Progression of retinal degeneration and immune cell response visualized by Hematoxylin & Eosin (H&E). H&E staining of cryosections to visualize progression of the ouabain-induced lesion and immune cell accumulation. A–C Saline-injected controls did not show significant changes in retinal structure; ramified microglia are not readily visualized due to their thin processes with little cytoplasm and integration into retinal layers. D–F By 24 h post-ouabain injection (24 hpi), the inner retina has begun to swell, pyknotic nuclei are present (asterisks), and ameboid immune cells can be detected within the GCL and INL (D, white arrows and G, black arrows). Immune cells are visible in the vitreal space at 24 hpi (black arrows, G), consistent with invasion of extra-retinal immune cells from the retinal vitreal face. Immune cells accumulate in degenerating retinal tissue, which contains many pyknotic nuclei (asterisks), through 48 and 72 hpi ouabain injection (E and F). However, immune cells are no longer observed in the vitreal space at 48 and 72 hpi ouabain (E, F, H, I). Immune cells present in the degenerating retinal tissue are highly phagocytic, demonstrated by cytoplasmic color, regions of space immediately surrounding them, and the progressive disappearance of extracellular matrix and debris over time (D, E, F). Immune cells appear to invade from structures apical to the retina at 48 and 72 hpi ouabain (white arrows, E and F). G–I Evidence of surviving blood vessels at all time points following ouabain injection: at 24 hpi, blood vessel structures at the anterior of the eye adjacent to the peripheral retina are present (dashed box, G). Sections of intact retinal blood vessels at the vitreal face of the retina are present at 48 and 72 hpi ouabain injection (dashed box and inset, H, and black arrows, I). Scale bar in A (applies to A–F) = 20 μm. Scale bar in G (applies to G–I) = 40 μm
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