Involvement of corneal nerves in the progression of keratoconus

Abstract

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte–epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to α-tubulin (nerves), α3β1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.