Retinal tissue preparation

The animals were euthanized using CO₂ inhalation, after which the eyes were enucleated and hemisected in Ringer’s solution (110 mM NaCl, 22 mM NaHCO₃, 2.5 mM KCl, 1.6 mM MgCl, 1 mM CaCl₂, NaH₂PO₄, and 10 mM Glucose) and then continuously oxygenated with 95% O₂ and 5% CO₂. The vitreous humor and lens were carefully removed from the opened eyecup using precision forceps. The retina was then hemisected and gently separated from the retinal pigment epithelium. The isolated rat retina was then mounted, with the photoreceptor side down, on a Multi-Electrode Array (MEA- 60MEA200/30iR-Ti-pr-T, Multi-Channel System Germany) for electrical stimulation via a current injection system (MEA 2001, Multi-Channel System Germany). Cathodic pulses ranging from 0.1 to 1 ms and 10–120 µA were applied at a frequency of 0.2 Hz, and the induced RGCs responses were analyzed.