Picrosirius Red staining and analysis were performed on 7 μm mid-coronal disc sections from 6-, 14- and 24-month-old animals as described before (45 (link)). The stained sections were imaged using Eclipse LV100 POL (Nikon, Tokyo, Japan) with a 10x/0.25 Pol/WD 7.0 objective Nikon’s Digital Sight DS-Fi2 camera. Images were analyzed in the NIS Elements Viewer software (Nikon, Tokyo, Japan). Under polarized light, stained collagen bundles appear as either green, yellow, or red pixels that correlate to fiber thickness indicating thin, intermediate, or thick fiber thickness respectively which can be quantified (46 (link)). In NIS Elements Viewer software fibers were quantified by thresholding for green, yellow, and red pixels over the selected region of interest (ROI). The ROIs were determined by selecting for NP or AF area using the ROI selection tool. ROIs were then thresholded for green, yellow, and red separately and the percentage area of staining was used to quantify the results for each compartment.