Phenylephrine

When imaging with the awake-behaving mouse, no additional procedures were applied in the eye except pupil dilation. Pupil dilation was applied in either the awake or anesthetized mice, which was achieved by putting in eye drops of 1% tropicamide (Sandoz, Basel, Switzerland) and 2.5% phenylephrine (Akorn, Lake Forest, Illinois, USA). Imaging was performed in a dark environment with a 10–15 min dark adaptation phase prior to the start of each imaging session. Because the adjustment of the roll rotation axis with the mouse was limited in our platform, the FOV of AOSLO imaging were mostly at the upper quadrant of the retina. When imaging with anesthetized mice, intraperitoneal injection with a mixture of ketamine hydrochloride (100 mg/kg dosage) and xylazine (10 mg/kg dosage) was applied. An external heating pad and rectal probe thermometer (Physiosuite, Kent) was used to monitor and maintain a body temperature of 37.0 ˚C for the anesthetized mice. To prevent ocular opacification induced by dehydration under anesthesia, a contact lens (Advanced Vision Technologies, Lakewood, Colorado, USA) was placed over the eye being imaged, and an artificial tear (GenTeal, Alcon Laboratories, Inc, Fort Worth, Texas, USA) was routinely applied every 20 minutes during imaging.

Nine hyperglycemic male mice and 10 age-matched euglycemic male littermates were used for this study. Mice were anesthetized using an intraperitoneal injection of ketamine and xylazine (100 mg/kg ketamine, 10 mg/kg xylazine). Anesthesia was then sustained throughout the imaging session by delivering 1% v/v isoflurane with supplemental oxygen through a nose cone. Pupil dilation was achieved with eye drops of 1% tropicamide (Sandoz, Basel, Switzerland) and 2.5% phenylephrine (Akorn, Lake Forest, IL). Mice were placed on a stereotactic stage with five degrees of freedom that allowed both the centration of the pupil and the navigation towards the desired retinal location. A +10 D rigid contact lens with base curvature of 1.6 mm (Advanced Vision Technologies, Lakewood, CO) was placed over the cornea to maintain eye hydration while providing an optical interface for retinal imaging. GenTeal (Alcon Laboratories, Inc, Fort Worth, TX) was administered around the contact lens every 20 minutes during the imaging session to sustain eye hydration. A heat pad was used to maintain mouse body temperature at 37°C.