Corneal alkali burn in mice: an update to adapt the model for comparative investigation of healing outcomes.

Abstract

We set a goal to establish a robust model of alkali burn by various NaOH concentrations that would induce either haze-free healing or pathological remodeling for further use in comparative animal studies. Using C57/BL6 mice as a model object we modified existing protocols that utilize filter paper disc as a carrier for alkali solution application and established that 30 seconds of burn by 0.5M or 1M NaOH result in desired outcomes of haze-free healing or haze formation respectively. Using these concentrations we obtained whole mount corneas at 3 hours, 7 and 21 days after injury and immunolabeled myofibroblasts, blood and lymphatic vessels for subsequent confocal microscopy. We found that both concentrations induce full thickness injury with anuclear zone being filled by cells by day 7 with a decline of cell count on day 21. After injury by 0.5M myofibroblasts were detected at day 7 which was followed by their vanishment by day 21. After 1M alkali injury myofibroblast count was higher at day 7 (vs. 0.5M) and at day 21 their quantity reduced, but αSMA+ cells persisted which was well-concordant with outcome to haze formation. Rapid re-epithelization was vivid in both groups and had no significant differences while endothelium was partially replaced by αSMA+ irregular cell patches observed after injury by 1M NaOH which was not observed in 0.5M group. Similarly, prominent neovascularization by blood and lymphatic branches was observed only after 1M injury and mature αSMA+ vessels were formed as early as day 7 penetrating to the central portion of the cornea. Thus, we have found persistence of myofibroblasts and full-scale neovascularization only followed by haze formation in 1M alkali burn, but not in 0.5M which resulted in haze-free healing after significant initial injury. We believe that application of this proposed updates provides a tool for comparative investigation of haze-free healing vs. pathological remodeling as a promising approach to identify novel molecular targets for therapies in ophthalmology.