Fig. 5

IR injury induced progressive changes in innate immune cell populations within the retina. A Representative scatter-graphs showing the flow-cytometric analysis used to quantify immune cell populations in the retina. After gating for single cells, events were gated into CD11b⁺/CD45^low (principally microglia), CD11b⁺/CD45^hi myeloid leukocytes, and CD11b^neg/CD45^hi lymphocytes. Myeloid leukocytes were further gated into CD11b⁺/CD45^hi/Ly6C^hi/Ly6G^neg classical monocytes, CD11b⁺/CD45^hi/Ly6C^neg/Ly6G^neg non-classical monocytes or MDM, and CD11b⁺/CD45^hi/Ly6C⁺/Ly6G⁺ granulocytes. Microglia-like cells were further gated to quantify CD11b⁺/CD45^low/Ly6C^neg/Ly6G^neg microglia. B–E At the indicated times following IR injury, flow-cytometric analysis was used to quantify microglia and leukocyte populations in Sham and IR-injured retinas, including: granulocytes (B), classical monocytes (C), non-classical monocytes/MDM (D), and microglia (E). For each analysis, 4 retinas were pooled and analyzed with n = 4 pools of retinas for each group at 1 day, 4 days, 1 week, and 4 weeks following IR injury. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 by one-way ANOVA with Bonferroni and Sidak multiple comparison test. F Representative images obtained by confocal microscopy of the superficial retinal vasculature of at the optic nerve head and periphery of Sham and IR retinas at 24 h after injury. Images show IB4 (green, note: binds to endothelium, with arterial > venus, and to some leukocytes), ColIV (red), and CD45 (magenta). Note that CD45⁺ leukocytes appear both within vessel lumen and within the retina. G Confocal microscopy images showing IB4 (green), ZO-1 (red), and CD45 (magenta) of a superficial vascular region of an IR-injured retina at 24 h after injury (Sham not shown). Arrows indicate regions where the vessel is exhibiting disorganization of endothelial TJ complexes coinciding with apparent extravasation of CD45⁺ leukocytes