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Irene Kochevar, Thomas Gisel, Erol Verter, Giuliano Scarcelli, Seok Yun, Robert Webb, Robert Redmond, Samir Melki; Crosslinking Corneal Collagen using Rose Bengal and Green Light. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5286.
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We have previously shown that Rose Bengal (RB) plus green light markedly stiffens the corneal stroma by a rapid collagen crosslinking process that is not toxic to keratocytes. The goals of this study were to determine the spatial localization of crosslinks, identify mechanisms and evaluate treatment safety.
RB (0.1% in PBS) was applied to de-epithelialized rabbit cornea ex vivo for 2 min. The diffusion distance of RB into the stroma was evaluated on frozen sections by fluorescence microscopy. After irradiation of RB-stained cornea with green (532 nm) light from a KTP laser (10 min, 150 J/cm2), the spatial distribution of increased elastic modulus was determined by Brillouin scattering microscopy and the average stromal stiffness was measured by uniaxial tensiometry. The availability of oxygen in the stroma for the crosslinking mechanism was evaluated by absorption spectroscopy during the irradiation. The irradiance and fluence on the retina of a model eye was measured using a newly designed light delivery system and compared to the ANSI standard damage thresholds.
RB penetration into the stroma was limited to ~120 µm from the anterior surface and did not increase over time. Consistent with this distribution, Brillouin microscopy demonstrated increased elastic modulus localized to the most anterior 120 µm of the stroma. Photo-crosslinking increased the average stromal stiffness by four-fold compared to untreated control cornea. Irradiation of RB-stained cornea in the absence of oxygen shifted the absorption maximum from 562 nm to 520 nm, a signature of photoproducts formed only under anoxic conditions. During photo-crosslinking the absorption maximum did not change suggesting an oxygen-dependent crosslinking mechanism. The irradiance and fluence at the retina of a model eye during the treatment were at least 20-fold below the ANSI damage threshold for thermal or photochemical damage.
The shallow penetration of RB into corneal stroma and formation of crosslinks within 120 µm of the anterior surface suggest that thin corneas of keratoconus or other ectatic cornea conditions can be treated. Photo-crosslinking rapidly and substantially increases cornea stiffness using safe retinal exposure levels of green light.
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