The full-field (Ganzfeld) scotopic electroretinogram (ERG) was used to assess retinal function at baseline and during acute IOP challenge according to the established protocols (Kong et al., 2009). In brief, animals were dark-adapted overnight (> 12 h) with experimental manipulation being performed using head-mounted night vision goggles (Scout2; Trivisio Prototyping GmbH, Dreieich, Germany). Animals were anesthetized with intraperitoneal injection of ketamine/xylazine (70: 7 mg kg−1; Troy Laboratories Pty Ltd., Smithfield, NSW, Australia) followed by supplementation with 20% of the initial dose every 30 min (Saszik et al., 2002; Peachey & Ball, 2003). Mydriasis was achieved with 1 drop of tropicamide (0.5%, Alcon Laboratories Inc., Fort Worth, TX, USA) and phenylephrine (2.5%, Minims; Chauvin Pharmaceuticals, Surrey, UK). Corneal anesthesia was achieved with a single drop of proxymetacaine hydrochloride (0.5%, Alcon Laboratories Inc.). Animals were lightly secured to a platform with wire loops across the upper back and nose. A circulating warm water heating pad was used to maintain body temperature (37–38 °C). Signals were recorded using silver/silver chloride electrodes (99.99% purity, 0.329 mm ¼ 29 G A&E Metal Merchants, Sydney, NSW, Australia) with a scleral coil reference and corneal active (He et al., 2006; Weymouth & Vingrys, 2008). Both of these were referenced to a stainless steel ground (F-E2-60, Grass Technologies, West Warwick, RI, USA) inserted into the tail. Electrode placement and anterior chamber cannulation with a 50-μm-diameter borosilicate needle were performed using monocular night vision scopes (NVMT1; Yukon, Mansfield, TX, USA) fitted on the eye piece of a dissecting microscope (MZ6; Leica). Light stimuli were brief (1 ms) white flashes (5 Watt white LEDs, 5500°K; Luxeon Calgary, Alberta, Canada) delivered via a Ganzfeld integrating sphere (Photometric Solutions International, Huntingdale, VIC, Australia). Signals were amplified 1000× and recorded with band-pass setting of −3 dB at 0.3–1000 Hz (P511 AC Preamplifier, Grass Telefactor, West Warwick, RI, USA) and digitized with a 4-kHz acquisition (Powerlab 8SP, ADIntruments, Bella Vista, NSW, Australia).
Following electrode placement, the anterior chamber of the left eye was cannulated using a borosilicate needle (approximately 50 μm, 1B100-6, WPI) connected via polyethylene tubing (0.97 mm inner diameter; Microtube Extrusions, North Rocks, NSW, Australia) to a pressure transducer (Transpac IV; Abbott Critical Care Systems, Sligo, Ireland), which was connected in series with a sterile Hanks’ balanced salt solution reservoir (JRH Biosciences, Lenexa, KS, USA). By altering the height of the reservoir, the level of intraocular pressure in the eye can be altered. Patency of the needle was determined by observation for anterior chamber distension under microscope. Previous work by our laboratory showed that the needle aperture has low resistance and thus a negligible effect on pressure equilibration within the anterior chamber (Kong et al., 2009).
Following cannulation of the anterior chamber of the left eye, intraocular pressure was raised in a stepwise manner starting from 25 mmHg up to 50 mmHg at 5 mmHg increments, and from there up to 80 mmHg in 10 mmHg increments. At each step, IOP was stabilized for 10 min before ERG recording. ERG recordings were made at 10-min intervals at baseline and during IOP elevation. Stimuli to elicit the scotopic threshold response (STR, −4.54 log cd.s.m−2, 30 averages, 3-second interstimulus interval), the ON-bipolar cell response (P2, −2.23 log cd.s.m−2, single flash), and a photoreceptoral P3 response (single flash, 0.34 log cd.s.m−2) were employed. All procedures (ERG and IOP elevation) were performed with the genotype of animals masked to the investigator at the time of the procedure.