Calcitonin gene-related peptide and corneal innervation: a developmental study in the rat

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.