SD-OCT was performed using the ophthalmic imaging system (Bioptigen Spectral Domain Ophthalmic Imaging System; Bioptigen, Inc., Durham, NC). The system has a probe and a platform designed for mice that can easily orient and align the subject. Animals were anesthetized by intraperitoneal injection with a solution of ketamine (120 mg/kg) and xylazine (20 mg/kg). Pupils were dilated using 1% tropicamide (Akorn, Lake Forest, IL). Lubricant gel drops (Novartis Pharmaceuticals Corp, East Hanover, NJ) were applied to the animal eyes every 3 minutes to prevent cornea dehydration.
Imaging and data process were performed with image analysis software (InVivoVue Clinic; Bioptigen). Briefly, radial volume scan (centered on optic disc, diameter 1.3 mm) was used, each volume consisting of 100 B-scans (1,000 A-scans per B-scan). Four scans (scan 1, 26, 51, and 76 at 0°, 45°, 90°, and 135° in en-face image) were selected for the ONL thickness measurement. Four vertical calipers (defining the ONL, from the outer plexiform layer [OPL] to the outer limiting membrane [OLM]) were placed on each scan, with two on each side of the optic disc, 250 μm and 500 μm away from the optic disc, respectively.