Assessment of Thrombus Stage by ‘Multicolor’ 19F MRI
Sebastian Temme1, Christoph Jacoby2, Christoph Owenier1, Christoph Grapentin3, Xiaowei Wang4, Rolf Schubert3, Karlheinz Peter4, Jürgen Schrader1, and Ulrich Flögel1,2

1Molecular Cardiology, University of Düsseldorf, Düsseldorf, Germany, 2Department of Cardiology, Pneumology and Angiology, University Hospital Düsseldorf, Düsseldorf, Germany, 3Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg i. Br., Germany, 4Atherothrombosis and Vascular Biology, Baker IDI Heart and Diabetes Institute, Victoria, Australia

Synopsis

The present study was aimed at developing a non-invasive approach for direct assessment of thrombus stage by ‘multicolor’ 19F MRI. To this end, we used ligands binding specifically during different phases of thrombosis and coupled them to perfluorocarbons (PFCs) with indvidual spectral signatures. Discrimination of the targeted agents was achieved by a novel multi chemical shift selective imaging technique for simultaneous, artifact-free detection of different PFCs. The results show that this technique holds the potential to differentiate thrombi in the acute, subacute and chronic phase and may also be used for in situ labeling of a variety of other targets.

Abstract

Background: Exact localization of deep venous thrombi as well as determination of thrombus age is still a serious problem in the clinical setting and has significant implications for antithrombotic treatment regiments, since it will help to adequately adjust doses in lysis schemata. Thrombus detection by conventional 1H MR methods, like angiography or T1/T2-weighted techniques can be difficult, because small, non-occlusive thrombi have only minor impact on blood flow and may not give rise to a clear signal in weighted images. To overcome this limitation, we have recently used 19F MRI for detection of early thrombi utilizing a targeted perfluorocarbon nanoemulsion (PFC) [1]. Due to the fact that 19F is nearly absent from biological tissue, 19F-containing agents result in an unequivocal positive contrast without any background signal. In order to further distinguish between acute, subacute, and chronic thrombi, we now used additional probes binding specifically at different stages to thrombi and coupled them to PFCs with indvidual spectral signatures. Discrimination of the targeted agents was achieved by a novel multi chemical shift selective imaging technique (mCSS-RARE [2]) which was used for simultaneous detection of three different PFCs.

Methods: For ‘multicolor’ 19F MRI, we generated nanoemulsions which contained 10% perfluoro-15-crown-5 ether (PFCE), 40% perfluorooctyl bromide (PFOB) or 40% perfluoro-1,3,5-trimethylcyclohexane (PFCH). For PFOB and PFCH a diblock (semifluorinated alkane) was added to stabilize the nanoemulsions. The diameter of the emulsion droplets was in the range of 100-200 nm, displayed a narrow size distribution and a strongly negative ζ-potential. To target the emulsions to specific stages of thrombi, we used ligands for three different components of thrombi: the peptide α2-antiplasmin (α2AP) which is cross-linked by factor XIIIA to the fibrin network in the very early phase of thrombus formation [3], EP2104R, a peptide which binds to the fibrin network of early as well as chronic thrombi [4], and the single chain antibody anti-LIBS which binds to the activated gpIIb/IIIa receptor of platelets [5], which is predominantly invoked in the early and intermediate stage of thrombus development. Ex vivo experiments were carried out with thrombi obtained by platelet rich plasma of human blood which was incubated with targeted PFCs at different times after thrombus formation. For in vivo experiments, a murine model of deep venous thrombosis was used in that a filter paper soaked with 10% FeCl3 was applied for 8 min to the external side of the vena cava inferior which resulted in induction of non-occlusive thrombi. Combined 1H/19F MRI was performed at a 9.4T Bruker AvanceIII WideBore NMR spectrometer using a microimaging unit (Micro 2.5) with actively shielded 40-mm gradients (1 T/m maximum gradient strength, 110 µs rise time at 100 % gradient switching). Mice were placed in a 25-mm 1H/19F birdcage resonator and after acquisition of anatomical 1H reference images, a 19F mCSS-RARE sequence with the following parameters was used: 2.56x2.56 cm2 FoV, 64x64 matrix, 1 mm slice thickness, TR 2500 ms, RARE factor 32, 256 averages, 20 min acquisition time.

Results: In a first step, 19F MR spectroscopy was used to identify the resonance frequencies specific for the individual PFCs, which were subsequently used for 19F mCSS-RARE to acquire artifact-free images of multiresonant PFCs (Fig. 1). Next, all ligands were successfully coupled to PFCE using sterol-based post-insertion [1] or a biotin/avidin system and were shown to specifically label ex vivo generated human thrombi. In contrast, corresponding control emulsions did not result in any detectable 19F signal within thrombi. Thereafter, EP2104R-coupled PFOB and PFCH nanoemulsions were demonstrated to be as well suitable for identification of thrombi as 19F hot spots (Fig. 2A) confirming that all PFCs (PFCE, PFOB, and PFCH) can be equipped with targeting ligands for thrombi and differentiated by 19F mCSS-RARE. Using a deep venous thrombosis mouse model, we proved that α2AP-PFCE and -PFOB can also be detected in thrombi by 19F mCSS-RARE in vivo. In separate experiments, we were able to show that early thrombi are labelled by both α2AP-PFCE and EP2104R-PFOB, whereas subacute thrombi are exclusively labelled with EP2104R-targeted PFOB or PFCH only (Fig. 2B).

Conclusions: The results demonstrate that the presented approach holds the potential to assess the current stage of thrombosis via ‘multicolor’ 19F MRI by simultaneous visualization of FXIIIa activity, fibrin and activated platelets. Since this technique is not restricted to thrombi, it may also be used for in situ labeling of a variety of other targets, e.g. different cell populations like immune or stem cells, and can thus substantially extend the field of application of 19F MRI for basic research and possible clinical applications.

Acknowledgements

We like to thank Bodo Steckel and Sabine Barnert for excellent technical assistance.

References

[1] Temme S et al. Noninvasive imaging of early venous thrombosis by 19F magnetic resonance imaging with targeted perfluorocarbon nanoemulsions. Circulation. 2015 131:1405-14.

[2] Jacoby C et al. Simultaneous 19F MR imaging at different resonance frequencies using multi chemical shift selective RARE. ISMRM Milan. 2014:2927

[3] Robinson BR et al. Catalytic life of activated factor XIII in thrombi. Implications for fibrinolytic resistance and thrombus aging. Circulation. 2000;102:1151-7.

[4] Sirol M et al. Chronic thrombus detection with in vivo magnetic resonance imaging and a fibrin-targeted contrast agent. Circulation. 2005;112:1594-600.

[5] Wang X et al. Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice. Circulation. 2012; 125:3117-26.

Figures

Figure 1: Structures and 19F MR spectra of the PFCs investigated indicating the five specific resonance frequencies used for 19F mCSS-RARE (top left) and the resulting artifact-free 19F MR images (right).

Figure 2: A) 19F mCSS-RARE visualization of ex vivo generated human thrombi with EP2104R (top) coupled to PFCs with three different spectral signatures (PFCE, PFOB and PFCH). PFCs labelled with a nonspecific control peptide did not bind to the thrombi (bottom). B) Discrimination of acute and subacute thrombi by α2AP-PFCE and EP2104R-PFCH. Acute thrombi are labelled by α2AP as well as EP2104R whereas subacute thrombi only bind EP2104R-PFCs.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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