Retinal Vascular Damage Assessment

To assess vascular damage in the retina, fundus imaging and fluorescein angiography were performed using the Micron III system (Phoenix Research Laboratories, Pleasanton, CA, United States) as described (Koirala et al., 2013 (link); Chakraborty et al., 2020 (link)). Mice were anesthetized with ketamine/xylazine (84, 7 mg kg^–1)) and eyes were dilated with 1% cyclopentolate eye drops (Family Medicine Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States). One drop of 2.5% Gonak (McKesson Medical Surgical, Richmond, VA, United States) was applied to each eye. Bright field images were collected, and then animals were injected intraperitoneally with 100 μL of 1% (w/v) fluorescein sodium (Sigma-Aldrich, St. Louis, MO, United States) for angiography. Angiogram images were captured using a GFP filter. All fundus images were captured using StreamPix software (Phoenix Research Labs, Bend, OR, United States). We observed repeated patterns of constriction in some retinal vessels, to semiquantitatively assess this phenotype, the number of affected vessels in each image was counted by an observer blinded to age and genotype.