NETs and PAD4 contribute to tPA-mediated BBB disruption and cerebral hemorrhage after ischemic stroke. (A-B) Representative immunoblots and quantification of PAD4 levels in the ischemic cortex 24 hours after stroke in mice treated with vehicle or tPA compared with mice undergoing sham surgery (n = 5). (C) Representative images of the dorsal surface (upper panel) and a coronal section (bottom panel) show Evans blue extravasation 24 hours after stroke in WT and Pad4^−/− mice treated with tPA. (D) Quantification of Evans blue fluorescence intensity for each group (n = 8). (E-F) Representative immunoblots and quantification of IgG levels in capillary-depleted brain tissue at 24 hours (n = 5). (G-K) Representative immunoblots and quantification of zonula occludens-1 (ZO-1), occludin, claudin-5, and vascular endothelial–cadherin (VE-cadherin) in isolated brain microvessels (n = 5). (L) Representative images of the dorsal surface (upper panel) and a coronal section (bottom panel) show cerebral hemorrhage 24 hours after stroke in WT and Pad4^−/− mice treated with tPA. (M) Quantification of cerebral hemorrhage by spectrophotometric hemoglobin assay (n = 8). (N-P) Effects of PAD deficiency on forelimb force test and beam walking test 24 hours after stroke (n = 10). Values are means ± standard deviation. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.