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C. L. Springs, M. Balasubramanian, S. Cheluvaraja, R. S. Pinapati, S. P. Srinivas; Phototoxic Effects on the Corneal Endothelium During (Riboflavin/UV-A)-Mediated Collagen Cross-Linking. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6422.
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Photochemical cross-linking of stromal collagen to halt progression of corneal ectasia is a new approach to treat keratoconus. The treatment consists of irradiation of the affected cornea with UV-A (365 nm; 2 mW/cm2) concomitant with topical riboflavin (Rf; 0.1%). We have characterized the phototoxicity of the treatment on corneal endothelium using electrical-cell substrate impedance sensing (ECIS).
Bovine cells were grown to confluence on gold electrodes for ECIS-based measurements of trans-endothelial electrical resistance (TER), which decreases upon cell death and stress-induced loss of integrity of the apical junctions. After steady state in TER in 24 hrs, cells were challenged with Rf and UV-A light (30 min, 0.5 mW/cm2). Lethal dose for 50% loss in TER was calculated from response to 1-200 µM of Rf. UV-A transmittance across the bovine cornea, with and without topical Rf, was assessed using a fiber-optic spectrometer.
UV-A alone did not induce a loss in TER except for a small transient decrease that lasted for the duration of UV-A exposure (i.e., 30 min). In the presence Rf (> 30 µM), similar exposure to UV-A led to an acute fall followed by a sustained loss for > 20 hrs. This biphasic response obtained was similar to that produced by rose bengal (40 nM)/white light (0.1 mW/cm2; 30 min) exposure.LD50 for Rf was 160, 80, and 90 µM for loss in TER at 8, 12, and 16 hrs, respectively. When the medium contained catalase (7000 units/mL), Rf/UV-A had no effect on TER. Rf/UV-A produced both apoptosis and necrosis as measured by flow cytometry. Upon topical Rf, transmittance at 375 nm reduced by 50% immediately, but recovered to ~ 50% in 30 min.
At Rf levels > 30 µM, UV-A irradiation employed for CxL is phototoxic to corneal endothelial cells. Although Type-1 reaction mediated singlet oxygen is implicated in collagen cross-linking, catalase effect suggests that the toxicity involves H2O2 production (i.e., Type II reaction). The immediate loss in UV transmittance is secondary to formation of a thick film of Rf on the stromal surface upon administration of the drop.
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