Scleral collagen remodeling and repair assessed in intact eyes through Second Harmonic Generation

Abstract

Purpose : The remodeling of the sclera during myopia progression is initiated through the activation of matrix metalloproteases (MMPs), which play a pivotal role in collagen digestion. Recently, we identified reparative capabilities by collagen-mimetic peptides (CMPs) within collagen structures in excised sclera. Here, we used Second Harmonic Generation (SHG) Microscopy to study the structural alterations of the scleral collagen network induced by MMP-1 digestion and CMP-induced restoration in intact eyes.

Methods : A custom-built two-photon SHG microscope was used to measure the direct backward scattering signal of collagen fibers in the posterior sclera. This system was coupled with a compressive fixture (3 mm height) enabling ex vivo whole-eye imaging of male C57BL/6J mice, postnatal age 34-43 days (n=8 eyes). Stacks of images were captured along the optical axis (1 μm intervals) in two field of view configurations (FoV): 200μmx200μm (0.7 μm/px) and 500μmx500μm (1.7 μm/px). Sample eyes were imaged at 3 timepoints: Immediately after enucleation (virgin), MMP-1 treatments (TDzyme; 10μL drop, 200μg/mL, 30 min), and CMP treatment “ST-103” (Stuart Therapeutics; 10μL drop, 250μg/mL, 30 min). Custom processing algorithms were used to assess anisotropy (FoV1; order coefficient: OC) and collagen density (FoV2; normalized Irradiance: NI).

Results : Control experiments in contralateral eyes did not show differences in collagen organization between untreated eyes and sham controls (n=5). Collagen structure was observed to a depth of at least 9 µm in virgin and CMP-treated eyes and 15 µm in MMP-treated eyes. Virgin sclerae showed high degree of collagen interweaving and density throughout the entire depth (n=3: OC=0.26±0.02 and NI=0.96±0.06, respectively). MMP-1 produced a qualitative reduction of collagen interweaving (OC=0.32±0.04) and density (NI=0.46±.07). CMP restored collagen anisotropy (OC=0.26±0.02) and density (NI=0.79±0.04, respectively) to values close to virgin levels.

Conclusions : SHG microscopy allowed characterization of scleral collagen organization in intact eyes in murine models, avoiding the need of excising tissue. This method is well suited to future characterization of scleral collagen remodeling in murine and other animal myopia models, which can be mimicked by MMP enzymatic digestion. CMP appeared to restore scleral collagen ex vivo, holding promise as a therapeutic agent for myopia.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.