Progression of ouabain-induced retinal cell death and accumulation of immune cells in damaged retinas. Cryosections (5 μm thick) from retinas at 24 and 48 h post-intravitreal injection (24 and 48 hpi) of saline (A and B) or 2 μM ouabain (C and D) were stained using the TUNEL cell death detection process (green), the immune cell marker L-plastin (magenta), and DAPI (blue). A–B Saline injection did not induce cell death (absence of TUNEL staining), and immune cells remain ramified (arrows). C–D TUNEL+ nuclei and debris (green) are present in the ganglion cell layer (GCL) and inner nuclear layer (INL) following intravitreal injection of 2 μM ouabain, and immune cells assume ameboid morphologies (arrows). As the lesion progresses over time, immune cells accumulate in regions of cell death, and the clearance of TUNEL+ nuclei and debris corresponds to this immune cell accumulation. Few immune cells contained TUNEL+, DAPI+ nuclei (quantification in the “Results” section), suggesting that immune cell death was not significantly induced by ouabain. E–G High magnification images of immune cells revealed immune cells phagocytosing TUNEL+ nuclei (E), immune cells with cytoplasmic TUNEL+ material (F, arrow), and immune cells with multiple nuclei, some of which are TUNEL+ (G, asterisks), suggesting that the accumulating immune cells are highly phagocytic and important for clearing dead cells and debris from the lesioned retina. Scale bar in A (applies to A–D) = 20 μm. Scale bars in E, F, and G = 5 μm. ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer
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