Animals were dark adapted overnight (>12 hours) and prepared for recording under dim red light (λ > 600 nm). Anesthesia was initially induced with an intramuscular injection of ketamine (55 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA), xylazine (5 mg/kg, X-ject E; Phoenix Scientific Inc., St. Joseph, MO) and acepromazine maleate (1 mg/kg, Aceproject; Phoenix Scientific, Inc.). Supplemental anesthesia was provided approximately 50 minutes after initial induction using a mixture of ketamine, xylazine, and acepromazine (30:2:1 mg/kg, intramuscularly). Pupils were dilated with 1 drop each of 0.5% tropicamide (Alcon Laboratories, Inc.) and phenylephrine (2.5%, Bausch and Lomb Pharmaceuticals, Inc., Tampa, FL). Corneal anesthesia was achieved with 1 drop of 0.5% proparacaine hydrochloride (Alcon Laboratories, Inc.). Animals were lightly secured to a stage with Velcro strips across the upper and lower back to ensure a stable, reproducible position for ERG recording. Body temperature was maintained between 37°C and 38°C with a pumped-water heating pad (TP500 T/Pump; Gaymar Industries, Orchard Park, NY) that was fixed to the top of the stage. The duration of the ERG recording session was 75 minutes for each animal, after which animals fully recovered from anesthesia while resting on a heated pad.
Full-field ERGs were recorded (UTAS-E3000; LKC Technologies, Gaithersburg, MD) in both eyes simultaneously with custom silver-chloride electrodes. The tip of the active electrode was placed at the corneal apex and was referenced to a ring-shaped electrode positioned against the scleral conjunctiva around the equator of the eye. Eyes were lubricated after electrode placement and periodically throughout the session with 1.0% carboxymethylcellulose sodium (Allergan, Irvine, CA). A platinum electrode (Grass-Telefactor, West Warwick, RI) placed in the tail served as the ground. Simultaneous recording from both eyes effectively halved the recording time and allowed ERGs to be obtained from the control and treated eyes under identical states of anesthesia and adaptation.
Stimuli were brief white flashes (xenon arc discharge, x = 0.32, y = 0.33) delivered through a Ganzfeld integrating sphere (UTAS-3000; LKC Technologies). Stimulus intensities were measured with a calibrated photometer (Spectra Pritchard PR-1980B; Photo Research, Chatsworth, CA) with a (human) scotopic luminosity filter in place. STR responses were obtained for flash intensities ranging from −6.64 to −3.30 log cd-s/m2 in 0.2-log-unit increments, by averaging 20 to 60 responses per intensity (60 for the dimmest and 20 for the higher intensities), with an interstimulus interval of 2 seconds. Scotopic ERGs obtained for all intensities above −3.30 log cd-s/m2 were recorded as single flash responses. For stimulus intensities between −3.04 to 2.72 log cd-s/m2 the interval between flashes was progressively lengthened from 10 to 120 seconds to allow complete recovery of b-wave amplitude. After completion of the scotopic ERG intensity series, animals were light-adapted for 15 minutes to a steady white background (150 cd/m2, x = 0.44, y = 0.41). Photopic flash responses were recorded for intensities between 0.97 and 2.72 log cd-s/m2 in 0.25-log-unit increments. Each record was an average of 20 responses obtained with a 2-second interstimulus interval. STR records were acquired at 1 kHz with high- and low-pass filters set to 0.3 and 30 Hz, respectively. All other ERG records were acquired at 2 kHz with high- and low-pass filters set to 0.3 and 500 Hz, respectively.