Retrograde Labeling of Retinal Ganglion Cells

These procedures have been further explained in our previous studies [34 (link),35 (link)]. At day 21 after rAION induction, rats were anesthetized and placed in a stereotactic apparatus (Stoelting, Wood Dale, IL, USA). The skull was exposed, and 2 μL of 5% FG was injected into the superior colliculus on each side via a Hamilton syringe. After one week, the eyeballs were obtained after the rats had been euthanized. The eyeballs were incubated in 10% formalin for 2 h. Each retina was dissected and flat-mounted on a slide. The morphometry of the RGCs was detected by fluorescence microscopy (Axio Scope A1, Zeiss, Oberkochen, Germany) with a filter set (excitation filter = 350–400 nm, emission filter = 515 nm) and photographed with a digital camera (Axiocam 305 color, Zeiss, Oberkochen, Germany). The number of RGCs at a distance of 1 or 3 mm from the ONH was counted for determination of the central and mid-peripheral RGC densities. Eight random regions per slide were imaged in both the central and mid-peripheral areas. RGC density was calculated by ImageJ software.