Standardized Rat Electroretinography Protocol

The animals were dark adapted overnight before the ERG recordings, and their manipulation was done under dim red light (λ>600 nm). The rats were anaesthetized and bilateral pupil mydriasis was induced by applying a topical drop of 1% tropicamide (Colircusi tropicamida 1%®; Alcon-Cusí, S.A., El Masnou, Barcelona, Spain) to both eyes. The light stimulation device used was a Ganzfeld dome, which ensures a homogeneous illumination anywhere in the retina, with multiple reflections of the light generated by light emitting diodes (LED), which provided a wide range of light intensities. For high intensity illuminations, a single LED placed close (1 mm) to the eye was used. Light intensity was calibrated by a dual-biosignal generator device specifically adapted for ERG responses. The recording system comprised Burian-Allen bipolar electrodes (Hansen Labs, Coralville, IA) with a corneal contact shape; a drop of methylcellulose, 2% (Methocel 2%®; Novartis Laboratories CIBA Vision, Annonay, France) was placed between the eye and the electrode to maximize conductivity of the generated response. The reference electrode was placed in the mouth and the ground electrode in the tail. Electrical signals generated in the retina were amplified (x1000) and filtered (band pass from 1 Hz to 1000 Hz) by a commercial amplifier (Digitimer Ltd, Letchworth Garden City, UK). The recorded signals were digitized (Power Lab; ADInstruments Pty. Ltd., Chalgrove, UK) and displayed on a PC computer. Bilateral ERG recording was performed simultaneously from both eyes. Light stimuli were calibrated before each experiment, and the calibration protocol was set to assure the same recording parameters for both eyes. The ERG responses were recorded by stimulating the retina with light intensities ranging between10⁻⁶ and 10⁻⁴ cd·s·m⁻² for the scotopic threshold response (STR), 10⁻⁴ and 10⁻² cd·s·m⁻² for the rod response, and 10⁻² and 10² cd·s·m⁻² for the mixed (rod and cone) response. For each light intensity level, a series of ERG responses were averaged (from 40 ERG responses for the dimmest stimulus intensities to 5 for the brightest stimulus), and the interval between light flashes was adjusted to ensure a timing that allowed response recovery (from 5 s for the dimmest stimulus intensities to 60 s for the brightest stimulus). At the end of each session the animals were treated with topical tobramicine (Tobrex®; Alcon-Cusí, S.A.) in both eyes. The analysis of the different recordings was performed with the normalization criteria established for the International Society for Clinical Electrophysiology of Vision (ISCEV) for the measures of the amplitude and implicit time of the different waves studied.