Fig. 6

Retinal tissue invasion of CD45⁺ leukocytes following ischemia-reperfusion (IR). Mice eyes were subjected to IR for 90 min or needle puncture only (Sham), followed by natural reperfusion. At 1 day (1d) or 2 days (2d) after IR, mouse retinal whole mounts were analyzed for A IF for CD45 (green) and IB4 (magenta) at both the superficial vascular plexus (left panel) and deep vascular plexus (right panel) of retinal whole mounts from IR retinal at 1 d after reperfusion. Scale bars: 40 μm. B Imaris software processed images showing 3D surface re-construction for identifying the vessels stained with IB4 (magenta) and the number of CD45⁺ cells (green with grey dots). Scale bars: 40 μm. C Representative Imaris software 3D re-constructed image (left panel) showing the location of luminal (yellow) and extravascular (green) CD45⁺ cells in relation to the IB4-positive vessels (magenta). The magnified portion of the boxed region is shown on the right panel. Scale bars: 10 μm (left panel) and 2 μm (right panel). D Results of quantification of CD45⁺ cell localization in relation to IB4⁺ vessels in the superficial vascular plexus. E Quantification of CD45⁺ cell localization in relation to IB4 stained vessels in the deep vascular plexus. Cell numbers were normalized to the calculated volume (mm³) of IB4⁺ vascular plexus in each image to obtain relative cell densities. For each condition, two representative image fields from each retina of Sham (n = 1) and IR (n = 3) were quantified