Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model

Abstract

Citation: Gore DM, O'Brart D, French P, Dunsby C, Allan BD. Transepithelial riboflavin absorption in an ex vivo rabbit corneal model. Invest Ophthalmol Vis Sci. 2015;56:5006-5011. DOI:10.1167/iovs.15-16903 PURPOSE. To measure depth-specific riboflavin concentrations in corneal stroma using twophoton fluorescence microscopy and compare commercially available transepithelial corneal collagen cross-linking (CXL) protocols. METHODS.Transepithelial CXL riboflavin preparations-MedioCross TE, Ribocross TE, Paracel plus VibeX Xtra, and iontophoresis with Ricrolinþ-were applied to the corneal surface of fresh postmortem rabbit eyes in accordance with manufacturers' recommendations for clinical use. Riboflavin 0.1% (VibeX Rapid) was applied after corneal epithelial debridement as a positive control. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35-lm thick were cut on a cryostat, mounted on a slide, and imaged by two-photon fluorescence microscopy. Mean (SD) concentrations were calculated from five globes tested for each protocol.RESULTS. Peak riboflavin concentration of 0.09% (60.01) was observed within the most superficial stroma (stromal depth 0-10 lm) in positive controls (epithelium-off). At the same depth, peak stromal riboflavin concentrations for MedioCross TE, Ricrolinþ, Paracel/Xtra, and Ribocross TE were 0.054% (60.01), 0.031% (0.003), 0.021% (60.001), and 0.015% (60.004), respectively. At a depth of 300 lm (within the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (60.006), while at the same depth MedioCross TE and Ricrolinþ achieved 0.018% (60.006) and 0.016% (0.002), respectively. None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300-lm depth. Overall, MedioCross TE was the best-performing transepithelial formulation.CONCLUSIONS. Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. Existing commercial transepithelial CXL protocols achieve relatively low riboflavin concentrations in the anterior corneal stroma when compared to gold standard epithelium-off absorption. Reduced stromal riboflavin concentration may compromise the efficacy of riboflavin/ultraviolet corneal CXL.

A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration

“…9,29,30 This has been confirmed by a series of experiments using spectrophotometry as an indirect measure of stromal riboflavin concentration. Using our TPF methodology, which can directly quantify riboflavin concentration through the entire depth of the cornea, 13,28,31 we previously confirmed these findings, demonstrating very limited riboflavin diffusion through an intact epithelium with any of the several current commercially available transepithelial riboflavin formulations and protocols.…”

“…[9][10][11][12] A number of formulations of riboflavin designed to facilitate penetration across an intact corneal epithelium are currently marketed. 13 Most contain toxic agents to increase epithelial permeability, including benzalkonium chloride (BAC), highconcentration sodium chloride, sodium ethylenediaminetetraacetic acid (EDTA), or trometamol. Despite these chemical enhancers, results in clinical studies with transepithelial riboflavin preparations have been equivocal: Some studies report similar efficacy to epithelium-off CXL, 14,15 but most report inferior results.…”

“…13 Here, using the same method, we investigated whether modified iontophoresis protocols designed by one of the authors (DPO) can enhance riboflavin penetration across an intact epithelium.…”

“…13,27 Briefly, following iontophoresis, the globes were immediately immersed in liquid nitrogen. Corneal cross sections (35 lm) were cut on a cryostat 1 mm apart (including the central meridian) and then mounted on a slide and covered with fluorescencefree immersion oil (Immersol 518 F; Carl Zeiss Ltd., Cambridge, UK) to prevent any leakage of riboflavin out of the tissue.…”

“…To avoid overlap with the absorption spectrum of riboflavin, fluorescence was detected in the range between 525 and 650 nm. (We previously investigated 13 the presence of endogenous fluorescence within this spectral band by imaging negative controls without riboflavin and detected negligible TPF signal). …”