Retinal Vasculature Trypsin Digestion

The retinal vasculature was prepared through trypsin digestion, as previously described [17 (link)]. Briefly, retinas were fixed overnight using 4% paraformaldehyde, followed by incubation in 3% trypsin 250 (BD Biosciences, San Jose, CA, USA) on a gentle shaker at 37 °C. After each 30-min trypsin incubation interval, the retinas were rinsed with PBS until the internal limiting membrane was digested, leaving only the retinal vasculature. Subsequently, the retinal vasculature was meticulously mounted onto a glass slide and stained using periodic acid-Schiff’s base (PAS)-hematoxylin (Sigma-Aldrich, MO, USA). A Leica DM300 microscope was employed to capture retinal images, with acellular capillaries quantified across 10 randomly selected fields by two independent blinded investigators.