Neuroanatomy and neurochemistry of rat cornea: Changes with age

Purpose: To characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution. Methods: Sprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis. Results: Thick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% +/- 3.5% and 34.9% +/- 5.1%, respectively, of the total nerves. VIP was 18.4% +/- 5.7%, and NPY and TH positive fibers took up 6.92% +/- 2.66% and 2.92% +/- 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks. Conclusion: This study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.