NETs promote lung vaso-occlusion in mice with SCD in vivo. (A) Lung histology (Hematoxylin and Eosin staining; 10× magnification) of control mice and mice with SCD, 3 hours after IV administration of 10 µmol/kg oxy-Hb. Refer to supplemental Figure 1 for larger FOVs and lung injury quantification. (B) Experimental scheme used in panels C to H: Control mice and mice with SCD were IV administered 10 µmol/kg oxy-Hb or saline, and qFILM was used to assess the absence or presence of platelet-neutrophil aggregate mediated pulmonary vaso-occlusion (PVO). Pulmonary microcirculation (pseudo-colored purple), neutrophils (red), and platelets (pseudo-colored green) were labeled in vivo by IV administration of fluorescein isothiocyanate (FITC) dextran, AF546-anti-Ly6G Ab, and V450-anti-CD49b Ab, respectively. Representative qFILM images are shown in panels C to D. (C) IV oxy-Hb led to minimal lung vaso-occlusion in a control mouse. (D) IV oxy-Hb led to occlusion of arteriolar bottlenecks in the lung of a mouse with SCD by large neutrophil-platelet aggregates (marked by dotted white ellipse). Magnified cropped images of the individual neutrophil-platelet aggregates are shown on the right. Colocalization of neutrophils (red) with platelet (green) appears yellow. (E-H) PVOs were quantified using strategy described in supplemental Methods. IV oxy-Hb led to significantly more (E) PVOs per FOV, (F) percent FOVs with PVOs, (G) large PVOs (with area >1000 µm²) per FOV, and (H) both neutrophil-rich and platelet-rich PVOs per FOV, in mice with SCD compared with control mice. Control IV saline (n = 3 mice; 38 FOVs), SCD IV saline (n = 4 mice; 49 FOVs), control IV oxy-Hb (n = 5 mice; 71 FOVs), SCD IV oxy-Hb (n = 5 mice; 75 FOVs). (I) Experimental scheme used in panels J to N: Control mice and mice with SCD were IV administered 10 µmol/kg oxy-Hb, and qFILM was used to assess the absence or presence of NETs within the pulmonary microcirculation. Pulmonary microcirculation (pseudo-colored purple), neutrophils (blue), extracellular DNA (green), and citrullinated histones (H3-Cit; red) or NE (red) were labeled in vivo by IV administration of Evans blue, Pacific blue–anti-Ly6G Ab, Sytox green, and AF546–anti-H3-Cit Ab or AF546–anti-NE Ab, respectively. (J-K) NETs were quantified as described in supplemental Methods. Number of NETs per FOV (#NETs/FOV) was significantly higher in mice with SCD administered IV oxy-Hb (n = 4 mice; 44 FOVs) than (J) mice with SCD administered IV saline (n = 4 mice; 49 FOVs) or (K) control mice administered IV oxy-Hb (n = 4 mice; 43 FOVs). Representative qFILM images (L-N) reveal NETs (marked by dotted white ellipse) in the pulmonary arteriole bottlenecks of mice with SCD administered IV Oxy-Hb. NETs were identified based on colocalization of Ly6G (blue) with exDNA (green) and (L) H3-cit (red) or (M,N) NE (red). “X” (in panel N) denotes loss of blood flow (purple dye absent) downstream of NETs-associated lung vaso-occlusion (marked by white ellipse). White arrows denote the direction of blood flow within the pulmonary arterioles. Alveoli are marked with white asterisks. Scale bars, 200 µm, in panel A and 20 µm in panels C-D, L-N. qFILM FOV size ∼65 536 µm². *P < .05 for SCD compared with control. #P < .05 for IV oxy-Hb compared with IV saline. Means in panels E,G compared using Student t test with Bonferroni correction. Percentages in panel F compared using fourfold table analyses with Bonferroni χ² statistics. Means in panels H, J-K compared using Student t test. Data in panels E, G, H, J, and K represent mean ± standard error (SE). The diameter of pulmonary arteriole in panels C, D, L, M, and N is ∼28 µm, 26 µm, 14 µm, 14 µm, and 28 µm, respectively.