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Theo G. Seiler, Ana Batista, Beatrice E. Frueh, Karsten Koenig; Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans. Invest. Ophthalmol. Vis. Sci. 2019;60(6):2140-2145. https://doi.org/10.1167/iovs.19-26686.
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To determine the riboflavin concentration in the posterior corneal stroma, Descemet's membrane, and endothelium prior to UV irradiation in corneal cross-linking (CXL) in humans.
Five human deepithelialized cadaver corneas were mounted into artificial anterior chambers. After the establishment of stable physiological hydration, two-photon imaging with a certified multiphoton tomograph was used to determine fluorescence intensity and second harmonic generation signals from collagen throughout each cornea by optical sectioning, with a step size of 2.5 μm. Afterward, 0.1% riboflavin solution was applied to the anterior corneal surface, similar to the standard CXL protocol. To determine the absolute riboflavin concentration immediately before UV irradiation, corneas were measured by two-photon imaging just at the end of the riboflavin imbibition and after riboflavin saturation.
The topical application of 0.1% riboflavin results in a riboflavin concentration that decreases to 0.035% in the posterior stroma. Inside Descemet's membrane and endothelium, the concentration drops further to only approximately 0.015% at the endothelial level. Local riboflavin distribution indicates a predominantly paracellular passive diffusion of riboflavin into the anterior chamber.
The experimentally determined riboflavin concentration of 0.015% at the endothelium shows a substantial discrepancy of a factor of 1.7 to the previously theoretically calculated 0.025%. A lower riboflavin concentration at the endothelium may enable higher radiant exposures and further improve the efficacy of CXL.
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