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James A Germann, Eduardo Martinez-Enriquez, Carmen Martinez-Garcia, Irene E Kochevar, Susana Marcos; Depth-resolved collagen order after in vivo rabbit corneal cross-linking. Invest. Ophthalmol. Vis. Sci. 2019;60(9):326.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal cross-linking (CXL) stiffens collagen fibers by creating additional intrafiber bonds between collagen molecules. As the photoinitiator in CXL must diffuse into the cornea, a depth dependence on CXL efficacy is expected. We used custom-built Second Harmonic Generation (SHG) microscopy and quantifying routines to measure the order of collagen fibers throughout the cornea following CXL.
Rabbit corneas were treated in vivo with two different CXL modalities; Riboflavin/UV illumination (UVX) and Rose Bengal/green light illumination (RGX). Twelve corneas were imaged by SHG 1 and 2 months after treatments (n=4 control, UVX, RGX). Collagen fiber images were taken through the entire stroma (350−400 μm) at 2 μm intervals in the forward scattering direction, with dimensions of 225×225 μm and resolution of 600×600 pixels. Order coefficient (OC) analysis was used to quantify the order of collagen fibers by measuring the variance of the highest amplitude points of the 2D Fourier transform between different angular windows. Total SHG signal from each image was determined by summing up the individual pixel values. OCs were obtained for the anterior, intermediate and posterior stroma (AS, IS, PS, respectively).
After 1&2 months, OC was higher in CXL than in control corneas. The percentage increases at 1 month/2 months were: 39/44 (AS), 38/52 (IS), and 27/43 (PS) in RGX corneas, and 35/40 (AS), 31/43 (IS) and 25/34 (PS) in UVX corneas. After 1&2 months the average SHG counts per image were higher in CXL than in control corneas by 18%/26% in RGX corneas, and by 1%/10% in UVX corneas. SHG counts per image followed a ballistic model of depth penetration in all treatments.
Collagen order coefficients seem to correlate with the mechanical strength in the cornea and with treatment depths. Both analyses suggest that in rabbits: (1) CXL has a deeper effect on the cornea than the penetration depth of the photoinitiator (120 μm in RGX, 70% of CXL in anterior 200 μm in UVX), (2) RGX is more efficient than UVX in re-ordering the collagen fibers and increasing SHG signal, (3) CXL-induced remodelling in the cornea continues at least 2 months after treatment. These findings are consistent to previous reports of differences in corneal stiffness between RGX and CXL and its change with time post-surgery.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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