Fig. 4

Restoration of the vascular barrier coincided with reorganization of TJ complexes. A At the indicated times following IR injury, retinas were removed and proteins in whole retinal lysates examined by Western blotting with antibodies to occludin phosphorylated on serine 490 (pS490), total occludin protein, ZO-1, ZO-2, and claudin-5. A representative blot is shown. B Quantification of 4 replicate Western blot experiments. Occludin pS490 immunoreactivity was normalized to that of total occludin. ZO-1, ZO-2, and claudin-5 immunoreactivities were normalized to that of β-actin. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by t test. C At various times following IR injury (2-day and 4-week samples shown), retinas were removed and immuno-probed with antibodies to occludin and ZO-1, followed by flat mounting and confocal microscopy. Arrows indicate discontinuities in TJ proteins at endothelial cell borders. D Histograms represent the evaluation of TJ continuity at endothelial cell borders using a blinded rank (1–5) scoring system. In the graph, green indicates completely continuous, yellow indicates 75% to 100% continuous, pink indicates 50% to 75% continuous, orange indicates 25% to 50% continuous, and red indicates 0% to 25% continuous border staining. For each retina, four images equidistant from the optic disc were averaged with n = 4 retinas for each condition. *p ≤ 0.05, ****p ≤ 0.0001 by t test