现，使用免疫组化的方式染色后，功能束所含有的特异性抗体的不同，能直观明确精准地区分运动神经纤维束及其他纤维束，见图1A，B。人坐骨神经中的运动神经纤维束经过染色后颜色变深，其他神经纤维束未染色。利用免疫荧光染色，观察了距股骨髁近端20 cm坐骨神经，1 cm各断面神经束的数目和性质。阳性对照选取纯运动神经束(腓深神经肌支)，经Anti-HB9/HLXB9 Antibody特异性染色，放大50倍镜下观察显示神经纤维内均有特异性染色，见图1C；阴性对照选取纯感觉神经束(腓肠神经)，经Anti-HB9/HLXB9 Antibody特异性染色，观察显示神经纤维内没有特异性染色，见图1D。而实际的人坐骨神经样本，区别于坐骨神经内运动神经束及其他神经束，观察发现，此段坐骨神经内大部分纤维束均有特异性染色，小部分未被染色，此段坐骨神经纤维束由被特异性染色的纯运动神经束、混合神经束及纯感觉神经束组成，见图1E，坐骨神经神经束的数量总共有50束。标本的坐骨神经平面位于大腿中段，坐骨神经在大腿中部该横切面的神经纤维束数目较多；纤维交叉较频繁，由于重建坐骨神经长度为1 cm，该段坐骨神经纤维束形态、大小变化频率不高；以混合束为主。神经束数目较大腿上部减增多。该横切面总共有50个神经束，其中运动及混合神经束44个，感觉神经束6个，神经束断面形状无规律，见图1F。 "

2.2 通过CINEMA 4D软件三维立体重建人坐骨神经可视化运用CINEMA 4D软件进行三维重组，重建人坐骨神经三维形态。根据假设，神经纤维束可看作管道结构，该管道的表面是由球心沿着中轴线的球包络滚动而成。用管道沿着横断面连续切片，获得该管道的某一段结构的连续200张平行切片，利用显微成像技术将管道横断面保留为可处理图像，记录管道与切片的多组信息，图像按照切片的先后顺序依次命名为0-199.bmp，为方便数据导入和图像编辑，均保留成BMP格式，分辨率宽和高均为512 pixel。每个球的半径固定；切片间距以及图像象素的尺寸均为1[13-14]。取垂直于切片的坐标系为Z轴，平面Z=0代表第1张切片，以此类推，平面Z=199代表第200张切片，那么，坐标所代表的Z=z切片在文件中的先后次序为：(-256，-256，z)，(-256，-255，z)，…(-256，255，z)，(-255，-256，z)，(-255，-255，z)，…(-255，255，z)。按照之前的假设，管道是由一组等径的圆球，其圆心按中轴线滚动所形成的包络。每张切面与中轴线有且仅有一个交点，并且这个交点就是切片图像上最大内切圆的圆心，也就是球心的位置。因此，所要寻找的中轴线与半径，就是找图像中最大内切圆的圆心和半径，其解决方案就是求任意一个骨架上的点到所有轮廓点的最小距离，再取所有骨架点对应的最小距离的最大值，此最大值即最大内切圆的半径，对应的点即为最大内切圆的圆心。求出了最大内切圆的半径和球心后，通过拟合的方式来获取中轴线的图像，将最大内切圆的半径取平均值，得到管道半径。在计算得到最大内切圆的球心和半径的基础上，通过拟合的方式获得中轴线的图像，再根据CINEMA 4D软件排列平面的投影图。按照以上思路，200张切片的最大内切圆圆心坐标及半径，结合体质量的信息，中轴线的z轴从1-200，得到中轴线在空间中的坐标。放样制作得到管道模型。此次用创建神经管道的模型，主要应用到放样命令的操作。整个神经模型就应用不同切片路径，按照一定到等比间隙进行排列，依次链接，形成某段神经到重建复原。打开CINEMA 4D导入切面图转化来到路径，将该路径转换为可编辑多边形，按照一定到等比间隙进行排列，依次链接，内部及轮廓做一个组，见图2A。先选取内部所有轮廓，在快捷菜单栏展开中找到“放样”工具，将轮廓线处于放样的子级中。工作区即可看到一个生成的实体模型，将运动纤维及混合纤维识别出并进行重新建，见图2B。同理生成没有被抗体特异性结合的感觉纤维，见图2C。同理生成此段坐骨神经内部的滋养血管，见图2D。此模型为实体模型无法看到内部结构，对该层赋予半透明材质，点击材质求，对参数进行调节，包含颜色，半透明，在透明图层上加上噪波纹理，使其更加具有真实感，见图2E。将得到的2组模型拼合到一起，配合灯光，见图2F；调节好渲染参数进行渲染，得到效果，见图2G。 "

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