Anatomy of the human corneal innervation

Abstract

The anatomy of the human corneal innervation has been the subject of much investigation; however, a comprehensive description remains elusive. The purpose of the present study was to provide a detailed description of the human corneal innervation using a novel approach involving immunohistochemically stained anterior-cornea whole mounts. Sixteen donor corneas aged 19–78 years were cut with a 6.0 mm trephine into a central plug and two peripheral rims. Each specimen was sectioned serially on a cryostat to produce several 100 μm-thick stromal sections and a 100–140 μm-thick anterior-cornea whole mount that contained the entire corneal epithelium and much of the anterior stroma. The corneal innervation was stained with a primary antibody against beta neurotubulin and subjected to rigorous quantitative and qualitative analyses. The results showed that a mean of 71.3 ± 14.3, uniformly spaced, main stromal nerve bundles entered the cornea at the corneoscleral limbus. The bundles averaged 20.3 ± 7.0 μm in diameter, were separated by a mean spacing of 0.49 ± 0.40 mm, and entered the cornea at a mean distance of 293 ± 106 μm from the ocular surface. Each stromal bundle gave rise through repetitive branching to a moderately dense midstromal plexus and a dense subepithelial plexus (SEP). The SEP was comprised of modest numbers of straight and curvilinear nerves, most of which penetrated Bowman's membrane to supply the corneal epithelium, and a more abundant and anatomically complex population of tortuous, highly anastomotic nerves that remained largely confined in their distribution to the SEP. SEP density and anatomical complexity varied considerably among corneas and was less dense and patchier in the central cornea. A mean of 204 ± 58.5 stromal nerves penetrated Bowman's membrane to supply the central 10 mm of corneal epithelium (2.60 nerves/mm²). The density of Bowman's membrane penetrations was greater peripherally than centrally. After entering the epithelium, stromal nerves branched into groups of up to twenty subbasal nerve fibers known as epithelial leashes. Leashes in the central and intermediate cornea anastomosed extensively to form a dense, continuous subbasal nerve plexus, while leashes in the peripheral cornea demonstrated fewer anastomoses and were less complex anatomically. Viewed in its entirety, the subbasal nerve plexus formed a gentle, whorl-like assemblage of long curvilinear subbasal fibers, 1.0–8.0 mm in length, that converged on an imaginary seam or gentle spiral (vortex) approximately 2.51 ± 0.23 mm inferonasal to the corneal apex. Mean subbasal nerve fiber density near the corneal apex was 45.94 ± 5.20 mm/mm² and mean subbasal and interconnecting nerve fiber diameters in the same region were 1.51 ± 0.74 μm and 0.69 ± 0.26 μm, respectively. Intraepithelial terminals originated exclusively as branches of subbasal nerves and terminated in all epithelial layers. Nerve terminals in the wing and squamous cell layers were morphologically diverse and ranged in total length from 9 to 780 μm. The suprabasal layers of the central corneal epithelium contained approximately 605.8 terminals/mm². The results of this study provide a detailed, comprehensive description of human corneal nerve architecture and density that extends and refines existing accounts. An accurate, detailed model of the normal human corneal innervation may predict or help to understand the consequences of corneal nerve damage during refractive, cataract and other ocular surgeries.

Introduction

The human cornea is the most densely innervated surface tissue in the body. In addition to their important sensory functions, corneal nerves help maintain the functional integrity of the ocular surface by releasing trophic substances that promote corneal epithelial homeostasis and by activating brainstem circuits that stimulate reflex tear production and blinking. Consequently, damage to corneal nerves as the result of surgery, trauma or disease leads to diminished corneal sensitivity and possible transient or long-term alterations in the functional integrity of the ocular surface.

The anatomy of the human corneal innervation has been studied for many years by a variety of methods, including light and electron microscopy, immunohistochemistry and in vivo confocal microscopy (IVCM). Despite these efforts, a detailed, comprehensive description of human corneal nerve architecture remains elusive. Light and electron microscopic investigations of human corneal nerve distribution, density, and ultrastructure (Al-Aqaba et al., 2009, Muller et al., 1996, Muller et al., 1997, Schimmelpfennig, 1982, Ueda et al., 1989, Zander and Weddell, 1951) have generated most of the data on which current models of corneal innervation are based (Muller et al., 2003). More recently, IVCM has been used to image the innervation in healthy and diseased corneas and has provided considerable new information on the morphology, density, and disease- or surgical-induced alterations of corneal nerves, with special emphasis on the subbasal nerve plexus (Oliveira-Soto and Efron, 2001, Patel and McGhee, 2005; see Patel and McGhee, 2009, for review and additional references). IVCM is especially useful for imaging corneal nerves near the apex because of the relative ease of obtaining good quality tangential images in this region; however, although IVCM provides excellent resolution, it is often incapable of imaging reliably corneal epithelial terminals and very small diameter subbasal and stromal nerves.

The purpose of the present study was to provide a detailed and comprehensive description of the human corneal innervation using a novel approach that involved immunohistochemical staining of 100–140 μm-thick “anterior-cornea whole mounts” and stromal sections. This method provides excellent visualization of the main stromal bundles, midstromal plexus, subepithelial plexus, subbasal nerve plexus, and intraepithelial terminals throughout the entire cornea. The results of this study were presented in preliminary fashion at ARVO (Marfurt et al., 2008).