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Structural Organization of the Sclera in Chicks after Deprivation Myopia Measured Using Second Harmonic Generation Microscopy


Purpose : The outer layer of the sclera is a dense connective tissue mainly composed of collagen fibers. Abnormal eye growth during myopia progression is associated with extracellular matrix (ECM) restructuring and scleral thinning. Second Harmonic Generation (SHG) microscopy allows visualizing collagen-based tissues without the use of markers. In this work, we obtained SHG images from scleral tissues to objectively quantify possible changes in collagen organization as a function of different amounts of induced deprivation myopia in chicks.

Methods : A SHG microscope operating in backscattering-mode was used to image chick scleral tissues in 26 eyes from areas close to the optic nerve head. Unstained scleral punches of both control and deprived (mean refraction=-8D) eyes were used in the experiment. Myopia was induced by placing a translucent diffuser in one of the chickens’ eyes for one week. Two SHG imaging modalities were employed: Tomographic (XZ or YZ), and regular XY images. The former allowed direct estimation of the scleral thickness, while the latter were used to characterize the collagen fiber structural dispersion by means of Fourier and Radon transforms.

Results : Scleral collagen fibers provided strong SHG signals for analysis. We did not find differences in scleral thickness as a function of ocular refraction. On the opposite, fiber structural dispersion significant increased with refraction (R=0.48, p=0.01) and decreased with axial length (R=0.67, p=0.0002). The structure of scleral collagen fibers ranged between a non-organized and a quasi-aligned (with a dominant direction of the fibers) distribution.

Conclusions : SHG microscopy imaging provided a high-resolution visualization of individual collagen fibers of the chicken sclera with different levels of induced myopia. A significant decrease in fiber structural dispersion was associated to a higher myopic refraction. Nevertheless, the thickness of the control sclerae and that of deprived eyes were similar. This technique allows quantifying alterations during myopia induction, providing a useful tool for a better understanding of the emmetropization processes.

This abstract was presented (poster) at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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