Figure 1.
tPA activates neutrophils to release NETs after ischemic stroke. (A-B) Representative immunoblots and quantitative determinations of the amount of neutrophils in the ischemic cortex at 24 hours after stroke in mice treated with vehicle or tPA compared with mice undergoing sham surgery (n = 5). (C) Quantification of the numbers of infiltrating neutrophils in the ischemic cortex at 24 hours after stroke (n = 6). (D) Quantification of MPO activity in the ischemic brain (n = 6). (E-F) Representative immunoblots and quantitative determinations of H3Cit levels in the ischemic cortex (n = 5). (G) Representative confocal images of H3Cit+ neutrophils in the peri-infarct cortex. Scale bar, 50 μm. (H) Quantification of plasma DNA (n = 6). (I) Representative images of isolated peripheral blood neutrophils from mice undergoing sham surgery and ischemic mice treated with vehicle or tPA. Neutrophils were incubated with LPS for 2.5 hours. DNA was stained with Hoechst 33342 (blue), and neutrophils were stained with H3Cit (green). Arrows indicate NETs. Scale bar, 50 μm. (J-K) Quantification of the percentage of H3Cit+ neutrophils and NETs in unstimulated (US) and LPS-stimulated peripheral blood neutrophils (n = 8). (L-M) Quantification of the percentage of H3Cit+ neutrophils and NETs in peripheral blood neutrophils isolated from naïve mice, mice undergoing sham surgery, or ischemic mice (n = 5-8). Neutrophils from naive mice or mice undergoing sham surgery were treated with vehicle or 100 μg/mL of tPA. Neutrophils from ischemic mice were treated with vehicle or 25, 50, or 100 μg/mL of tPA. (N) Representative immunoblots of PAD4 expression in neutrophils isolated from ischemic mice. Neutrophils were treated with vehicle or 100 μg/mL of tPA. (O-P) Quantification of the percentage of H3Cit+ neutrophils and NETs in neutrophils isolated from ischemic mice (n = 8). Neutrophils were treated with vehicle, 100 μg/mL of tPA, or tPA in combination with the PAD inhibitor Cl-amidine. (Q) Representative immunoblots of lipoprotein receptor–related protein 1 (LRP-1) expression in neutrophils isolated from ischemic mice. Neutrophils were treated with vehicle or 100 μg/mL of tPA. (R) Representative immunoblots of PAD4 expression in neutrophils isolated from ischemic mice. Neutrophils were treated with 100 μg/mL of tPA or tPA in combination with the LRP antagonist RAP. (S-T) Quantification of the percentage of H3Cit+ neutrophils and NETs in neutrophils isolated from ischemic mice (n = 8). Neutrophils were treated with vehicle, 100 μg/mL of tPA, or tPA in combination with the LRP antagonist RAP. Values are means ± standard deviation. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

tPA activates neutrophils to release NETs after ischemic stroke. (A-B) Representative immunoblots and quantitative determinations of the amount of neutrophils in the ischemic cortex at 24 hours after stroke in mice treated with vehicle or tPA compared with mice undergoing sham surgery (n = 5). (C) Quantification of the numbers of infiltrating neutrophils in the ischemic cortex at 24 hours after stroke (n = 6). (D) Quantification of MPO activity in the ischemic brain (n = 6). (E-F) Representative immunoblots and quantitative determinations of H3Cit levels in the ischemic cortex (n = 5). (G) Representative confocal images of H3Cit+ neutrophils in the peri-infarct cortex. Scale bar, 50 μm. (H) Quantification of plasma DNA (n = 6). (I) Representative images of isolated peripheral blood neutrophils from mice undergoing sham surgery and ischemic mice treated with vehicle or tPA. Neutrophils were incubated with LPS for 2.5 hours. DNA was stained with Hoechst 33342 (blue), and neutrophils were stained with H3Cit (green). Arrows indicate NETs. Scale bar, 50 μm. (J-K) Quantification of the percentage of H3Cit+ neutrophils and NETs in unstimulated (US) and LPS-stimulated peripheral blood neutrophils (n = 8). (L-M) Quantification of the percentage of H3Cit+ neutrophils and NETs in peripheral blood neutrophils isolated from naïve mice, mice undergoing sham surgery, or ischemic mice (n = 5-8). Neutrophils from naive mice or mice undergoing sham surgery were treated with vehicle or 100 μg/mL of tPA. Neutrophils from ischemic mice were treated with vehicle or 25, 50, or 100 μg/mL of tPA. (N) Representative immunoblots of PAD4 expression in neutrophils isolated from ischemic mice. Neutrophils were treated with vehicle or 100 μg/mL of tPA. (O-P) Quantification of the percentage of H3Cit+ neutrophils and NETs in neutrophils isolated from ischemic mice (n = 8). Neutrophils were treated with vehicle, 100 μg/mL of tPA, or tPA in combination with the PAD inhibitor Cl-amidine. (Q) Representative immunoblots of lipoprotein receptor–related protein 1 (LRP-1) expression in neutrophils isolated from ischemic mice. Neutrophils were treated with vehicle or 100 μg/mL of tPA. (R) Representative immunoblots of PAD4 expression in neutrophils isolated from ischemic mice. Neutrophils were treated with 100 μg/mL of tPA or tPA in combination with the LRP antagonist RAP. (S-T) Quantification of the percentage of H3Cit+ neutrophils and NETs in neutrophils isolated from ischemic mice (n = 8). Neutrophils were treated with vehicle, 100 μg/mL of tPA, or tPA in combination with the LRP antagonist RAP. Values are means ± standard deviation. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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