Mechanism and clinical significance of superficial micropunctate fluorescein staining of the cornea

Abstract

It is for well over a century that fluorescein has been used as a preferred dye for identifying and enhancing corneal surface change. Superficial micropunctate staining of the corneal epithelium is the most commonly encountered clinical manifestation of fluorescein staining. Despite its well accepted use, neither the cellular basis nor the clinical significance of this regularly seen phenomenon are yet clear. Despite this uncertainty, the presence of corneal staining has traditionally been used as a clinical tool to identify corneal surface changes. Recent literature reports have argued for more thorough scientific evidence to be produced attesting to the exact status of the corneal epithelium when it appears to have stained with fluorescein. In this thesis we systematically evaluated the mechanism and clinical significance of superficial micropunctate fluorescein staining of the cornea. A series of experiments were designed to determine the cellular basis of corneal staining. Six mechanisms were identified from literature reports, these were pooling of fluorescein in voids on the ocular surface, accumulation of dye solution in intercellular spaces, staining of live, damaged or dead cells, a response to exposed cells un-protected by the mucin layer and the possibility that the staining appearance is an artefact. Evaluations were aimed to determine which of these mechanisms dominates in the process. A staining model was developed to facilitate evaluation in human subjects and when this could not be used due to safety or ethical considerations, in vitro cell culture and ex vivo organ culture models were utilized. Observations were performed at high magnification and were aided by simultaneous staining with metabolic dyes to identify live, apoptotic and dead cells. The results of these studies suggest that healthy corneal epithelial cells take up fluorescein to a moderate level and dead cells stain minimally. Fluorescent intensities associated with both these situations are not typically observed with clinical slit-lamp bio-microscopy. Irrespective of the cause of insult, apoptotic cells were associated with bright florescence, i.e. hyper-fluorescence relative to the background of normal cells. We conclude that the bright fluorescence perceived as micropunctate dots on ocular surface when observed with slit-lamp bio-microscope indicates apoptotic, but not dead, cells. Such staining was also associated with an increase in the level of some, but not all inflammatory mediators in the tear film.