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Daniel M Gore, David O’Brart, Christopher Dunsby, Bruce D S Allan; Transepithelial riboflavin absorption in an ex-vivo rabbit corneal model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3013.
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© ARVO (1962-2015); The Authors (2016-present)
To measure riboflavin concentration in the stroma achieved through an intact epithelium
Rabbit heads transported in phosphate buffered saline were received within 6 hours post-mortem. Intact globes were enucleated and different commercially-available riboflavin solutions applied to the corneas according to manufacturers’ protocols: MedioCross TE 30 minutes (0.25% riboflavin, hydroxypropyl methylcellulose [HPMC], benzalkonium Chloride [BAC]); Ribocross TE 30 minutes (0.125% riboflavin, vitamin E); Ricrolin + (0.1% riboflavin, trometamol, sodium edetate) 5 minutes 1mA iontophoresis; Paracel 4 minutes (0.25% Riboflavin, HPMC, BAC) followed by VibeX Xtra 6 minutes (0.25% riboflavin, saline). Epithelial-debrided globes soaked with 0.1% riboflavin in HPMC (VibeX Rapid) served as positive controls.<br /> At the end of the soak, the globes were immediately frozen in liquid nitrogen. 35µm corneal cross-sections were cut on a cryostat, mounted on a slide and imaged by two-photon fluorescence (TPF) microscopy. Riboflavin was excited by two-photon light of 890nm wavelength, with fluorescence signal detected between 525-650nm. TPF signals were converted to riboflavin concentration by normalizing to the TPF signal achieved in a well-slide reservoir of 0.1% riboflavin solution. Mean (SD) concentrations were calculated from 5 globes tested for each protocol.
Peak riboflavin concentration of 0.09% (±0.01) was observed within the anterior stroma in positive controls (epithelium-off). Peak riboflavin concentrations for Mediocross TE, Paracel/Xtra, Iontophoresis and Ribocross TE were: 0.054% (±0.01), 0.021% (±0.001), 0.020% (± 0.002) and 0.015% (±0.004) respectively.<br /> At a depth of 300µm (at the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (±0.006), while at the same depth MedioCross TE achieved only 0.018% (±0.006). None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300µm depth.
Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. None of the transepithelial protocols tested matched stromal concentrations achieved ‘epithelium-off.’
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