Introduction:
Inflammation following deep vein thrombosis (DVT) critically modulates thrombus resolution. However it is unknown whether the degree of inflammation
in vivo
predicts the magnitude of thrombus resolution. Here, we utilize serial multichannel intravital fluorescence microscopy (IVFM) to spatially map and quantify macrophage infiltration in thrombus, and then to determine the degree of thrombus resolution
in vivo
.
Methods:
C57Bl/6 mice (n=5) underwent topical ferric chloride injury to induce femoral venous thrombosis. On day 3, mice were i.v. injected with macrophage-targeted nanoparticles (CLIO-AF555, ex/em 555/565nm). On day 4, survival IVFM of thrombus macrophages was performed. In addition, venography was concomitantly performed (pre-injection of FITC-dextran 45 minutes before IVFM, MW 2000 kD, 490/520nm) to quantify DVT area and length in vivo at day 4 and 6. IVFM image analysis was performed using ImageJ. Mid-luminal z-stacks (40 μm thickness) were analyzed. Thrombus ROIs were outlined using FITC-dextran generated angiograms, and used calculating macrophage target-to-background ratios (TBR) and thrombus area and length. Contralateral sham femoral veins were imaged as controls. Imaging was followed by histopathology and fluorescence microscopy of the cryosectioned tissue.
Results:
At day 4, the baseline femoral DVT area and length were 0.22±0.08mm
2
and 1.29±0.16mm (mean±sd). The day 4 macrophage activity was noted to be heterogeneous with peripheral infiltration of the thrombus, and occasionally with some deeper penetration into the thrombus interior. At day 6, the thrombus area and length were reduced compared to day 4 (p<0.005 for both). The day 4 thrombus macrophage TBR significantly predicted the degree of thrombus reduction (correlation of initial macrophage TBR and Δthrombus area, r=0.97, p=0.001).
Conclusion:
This serial in vivo assessment of murine DVT resolution demonstrates that degree of macrophage infiltration at day 4 predicts the magnitude of thrombus resolution at day 6. Molecular imaging of DVT inflammation
in vivo
could provide new insights into DVT resolution and the development of the post-thrombotic syndrome, and also in evaluating inflammatory-modulating therapies for improved DVT resolution.
Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging
