鸡胚发育过程中角膜神经的分布和变化

Innervation of the chick embryo cornea during development

背景了解动物或人的角膜神经分布和发育过程对于角膜疾病的临床和基础研究具有重要意义，目前关于动物角膜神经发育和特异性定位的研究结果已有发表，但是有关胚胎发育阶段角膜神经纤维的分布规律和角膜神经纤维的定量研究结果尚少见。目的了解鸡胚发育过程中角膜神经纤维的分布，并定量评价随着鸡胚龄增长其角膜神经纤维长度和密度的变化规律。方法选取胚胎期6～20d（E6-E20）的鸡胚作为角膜胚胎发育模型，获取相应胚期的带有角膜缘的完整鸡角膜，使用β-tubulinⅢ抗体进行角膜免疫荧光染色，以标记角膜神经，将角膜上皮面朝上做角膜上皮的放射状切开，制备全角膜铺片，用含DAPI抗荧光淬灭缓冲甘油封片。利用正置荧光显微镜拍摄获取整个角膜的神经纤维图像，使用PhotoshopCS4测量不同胚龄的鸡胚角膜表面积和神经纤维束数量，采用Imaris x647．4．2软件测量不同胚龄的鸡胚角膜神经纤维总长度和密度。结果全角膜铺片显示，鸡胚E6～E8可见神经束从颞侧巩膜进入角膜缘，E9～E10时神经纤维在角膜缘呈环状分布，E11～E15时延伸进入角膜中央，E16～E20时角膜形成神经纤维丛。E6～E20期间，鸡胚角膜表面积、角膜神经纤维长度、角膜神经纤维密度均随着胚龄的增长而逐渐增加，总体比较差异均有统计学意义（F=127．007、227．051、67．748，均P〈0.01），鸡胚角膜表面积与角膜神经纤维长度间呈强正相关（r=0．863，P〈0．01）。鸡胚角膜神经纤维束从E13开始出现，为（59．00±1．14）／mm^2，此后缓慢增加，至E18达到高峰，数量为（576．75±29．16）／mm^2，至E20时角膜神经纤维束数量减少，为（299．67±25．46）／mm^2，不同胚龄期鸡胚角膜神经纤维束数量的总体比较差异有统计学意义（F=13．759，P=0．000）。结论鸡胚角膜神经发育从E9开始，随着发育鸡胚角膜表面积、角膜神经纤维总长度及密度均快速增加。

Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases. At present, some studies on cornea nerve development and location have been performed. However, the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported. Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo, and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo. Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days （E6-E20） , and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody. The corneas were radially cut into 4 parts,and the integrate corneal fiat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI. Fluorescence microscope was used to capture the nerve fiber images in cornea,and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4. Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software. Results Total cornea fiat mounts showed that the nerve bundles grew from temporal seleral forward cornea limbus at E6-E8, and the nerve fibers formed the ring surrounding by limbus during E9-E10. Then the fibers extended forward the central cornea in Ell to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20. During the period of E6-E20, the corneal surface area, the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo, showing statistically significant differences among different time points （F= 127. 007,227. 051,67. 748, all at P〈0. 01 ）. The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber （ r = 0. 863, P〈0.01 ）. Chick corneal nerve fiber bundle appeared at E13, with a number of （59.00±1.14）/mm^2 and then increased to a peak of （576.75±29. 16）/mm^2 at E18 and reduced to （299.67±25.46 ）/mm^2 at E20, with a significant difference among them （ F = 13. 759, P = 0. 000）. Conclusions Corneal nerve starts to develop in E9 of chick embryo,and the corneal surface area,the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo.