Epithelial innervation of human cornea - A three-dimensional study using confocal loser scanning fluorescence microscopy

Purpose: Evaluation of a new method to visualize distribution and morphology of human corneal nerves (A delta- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Methods: Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 mu m thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope Odyssey XL, step size between 0.5 and 1 mu m, and optical sections were digitally 3D-reconstructed. Results: Immediate staining of explanted corneas by the Life/DeadKit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, A delta- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. A delta-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, A delta-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Conclusions: Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.