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James Germann, Eduardo Martinez-Enriquez, Susana Marcos; Collagen arrangement from Second Harmonic Microscopy in corneas following cross-linking. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2907.
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© 2017 Association for Research in Vision and Ophthalmology.
Cross-linking (CXL) is a treatment for keratoconus that counteracts the weakening of the cornea by promoting the formation of links between the collagen fibers of the corneal stroma. How CXL affects the physical structure of the stroma and the organization of the collagen lamella is still precisely unknown. This study compares the orientation of collagen lamella of both cross-linked and untreated eyes and compares the average variance of lamella orientation to provide a quantitative measure of the changes induced by CXL.
Three freshly enucleated porcine eyes were obtained from a local abatoir and separated into 3 groups; untreated eyes, Riboflavin-UV (UVX) cross-linked eyes, and Rose Bengal-Green Light (RGX) cross-linked eyes. CXL treatments involved de-epithelization, photosensitizer instillation (0.125% Riboflavin/20% Dextran solution every 5 min for 30 min in UVX; Rose Bengal at 0.1% in PBS for 2 min and 30 s for RGX) and light irradiation (370 nm, 3 W/cm2, 30 min, in UVAX; 532 nm, 0.25 W/cm2, 400 s, in RGX). The corneas from the eyes were excised and placed under a custom built two-photon laser scanning microscope. A z-scan was performed with each sample, in 5 μm steps. The images were 2D Fourier transformed and the resulting magnitude and direction of the collagen lamella were calculated. The number of lamella was counted in angular sections and the variance between the numbers of lamella in each angular section was calculated.
The average orientation variance for the collagen lamella bundles within the 450 um anterior cornea was calculated to be 0.0462 ± 0.0136 for the untreated eyes, 0.0425± 0.0123 for the UVX treated eyes, and 0.0376 ± 0.083 for the RGX treated eyes. The Fourier transform of the images showed 17.6% of the untreated cornea had a random orientation while the cross-linked eyes had 26.3% for the UVX and 25.3% for the RGX. Of the random slices, approximately half were found within the first 100 um of the anterior surface for the untreated eye and the UVX eye. For the RGX eye, half of the random slices were located with the first 200 um.
The more random arrangement of collagen fibers in the stroma of the CXL-treated cornea, particularly within the treated volume, indicates microscopic changes introduced by the procedure, which should relate to mechanical and optical effects post-CXL.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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