Retinal Vasculature and Permeability Evaluation

To evaluate the retinal vasculature and permeability in vivo, one and six weeks after oral gavage/infection with Pg and biofilm, mice were anesthetized using intramuscular injection of the rodent anesthesia cocktail described above [74 (link)]. The pupils were dilated with 1% tropicamide eye drops, and the mice were placed on the imaging platform of the Phoenix Micron III retinal-imaging microscope (Phoenix Research Laboratories, Pleasanton, CA, USA). Goniovisc 2.5% (hypromellose; Sigma Pharmaceuticals, LLC, Monticello, IA, USA) was applied generously to maintain moisture in the eyes during imaging. Mice were administered 10 to 20 μL 10% fluorescein sodium (Apollo Ophthalmics, Newport Beach, CA, USA), and rapid acquisition of fluorescent images ensued for ∼5 min. Fluorescein leakage manifests as indistinct vascular borders progressing to diffusely hazy fluorescence. Fluorescein leakage (CNV area) was compared between different groups by quantifying the fluorescence intensities collected after 1, 2, and 3 min following fluorescein injection using ImageJ software (National Institutes of Health, Bethesda, MD, USA).