Changes in Collagen Structure and Permeability of Rat and Human Sclera After Crosslinking

Abstract

Purpose To use second harmonic generation imaging and fluorescence recovery after photobleaching to demonstrate alterations in scleral collagen structure and permeability after crosslinking in rat and human eyes. Methods Excised rat and human scleras were imaged ex vivo with an inverted two-photon excitation fluorescence microscope before and after photochemical crosslinking using riboflavin and 405-nm laser light. Fluorescence recovery after photobleaching was applied to measure the diffusion of fluorescein isothiocyanate–dextran across the sclera. Results Crosslinking caused scleral collagen fibers to become wavier and more densely packed, with surface collagen being more affected than deeper collagen fibers. Crosslinked sclera showed significantly decreased permeability in the irradiation zone and also extended as far as 250 µm outside the irradiation zone. Conclusions Photochemical crosslinking induced changes in scleral structure and permeability that extended to tissue even outside the irradiation zone. Translational Relevance Ultrastructural changes associated with the emerging clinical technique of photochemical scleral crosslinking have not been well characterized. We demonstrate not only changes in scleral collagen by second harmonic generation imaging but also the associated functional changes in tissue permeability by fluorescence recovery after photobleaching. We report the novel finding of reduced permeability extending well beyond the direct irradiation zone. This has implications for control in the clinical setting.

Changes in Rat Scleral Collagen Structure Induced by UVA–Riboflavin Crosslinking at Various Tissue Depths in Whole Globe Versus Scleral Patch

“…SHG sample preparation and imaging were performed as previously described. 12 Briefly, SHG images were generated with an inverted Olympus IX81 two-photon excited fluorescence microscope (FluoView FV-1000; Olympus Corporation of the Americas, Central Valley, PA, USA). The tissue (2 × 2 mm 2 ) was placed on a glass-bottom plate (Thermo Fisher Scientific, Waltham, MA, USA) with the outer scleral surface facing downward and cover-slipped with PBS.…”

Collagen Structural Changes in Rat Tarsus After Crosslinking

“…This was done for 10 randomly selected fibers in each tarsal plate, a methodology adapted from prior work . 9 , 18 , 19 From the experimental area (512 × 512 µm), these fibers were chosen randomly, separated by a set distance. The investigator analyzing the images was masked to the riboflavin concentration.…”

Ocular Barriers and Their Influence on Gene Therapy Products Delivery

“…In humans, surgical thinning increases the permeability to macromolecules. Cryotherapy and transscleral application of a diode laser had no significant impact [ 18 , 24 ], but crosslinking reduces scleral permeability up to 200 µm around the treated area [ 25 ].…”