Collagen Organization in the Posterior Rabbit Sclera Post Cross-Linking

Abstract

Purpose : Myopia is a growing global opthalmological concern. One preventative treatment of myopia under development is scleral cross-linking (SXL), where the posterior sclera is stiffened to retard axial length growth. The optimal location and microscopic effects of SXL are still under investigation.

Methods : A total of 18 freshly enucleated rabbit eyes were treated ex vivo with one of two treatments on either the nasal (N) or temporal (T) posterior sclera: (1) Rose Bengal/green irradiation (RGX, 0.25 W/cm², 6.6 min 532nm irradiation, 2.5 min installation) (N, n=6; T, n=6); (2) Riboflavin/UVA irradiation (UVX, 3mW/cm², 30 min 370nm irradiation, 1 hr installation) (N, n=3; T, n=3). The untreated N or T side of the globe was used as a control. After treatment, scleral pieces (1.25cm × 2.5cm) were extracted from the globe and optically cleared with Murray’s clear (1:2 Benzyl Alcohol-Benzyl Benzoate mix). Images were taken of the collagen fibers through the depth of the tissue (150μm × 150μm en face, 250-300μm depth) with a custom-built, second harmonic generation microscope and analyzed with an order coefficient (OC), which quantized how uniform the direction of the fibers was (1 for uniform direction and 0 for even distribution).

Results : The average OC values were 0.400±0.008 (N) and 0.413±0.007 (T) in untreated tissue, 0.421±0.007 (N) and 0.407±0.014 (T) in RGX-treated tissue and 0.417±0.012 (N) and 0.422±0.010 (T) in UVX-treated tissue. On average, in untreated sclera, OC was 3.2% larger in the temporal than in the nasal side (p<<0.01, student t-test). In the nasal sclera, RGX and UVX treatments produced a statistically significant increase in OC (p<<0.01), by 5.1% and 3.9% respectively (p<<0.01, t-test) and in the temporal sclera, RGX decreased OC by 1.5% and UVX increased by 2.2% (p<0.05, t-test), compared to untreated tissue. UVX produced a significantly larger increase in OC than RGX (by 3.4%, p<0.05) on the temporal side (p<0.05, t-test), but there were no significant differences between UVX and RGX treatments on the nasal side.

Conclusions : Scleral collagen organization varies geographically, with the posterior temporal sclera exhibiting higher order than the nasal sclera. Both RGX and UVX SXL treatments further significantly increased collagen order in the temporal sclera, but not consistently in the nasal sclera. These findings indicate that selecting the treatment area may be critical to optimize the efficiency of SXL.

This is a 2021 ARVO Annual Meeting abstract.