Contrast ultrasound for the quantification of deep vein thrombosis in living mice: effects of enoxaparin and P2Y12 receptor inhibition

Authors


  • Manuscript handled by: S. Watson
  • Final decision: D. Lane, 18 March 2013

Correspondence: Daniel Duerschmied, Department of Cardiology and Angiology I, University Heart Center Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.

Tel.: +49 761 270 3441; fax: +49 761 270 3200.

E-mail: daniel.duerschmied@uniklinik-freiburg.de

Summary

Background/Objectives

We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y12 receptor deficiency in vivo.

Methods

Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y12−/− mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles.

Results

Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15–25 min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y12−/− mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25 min was significantly smaller in enoxaparin- (0.3 ± 0.1 mm2) and ticagrelor-treated (0.5 ± 0.1 mm2) wild-type and in P2Y12−/− mice (0.4 ± 0.1 mm2) as compared with vehicle-treated wild-type mice (2.0 ± 0.3 mm2) in the maximal sagittal plane (P < 0.001, n = 5–10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT.

Conclusions

Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y12 receptor in early DVT formation.

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