Novel corneal crosslinking technique with eosin-Y and visible light

Abstract

Purpose: Keratoconus is a progressive disease which results thinning of the cornea and increase in the forward curvature of the cornea until corneal rupture. The first choice in clinical management of keratoconus is Ultraviolet-A(UV-A) crosslinking procedure with riboflavin. This procedure is painful, prolonged and has many side effects on the patient. Eosin-y is well known molecule and it has been used as crosslinker with visible light on hydrogels with various cells. Methods: Firstly, we have examined effects of sequential concentrations of eosin-y on proliferation and apoptosis of human corneal epithelial cells (HCECs). We have used MTT cytotoxicity assay, Xcelligence real-time cell analysis, Ki67 and Caspase-3 immunofluorescence staining. After these tests, visible light (525 nm) induced crosslinking has performed with Eosin-Y in fresh bovine corneas at selected doses. Corneas which applied eosin-y or riboflavin, or saline are observed under scanning electron microscopy for topographic, cross-sectional and elemental analysis. Their young moduli measured with hybrid rheometer. Lastly, resistance to osmotic stress and chemical digestion of the corneas compared with water swelling test and enzymatic digestion test, respectively. Results: We have demonstrated non-toxic doses of eosin-y with cytotoxicity and immunofluorescence experiments on HCECs. We determined LD50 concentration of eosin-y for HCECs. After that, we have showed significant crosslinking with eosin-y in fresh bovine corneas. Eosin-y and visible light procedure has created faster and stronger corneal crosslinking reaction on bovine corneas compared to riboflavin and UV-A procedure. Conclusions: We have observed, eosin-y initiate faster and stronger crosslinking in corneal stroma at subtoxic doses than standard procedure. It could be new photo-initiator agent for corneal crosslinking procedure in clinics. For next step, we will examine its toxicity on corneal stromal cells and corneal endothelial cells.