Abstract
Purpose :
Up-regulation of metalloproteinases have been shown in keratoconic corneas and in myopic sclerae, likely inducing remodeling of tissue organization and biomechanical changes. Second-harmonic generation microscopy (SHG) is a useful tool for imaging collagenous tissue. In this work we used SHG to evaluate the changes in collagen fibers in sclera in presence of digestive enzymes.
Methods :
A custom-built SHG microscope with a pulsed source (Fyla, Spain, 950-1150nm, 20fs, 80MHz) was used to collect images of excised scleral tissue (200 μm × 200 μm, 5 um z-steps). Eyes (n=8) were enucleated from 4 albino rats euthanized by potassium chloride injection. The sclerae from the eyes were separated from the rest of the globe and excessive tissue was scraped off using a scalpel. Stack of images were taken near the optic nerve head and 2mm anteriorly from it Images were taken in virgin sclerae, and following 30-min immersion in collagenase (MMP-1, TDzyme, BullDog Bio) with concentration of 200 μg/mL. The tissue samples were placed with the outer side facing down in the immersion bath so that the solution permeates through the tissue and rinsed with PBS prior to imaging. The tissue samples were imaged again at the same positions before and after treatment using similar imaging parameters. The collagen fiber images taken before and after treatment were compared using an order coefficient metric (OC) previously proposed [Germann et al., 2018] which measures the degree of organization of the collagen fibers, and OC averages across all images on the volume stack and both locations are presented.
Results :
The MMP1-collagenase treated scleral fiber tissues showed a sharp decrease in uniformity of orientation compared to their untreated state (figure 1). The average OC of virgin sclerae is 0.2905±0.0018, and the average OC collagenase-treated sclerae is 0.3361±0.0227 (15.7% difference, statistically significant, paired t-test p<0.05). The fibers were visibly disarranged, interwovenness is lost, and an increase in the intra-fiber gap can be seen.
Conclusions :
Collagenase type 1 (MMP1) had a significant digesting effect on rat scleral collagen fibers, producing changes in collagen fiber architecture and organization. The collagen organization obtained from SHG imaging in virgin and collagenase digested sclerae can be useful for modeling sclera at a microscopic level and input to constituent models of scleral mechanics.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.