In Vivo Imaging of Venous Thrombus and Pulmonary Embolism Using Novel Murine Venous Thromboembolism Model

Figures

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  Figure 1. Induction of Stasis- and Irradiation-Induced DVT in the Murine Femoral/Saphenous Vein (A) Surgical ligation of the femoral vein induced flow restriction. Dotted white line indicates the femoral vein. Bars = 5 mm. (B) Venous flow velocity at the saphenous vein in ligated mice showed significant decrease compared with in nonligated mice (median: 2.27 [interquartile range (IQR): 1.86 to 2.59] vs. 0.35 [IQR: 0.20 to 0.61] mm/s; p < 0.001; n = 8 to 16 per group). ***p < 0.001. Box-and-whisker plot: middle line represents median value, box indicates IQR (25th, 75th percentiles), and range bars show maximum and minimum. (C) Even after ligation of the femoral vein (V), venous flow in the saphenous vein persisted at a low level as flow diverted (white arrows) into its side branches. Bars = 500 μm. (D) Imaging of real-time thrombus formation (visualized as a filling defect [*] in fluorescein isothiocyanate [FITC]-dextran–based venography) in the flow-restricted saphenous vein following the light irradiation through excitation filter in FITC channel (475/35 nm). Thrombus formation was initiated from the downstream of blood flow within seconds after the irradiation and extended distally in…

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  Online Video 1 Time-lapse imaging of thrombus formation. Time-lapse imaging of thrombus formation at saphenous vein induced by light irradiation of epifluorescence microscopy. Blood flow was visualized by injection of FITC-dextran.

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  Figure 2. Formed Thrombus in Our New DVT Model Has Common Features With Clinical DVT (A) Thrombus positive for glycophorin A (red blood cell [RBC] marker) with low accumulation of rhodamine 6G indicates the erythrocyte- and fibrin-rich DVT. Both ferric chloride and rose bengal models induced dotlike platelet (Plt)-rich thrombus, indicating arterial thrombus. Continuous (>10 min) excitation light irradiation without ligation induced platelet-rich thrombus, similar to a previous laser-induced thrombus model (bottom). Real-time imaging of thrombus formation using FITC-labeled RBC is shown in Video 2. (B) Fibrin-rich thrombus with few platelets was observed in our new model. (C) Phlebotomy-induced anemic mice exhibited decreased thrombus size (median: 0.31 [IQR: 0.22, 0.48] vs. 0.19 [IQR: 0.14, 0.31] mm2; p = 0.017, n = 10 to 12 per group). (D) Macroscopic image of thrombus represented red-white thrombus, similar to clinical DVT. In vivo fluorescence imaging of same thrombus precisely visualized the localization of platelet-rich white thrombus lesion as rhodamine 6G–positive area. (E) Pathological analyses of formed thrombus in our model confirmed the erythrocyte- and fibrin-rich thrombus with…

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  Online Video 2 Real-time imaging of thrombus formation using FITC-labeled RBC. RBCs were ex vivo labeled with FITC, followed by injection to mice via jugular vein. Circulating RBCs were directly attached to venous wall during thrombus initiation.

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  Online Video 3 Real-time imaging of the moment of thromboembolism shortly after DVT formation. Movie of the initiation of thromboembolism using FITC-dextran was shown. The edge of DVT formed at lower extremity vein was separated and embolized.

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  Figure 3. Spontaneous DVT Embolization to Pulmonary Artery, as a Novel PE Model (A) Femoral vein was ligated and illuminated by excitation light to induce large thrombus size. DVT was visualized by pre-injection of fibrinogen-AF647 (red, left panel). Thirty min after the deligation of the femoral vein suture, the formed DVT had disappeared, indicating embolization (right panel). Bar = 500 μm. Dotted line indicates femoral vein. X indicates ligation site. (B) To confirm the femoral DVT embolized to the lungs, resected lungs were imaged using in vivo imaging system fluorescence reflectance imaging. Spotted fluorescence signal indicated embolized fibrin-rich DVT. Fibrinogen-AF647–injected mice without DVT exhibited no apparent fluorescent signal, suggesting fibrin-rich signal was not a postmortem clot. Bars = 2 mm. (C) HE staining of resected lung showed inflammatory cells infiltrated thrombus in the pulmonary artery. Right panel shows magnified image of dotted area in the left panel. Subsets of lung tissues were processed for frozen sections to visualize in vivo–injected fibrinogen-AF647 by fluorescence microscopy, confirming fluorescence signal (arrows) was fibrin-rich embolized DVT from…

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  Figure 4. Platelet-, Neutrophil-, and Oxidative Stress–Independent Thrombus Formation (A) Time-lapse multicolor real-time imaging of DVT formation revealed that rhodamine 6G–labeled leukocytes and platelets did not clearly accumulate and attach to the venous wall prior to DVT formation (arrows). Leukocytes and platelets (rhodamine 6G) accumulated after 30 s of irradiation, whereas DVT formed within 15 s. Bars = 200 μm. Delayed platelet accumulation was confirmed using antiplatelet antibody (right panel). Neither platelet-depletion (B) nor neutrophil-depletion (C) prevented thrombus formation nor changed thrombus size (median: 0.42 [IQR: 0.21 to 0.62] vs. 0.38 [IQR: 0.26 to 0.61]; p = NS; n = 10 per group for platelet depletion; and median: 0.34 [IQR: 0.26, 0.45] vs. 0.34 [IQR: 0.26, 0.55]; p = not significant [NS]; n = 6 per group for neutrophil depletion, respectively). (D) Pre-treatment with deoxyribonuclease also did not affect the DVT formation in our model, indicating neutrophil extracellular traps–independent thrombus initiation in this model (median: 0.39 [IQR: 0.30, 0.60] vs. 0.45 [IQR: 0.33, 0.61]; p = NS; n = 5 per group). (E) Reactive oxygen species (ROS) did not localize within…

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  Figure 5. Multiphoton Imaging of Formed DVT and Venous Valve Pocket (A) Representative image of DVT formed at saphenous vein. The vessel wall can be visualized by second harmonic generation (blue) without the need for exogenous labeling agents. (B) Magnified image of dotted linefromA is shown. Rhodamine 6G–labeled leukocyte and platelets can be distinguished by their size. (C) Venous valve leaflets were also visualized by second harmonic generation (blue, arrows). Sagittal image (D) and 3-dimensional image (E) confirmed that DVT formed within the valvular pocket sinus, which is the common DVT site in clinical situation. Bars = 50 μm. Abbreviations as in Figures 1 and 2.

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  Figure 6. Spatiotemporal Dynamics of Inflammatory Cells and Platelets in the DVT Organization (A) Serial imaging of DVT by epifluorescence allows visualization of rhodamine 6G–positive cells (leukocytes and platelets) infiltrating into DVT; however, discrimination between leukocytes and platelets is not possible due to low resolution. Bars = 100 μm. (B) Multiphoton imaging of DVT enabled clear visualization of spatiotemporal dynamics of leukocytes and platelets. Top left square shows magnified images from dotted line.Bars = 100 μm. (C) Zones of platelets aggregate in the circulating blood near the DVT (arrows), followed by platelets depositing at the edge of erythrocyte-rich DVT. The deposition of the platelets changed the rheology, resulting in the formation of multiples of platelet layers and erythrocyte layers (dotted line). Bar = 10 μm. (D) Blood-based leukocytes migrate into the DVT accompanied by platelets (Video 4). Lower panels show dynamics of leukocytes and platelets in dotted area in upper panel.Bars = 10 μm. (E) Tracked paths of migrating leukocytes over 30 min. White circle indicates tracking leukocytes, and white line shows tracked path (Video 4). Lower panel shows the moving…

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  Online Video 4 Time-lapse imaging of leukocyte migration into fibrin-rich DVT. Time-lapse imaging of leukocytes (red, rhodamine 6G) migration into fibrin-rich DVT (green, Fibrinogen-AF488). White lines indicate the migrating leukocytes path.