RGCs were retrogradely labeled using the double upper colliculus method. After anesthesia, the rats were fixed to a stereoscope, and the skin was disinfected with 0.5% iodide and cut in the middle to expose the skull. A hole 6 mm posterior and 2 mm lateral to the anterior fontanel was made using a dental drill. A microsyringe was then used to inject 2 μL of 3% Fluorogold into each location, and the needle was left in situ for 5 min. The eyeballs were quickly removed under heavy anesthetic on the 7th day after staining and fixed in 4% paraformaldehyde for an hour. The cornea and lens were removed, the eyeball was cut open 2 mm below the scleral edge of the horn, and the retina was separated and cut into the shape of a four-leaf clover. As seen in Figure 1 E, the retina was cut into four equal quadrants, and the ganglion cell layer was pressed against a cover glass slide. The number of RGCs in two locations (0.64 mm × 0.64 mm each) that were 1 and 3 mm from the optic disc were counted blindly in each quadrant. Thus, using laser scanning confocal microscopy (TCS SP8; Leica Microsystems, Germany) with a broadband ultraviolet excitation filter at 20× magnification, the number of RGCs was counted in 16 micrographs per retina.
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