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.

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.

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 flat 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.

Total cornea flat mounts showed that the nerve bundles grew from temporal scleral 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 E11 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)/mm² and then increased to a peak of (576.75±29.16)/mm² at E18 and reduced to (299.67±25.46)/mm² at E20, with a significant difference among them (F=13.759, P=0.000).

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.

Cornea/innervation; Nerve fibers, corneal/embryology; Development; Ophthalmic nerve/physiology; Chick embryo