Figure 6.
Figure 6. NETs propagate DVT in vivo. (A) Leukocyte accumulation in vivo at 6 h of flow restriction in the IVC of LysM-eGFP mice treated with control antibody or the anti-Ly6G mAb to deplete neutrophils. Arrowhead: aggregated neutrophils; arrows: single, adherent cells. Bars, 100 µm. (B) Thrombus weight 48 h after DVT induction in isotype and anti-Ly6G–treated WT mice (n = 6 per group). Dots represent individual experiments; lines show the mean of each group. (C) Representative image of n = 3 experiments of intravital microscopy 3 or 48 h after flow reduction showing Sytox Green+ NETs in the IVC. Bars, 50 µm. (D) Visualization of NETs in vivo by 2-photon microscopy. Ly6G-positive neutrophils (green, FITC anti-Ly6G antibody) attached to the vessel wall (blue) release Sytox orange–positive (red) NET structures inside the IVC 4 h after flow reduction (also see Video 7). Sytox orange–positive nuclei correspond to dying neutrophils, which have not (yet) exposed their DNA to the extracellular space. Arrowhead: extracellular DNA; arrow: neutrophil. Bar, 50 µm. (E) Immunohistochemical visualization of NETs by staining for DNA (Hoechst), MPO, NE, and histones (H2A-H2B-DNA, H3) in the IVC of WT mice 48 h after induction of DVT. Hoechst+ DNA originating from MPO+NE+ neutrophils (arrows) could be detected. Arrows, nuclei; arrowheads, NET fibers. Bars, 10 µm. (F) Number of NETs in neutropenic mice were quantified in thrombi after treatment with anti-Ly6G and isotype control antibody (n = 3 per group). Data are shown as mean ± SEM. (G) Representative images of WT thrombi stained with Hoechst after DNase1 treatment. Arrowhead, NET fiber. Bars, 10 µm. Shown is a representative of n = 3 experiments. (H) After injection of DNase1, thrombus weight (left) in the IVC was determined after 48 h of flow restriction. Dots represent individual experiments; lines show the mean of each group. Quantification of NETs is also shown (right) as mean ± SEM. Data were obtained in WT injected with normal saline i.v. (n = 14) or DNase1 (n = 6). (I) Quantification of NETs at 48 h in the enoxaparin-treated animals (n = 4 per group). Data are shown as mean ± SEM.

NETs propagate DVT in vivo. (A) Leukocyte accumulation in vivo at 6 h of flow restriction in the IVC of LysM-eGFP mice treated with control antibody or the anti-Ly6G mAb to deplete neutrophils. Arrowhead: aggregated neutrophils; arrows: single, adherent cells. Bars, 100 µm. (B) Thrombus weight 48 h after DVT induction in isotype and anti-Ly6G–treated WT mice (n = 6 per group). Dots represent individual experiments; lines show the mean of each group. (C) Representative image of n = 3 experiments of intravital microscopy 3 or 48 h after flow reduction showing Sytox Green+ NETs in the IVC. Bars, 50 µm. (D) Visualization of NETs in vivo by 2-photon microscopy. Ly6G-positive neutrophils (green, FITC anti-Ly6G antibody) attached to the vessel wall (blue) release Sytox orange–positive (red) NET structures inside the IVC 4 h after flow reduction (also see Video 7). Sytox orange–positive nuclei correspond to dying neutrophils, which have not (yet) exposed their DNA to the extracellular space. Arrowhead: extracellular DNA; arrow: neutrophil. Bar, 50 µm. (E) Immunohistochemical visualization of NETs by staining for DNA (Hoechst), MPO, NE, and histones (H2A-H2B-DNA, H3) in the IVC of WT mice 48 h after induction of DVT. Hoechst+ DNA originating from MPO+NE+ neutrophils (arrows) could be detected. Arrows, nuclei; arrowheads, NET fibers. Bars, 10 µm. (F) Number of NETs in neutropenic mice were quantified in thrombi after treatment with anti-Ly6G and isotype control antibody (n = 3 per group). Data are shown as mean ± SEM. (G) Representative images of WT thrombi stained with Hoechst after DNase1 treatment. Arrowhead, NET fiber. Bars, 10 µm. Shown is a representative of n = 3 experiments. (H) After injection of DNase1, thrombus weight (left) in the IVC was determined after 48 h of flow restriction. Dots represent individual experiments; lines show the mean of each group. Quantification of NETs is also shown (right) as mean ± SEM. Data were obtained in WT injected with normal saline i.v. (n = 14) or DNase1 (n = 6). (I) Quantification of NETs at 48 h in the enoxaparin-treated animals (n = 4 per group). Data are shown as mean ± SEM.

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