Abstract
Purpose :
Understanding scleral collagen remodeling in refractive eye development is essential to new therapeutical approaches for Myopia, and to correlate the structural, metabolical and mechanical changes occurring. We combine fluorescence lifetime measurements, and second harmonic generation (SHG) microscopy to investigate the links between molecular and structural changes following riboflavin mediated scleral crosslinking (SCXL).
Methods :
Measurements were performed in a porcine eye model (10 porcine eyes, Animal Biotech Industries) in control and treated regions, with riboflavin-UVA light cross-linking (UVX-SCXL) (Dresden protocol, 370nm, 3mW/cm2, 30min), using a commercial device (Avedro, Glaukos). After SCXL, scleral strips were cut from both treated and non-treated regions and measurements performed in the treated/non-treated riboflavin immersed strips. Porcine eyes were imaged using time-correlated single photon counting (TCSPC) and SHG. The fluorescence decay curves from riboflavin molecules in scleral tissues were acquired by using a picosecond laser (VisIR-765, PicoQuant, 382nm) with a time resolution of 35 ps, and the lifetime coefficients (T1 and T2) were determined using a conventional double exponential model. SHG measurements were performed using a two-photon microscope (IVM-MS2, IVIM Technology) using fluorescent microbeads as precise 3D landmarks.
Results :
Time constants, T1 and T2, were spatially uniform in the scleral tissues prior to SCXL (avg T1 4.87±0.26 ns; T2 3.30±0.60 ns; p<0.01). SCXL reduced T2 significantly (avg T2 2.18±0.28 ns, ↓34%, p<0.01; T1 5.22±0.47 ns). The reduction of T2 was lower in POSTERIOR than EQUATORIAL regions (EQ: ↓39%; POST: ↓22%). The SCXL treatment increased autofluorescence intensity with regional differences (POST>EQ). The spectral profiles of riboflavin fluorescence showed some changes after SCXL in the 400-490 nm region. The brightness of SHG signal in deeper tissue regions increased after SCXL (↑19%, p<0.01).
Conclusions :
The increase in both the fluorescence intensity and SHG might indicate an increase in the number of collagen crosslinks and densification after SCXL. The combination of fluorescence lifetime and SHG has potential to provide quantitative insights into the metabolic and structural changes of scleral collagen remodeling.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.