大鼠股神经分支基膜管差异的实验研究

A study on the difference of basement membrane of femoral nerve branches in rats

[目的]比较大鼠股神经分支基膜管结构和成分差异,为组织工程神经引导周围神经损伤后的精确再生提供依据。[方法]选取健康8周龄雄性SD大鼠30只,随机分为2组,实验组取股神经及其分支进行化学去细胞处理;对照组取新鲜股神经及其分支;观察指标为超微结构、组织学、免疫组织化学、拉曼光谱特征。[结果](1)超微结构:对照组股神经肌支髓鞘厚度大于皮支(1.28±0.41)μm vs (0.81±0.32)μm,(P<0.05),对照组股神经肌支基底膜厚度大于股神经皮支(34.55±2.23) nm vs (31.86±3.47) nm,(P<0.05);扫描电镜显示实验组去细胞处理后的取向性结构管状结构,无可见细胞结构;对照组扫描电镜显示可见明显分束的神经纤维及细胞结构;(2)组织学及免疫荧光显示:对照组股神经肌支乙酰胆碱酯酶染色阳性部位所占比例较大,皮支染色阴性部位所占比例较大;实验组去细胞处理后HE神经纤维呈均一红染管状细胞外基质结构,无蓝色可见细胞核结构;实验组神经去细胞处理后DAPI和Laminin染色为红色层粘连蛋白背景,无亮蓝色细胞核结构;(3)拉曼光谱分析显示:实验组神经去细胞处理后股神经肌支和皮支波峰出现位置无差异,主要为脂类和蛋白质及微量核酸。[结论]大鼠股神经肌支主要含运动神经纤维,皮支主要含感觉神经纤维;肌支和皮支的基膜管在结构上存在差异,其拉曼光谱特征基本相似。

[Objective] To observe the differences in the structure and components of the basement membranes of the branches of the femoral nerves in rats, and providing evidence for the precise regeneration of tissue-engineered nerves for peripheral nerve injury. [Methods] Thirty healthy 8-week-old male Sprague-Dawley rats were randomly divided into 2 groups.The animals in the experimental group were harvested femoral nerve and its branches for chemical decellularization, while those in the control group were gathered fresh femoral nerves and their branches. The ultrastructure, histology, immunohistochemistry,and Raman spectroscopy of the samples were observed. [Results] Regarding to the ultrastructure, the thickness of the myelin sheath of the femoral muscle branch was significantly greater than that of the cutaneous branch in the fresh samples(1.28±0.41μm versus 0.81±0.32 μm, P<0.05), whereas the thickness of the basement membrane of the femoral muscle branch proved significantly greater than that of the femoral cutaneous branch in the control group(34.55±2.23 nm versus 31.86±3.47 nm, P<0.05). Scanning electron microscopy showed that the samples in the experimental group treated with decellularization was oriented tubular structure without visible cell structure, while those in the control group had obviously split nerve fibers and cell structure. In term of histological staining and immunofluorescence, the proportion of positive acetylcholinesterase staining in the femoral muscle branches was larger, while the proportion of negative sites in the cutaneous branch was larger in fresh samples of the control group. By contrast, the nerve fibers in the experimental group showed some nonnuclear structure by HE staining,whereas only the red laminin background with no bright blue cell nuclear structure presented by DAPI and Laminin staining in the experimental group. In addition, the Raman spectroscopy showed that no a difference in the peak position was noticed between the muscle branch and the cutaneous branch after decellularization in the experimental group, and the main residual components were lipids, proteins and trace nu-cleic acids. [Conclusion] There are considerable differences in the basement membrane structure between the muscular branch and cutaneous branches of the rat femoral nerve, despite of similar characteristics in Raman spectroscopy.