tPA-associated BBB disruption and hemorrhage after ischemic stroke are rescued by loss of cGAS. (A) Representative immunoblots for STING, pTBK1 and total TBK1, and pIRF3 and total IRF3 expression in the ischemic cortex of WT and cGas^−/− mice treated with tPA. (B-D) Quantification of band intensities for each group (n = 5). (E-F) Quantification of IFN-β and IL-6 levels by enzyme-linked immunosorbent assay in the ischemic cortex (n = 6). (G-H) Representative in vivo multiphoton microscopic images of IV injected FITC-dextran (molecular weight, 40 KDa) leakage from cortical vessels and quantification of the permeability surface (P) product of FITC-dextran at 24 hours after stroke in WT and cGas^−/− mice treated with tPA (n = 6). Scale bar, 100 μm. (I-J) Representative immunoblots and quantification of IgG levels in capillary-depleted brain tissue (n = 5). (K) Representative images of the dorsal surface (upper panel) and a coronal section (bottom panel) show cerebral hemorrhage 24 hours after stroke. (L) Quantification of cerebral hemorrhage (n = 8). (M-O) Effects of cGAS deficiency on forelimb force test and beam walking test 24 hours after stroke in tPA-treated mice (n = 10). (P) Model figure showing how tPA stimulates NET formation and how NETs are sensed by microglia after ischemic stroke. Values are means ± standard deviation. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.