Live whole-eye, ex vivo imaging and laser-induced micro injury of the corneal basal epithelium and visualization of resident macrophage responses

Abstract

PurposeIn this paper, we describe a novel live-imaging approach to visualize the short-term response of the mouse cornea to basal epithelial cell damage. Laser scanning confocal microscopy was used to induce precisely-defined, localized corneal basal epithelial cell damage and the live macrophage response to this damage was visualized and analyzed. MethodsLipophilic fluorescent dyes, SGC5 or FM 4-64, were injected into the anterior chamber of enucleated eyes and imaged live in whole-mount using confocal laser scanning microscopy. Laser-induced damage was performed by focusing onto a defined region of the corneal basal epithelium for a brief period using a high laser power setting and then returning to low laser power for imaging. Eyes from CX3CR1+/GFP mice were used to observe macrophage responses to laser damage in real-time. ResultsSGC5 or FM 4-64 dyes injected into the anterior chamber readily enter the cornea and are taken up by the stromal layer and labeled the outer membranes of corneal epithelial cells and remained stable when visualized using low laser power. Subjecting a defined region of the basal epithelium to high laser power for 1 minute or longer led to a rapid internalization of dye in the exposed basal epithelium cells and overall increase in cellular fluorescence. This change in fluorescence was also accompanied by cell swelling and contraction. Cellular internalization of the non-lipophilic, dye Alexa 647 hydrazide, indicated that membranes were compromised indicating that exposure to high power laser stimulation causes cellular damage to the basal epithelium. Visualization of corneal resident macrophages close to the site of laser-induced damage showed that within minutes, projecting macrophage filopodia extended towards the damaged region at a rate of 0.75{micro}m/min for roughly 40 minutes. ConclusionWe have developed a novel approach to image the live cornea and its response to damage. Laser-scanning confocal microscopy can be utilized to induce localized damage to mouse corneal basal epithelium and elicit a macrophage morphological response. This approach represents a useful tool for studying corneal wound healing and cellular responses to damage using live whole-mount imaging.