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Pavel Kamaev, William Eddington, Sara Rood-Ojalvo, Marc Friedman, David Muller; Accelerated corneal cross-linking with pulsed light. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5288.
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Corneal collagen cross-linking (CXL) with riboflavin (RF) strengthens corneas affected by keratoconus. This treatment involves irradiation with continuous ultraviolet-A (UVA) which de-oxygenates the cornea and makes keratocytes more susceptible to the action of light. In order to avoid this self-limiting factor of CXL, we propose to apply pulsed UVA to allow the tissue to re-oxygenate and to increase efficiency of CXL.
Porcine eye globes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA). The corneas were then de-epithelialized and 0.1% riboflavin-5’-phosphate solution was applied topically during 20 min at 37°C. Eyes were placed in a beaker filled with oxygen and irradiated for 3 min continuously or for 6 minutes with pulsed UVA light (365 nm, 1.5 sec on / 1.5 sec off) at 30 mW/cm2 using a shutter (Sutter Instrument, Novato, CA). Corneal flaps (200 µm thick) were excised from the eyes using an Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA). The flaps were placed into a biaxial extensometer (CellScale Biotester 5000, Waterloo, ON), using biorake attachments with 5 tines spanning a width of 3mm. Each sample was stretched at a constant rate of 4 µm/s in saline at 37°C until sample failure. The flaps were then washed with distilled water, dried in a vacuum and digested at 65°C with 2.5 units/ml of papain in 1x PBS (pH 7.4) containing 2 mM L-cysteine and 2 mM EDTA. Fluorescence of the papain digests was recorded at 450 nm in a QM-40 Spectrofluorometer (Photon Technology Int., London, Ontario, Canada) using excitation of 360 nm.
Continuous irradiation of the corneal tissue in presence of RF resulted in a quick depletion of dissolved oxygen. This happens via the process of generation of reactive oxygen species (including singlet oxygen) and following cross-linking, mediated by free radicals. With pulsed fractionation of UVA radiation, cross-linking efficiency is improved by allowing the re-diffusion of oxygen during pauses in exposure.
Our work confirmed that re-oxygenation of cornea during UVA pulsation increases efficiency of the CXL. From a clinical perspective, accelerated CXL with pulsed light would lead to a reduced dose for the same amount of corneal stiffening and therefore a better safety profile.
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