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N. schrage, S. Langefeld, M. Frentz, M. Reim, J. Becker; The Flexible Aachen Keratoprosthesis in First Clinical Application . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3943.
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Within research on eye irritation the limitations of Draize's test have been recognized and new test systems have been adressed by strategies to replace experiments on living rabbits eyes by means of contructs such as cell cultures, bovine eyes, red blood cell lyses and synthetic corneal constructs. The knowledge of eye banking and rabbit eye burn lead us to construct a new system of keeping alive the isolated rabbit cornea in a special culturing device allowing media sampling and microscopical examination from epithelial and endothelial side.
Freshly taken eyes from deceased rabbits were prepared and placed into a continous flow cultivation chamber. This chamber provides nutrition by serum free DMEM Medium from the endothelial side and incubation once daily with the medium of the epithelial airlifted side. During experiments we exposed each 16 corneas to mechanical abrasion, H2O2, NaOH, SLS. Exposured corneas were monitored on pH ,lactate and glucose in the outflow media giving insight in corneal biochemistry. Samples of early mid and late phase of experiments were analysed on cytokines such as VEGF, FGF, IL8 and IL1 by means of Luminex R&D micro–Elisa assays. Endothelial microscopy was provided to know the damage of the most susceptible structure of the cornea
We were able to maintain corneas in this system for up to 21 days. Corneal epithelial healing was achieved regularly at day 7 in culture after mechanical abrasion. We found high levels of VEGF expression in exposures towards H2O2 up to 80 pg at day 2 and 5 after exposure. In sodiumhydroxide exposure IL–8 decreased within the medium and rose from 280 +– 220 pg/ml to 780 +– 270 pg/ml in supernatants. In abraded corneas IL 8 290+–30 pg/ml rose to 375 +– 50 pg/ml post expositionem. Negative controls remained stable. IL–1 alpha and beta were measured high after Sodiumhydroxide exposition and less in case of corneal abrasion. Endothelail damage was seen mostly in detergents and in H2O2 exposures with late onset and severe depletion of the endothelial cells.
The EVEIt system that we present here gives insight in the clinically well known patterns of inflammations, endothelial damage, cytokine release of the inflamed cornea in the early phase of eye irritation. We know that our system is self healing and gives biochemical reactions comparable to living animals. Therefore we are certain that the ex vivo eye irritation assay EVEIT is a valuable method and test system for evaluation of drugs applied on the cornea as well as method for toxicity evaluation.
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