Retrograde Tracing of Retinal Ganglion Cells

Rats from the different groups were anesthetized and injected with the fluorescent tracer 3% FluoroGold (2 μL; Sigma) diluted in saline into the bilateral superior colliculi (6.0 mm posterior and 2.0 mm lateral to bregma and 4–4.5 mm deep) via a microsyringe as previously described (Zhou et al., 2017a (link)) 5 days before sacrifice. Five days after FluoroGold injection, the retinas were isolated, dissected, divided into four quadrants, and flat-mounted on glass slides with the GCL facing up. Each quadrant was further divided into central (1.5 mm from the optic disk) and peripheral regions (3 mm from the optic disk). Two fields in each region were randomly selected and counted. Totally 16 microscopic fields in each retina were counted and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems) at a final magnification of 3,200 (scale bar, 50 μm). For each image taken, the number of labeled cells was counted two times by an investigator who was blinded to the grouping, and the average was used as the final result.