Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

Department of Pharmacology and Toxicology Masaryk University Žerotínovo náměstí 617 9 601 77 Brno Czech Republic

Department of Pharmacology and Toxicology Veterinary Research Institute Hudcova 296 70 621 00 Brno Czech Republic

Institute of Physics Czech Academy of Sciences Na Slovance 1999 2 182 21 Prague Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences Královopolská 147 612 64 Brno Czech Republic

Laboratory of Ligand Engineering Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Stroke Research Program International Clinical Research Center St Anne's University Hospital Pekařská 53 656 91 Brno Czech Republic

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Townsend N., Wilson L., Bhatnagar P., Wickramasinghe K., Rayner M., Nichols M. Cardiovascular disease in Europe: Epidemiological update. Eur. Heart J. 2016;37:3232–3245. doi: 10.1093/eurheartj/ehw334. PubMed DOI

Roger V.L. Epidemiology of myocardial infarction. Med. Clin. N. Am. 2007;91:537–552. doi: 10.1016/j.mcna.2007.03.007. PubMed DOI PMC

Powers W.J., Rabinstein A.A., Ackerson T., Adeoye O., Bambakidis N.C., Becker K., Biller J., Brown M., Demaerschalk B.M., Hoh B., et al. Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2018;49:e46–e110. doi: 10.1161/STR.0000000000000158. PubMed DOI

Kraft P., Meyer S.F.D., Kleinschnitz C. Next-Generation Antithrombotics in Ischemic Stroke: Preclinical Perspective on ‘Bleeding-Free Antithrombosis’. J. Cereb. Blood Flow Metab. 2012;32:1831–1840. doi: 10.1038/jcbfm.2012.108. PubMed DOI PMC

Kang K., Oh S., Yi H., Han S., Hwang Y. Fabrication of truly 3D microfluidic channel using 3D-printed soluble mold. Biomicrofluidics. 2018;12:014105. doi: 10.1063/1.5012548. PubMed DOI PMC

Hodge R.D., Bakken T.E., Miller J.A., Smith M.D., Barkan E.R., Graybuck L.T., Close J.L., Long B., Johansen N., Penn O., et al. Conserved cell types with divergent features in human versus mouse cortex. Nature. 2019;573:61–68. doi: 10.1038/s41586-019-1506-7. PubMed DOI PMC

Smith A.G., Rowland Hill C. Imaging assessment of acute ischaemic stroke: A review of radiological methods. Br. J. Radiol. 2017;91:20170573. doi: 10.1259/bjr.20170573. PubMed DOI PMC

Ciesienski K.L., Caravan P. Molecular MRI of Thrombosis. Curr. Cardiovasc. Imaging Rep. 2011;4:77–84. doi: 10.1007/s12410-010-9061-5. PubMed DOI PMC

Kim J., Park J.E., Nahrendorf M., Kim D.-E. Direct Thrombus Imaging in Stroke. J. Stroke. 2016;18:286–296. doi: 10.5853/jos.2016.00906. PubMed DOI PMC

Koudelka S., Mikulik R., Mašek J., Raška M., Knotigová P.T., Miller A.D., Turánek J. Liposomal nanocarriers for plasminogen activators. J. Control. Release. 2016;227:45–57. doi: 10.1016/j.jconrel.2016.02.019. PubMed DOI

Hosokawa K., Ohnishi-Wada T., Sameshima-Kaneko H., Nagasato T., Miura N., Kikuchi K., Koide T., Maruyama I., Urano T. Plasminogen activator inhibitor type 1 in platelets induces thrombogenicity by increasing thrombolysis resistance under shear stress in an in-vitro flow chamber model. Thromb. Res. 2016;146:69–75. doi: 10.1016/j.thromres.2016.09.002. PubMed DOI

Janis A.D., Buckley L.A., Nyara A.N., Prahl S.A., Gregory K. A reconstituted in vitro clot model for evaluating laser thrombolysis. J. Thromb. Thrombolysis. 2002;13:167–175. doi: 10.1023/A:1020431007864. PubMed DOI

Ouriel K., Welch E.L., Shortell C.K., Geary K., Fiore W.M., Cimino C. Comparison of streptokinase, urokinase, and recombinant tissue plasminogen activator in an in vitro model of venous thrombolysis. J. Vasc. Surg. 1995;22:593–597. doi: 10.1016/S0741-5214(95)70045-5. PubMed DOI

Roessler F.C., Ohlrich M., Marxsen J.H., Schmieger M., Weber P.-K., Stellmacher F., Trillenberg P., Eggers J., Seidel G. Introduction of a new model for time-continuous and non-contact investigations of in-vitro thrombolysis under physiological flow conditions. BMC Neurol. 2011;11:58. doi: 10.1186/1471-2377-11-58. PubMed DOI PMC

Stroughton J., Ouriel K., Shortell C.K., Cho J.S., Marder V.J. Plasminogen acceleration of urokinase thrombolysis. J. Vasc. Surg. 1994;19:298–303. doi: 10.1016/S0741-5214(94)70105-9. PubMed DOI

Wang S.-S., Chou N.-K., Chung T.-W. The t-PA-encapsulated PLGA nanoparticles shelled with CS or CS-GRGD alter both permeation through and dissolving patterns of blood clots compared with t-PA solution: An in vitro thrombolysis study. J. Biomed. Mater. Res. Part A. 2009;91:753–761. doi: 10.1002/jbm.a.32234. PubMed DOI

Fahy P., Malone F., McCarthy E., McCarthy P., Thornton J., Brennan P., O’Hare A., Looby S., Sultan S., Hynes N., et al. An In Vitro Evaluation of Emboli Trajectories within a Three-Dimensional Physical Model of the Circle of Willis under Cerebral Blood Flow Conditions. Ann. Biomed. Eng. 2015;43:2265–2278. doi: 10.1007/s10439-015-1250-6. PubMed DOI

Yohannes F.G., Hoffmann A.K. Non-invasive low frequency vibration as a potential emergency adjunctive treatment for heart attack and stroke. An in vitro flow model. J. Thromb. Thrombolysis. 2008;25:251–258. doi: 10.1007/s11239-007-0054-4. PubMed DOI

Mašek J., Bartheldyová E., Turánek-Knotigová P., Škrabalová M., Korvasová Z., Plocková J., Koudelka S., Škodová P., Kulich P., Krupka M., et al. Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: Preparation, structural study and immune response towards rHsp90. J. Control. Release. 2011;151:193–201. doi: 10.1016/j.jconrel.2011.01.016. PubMed DOI

Mašek J., Bartheldyová E., Korvasová Z., Škrabalová M., Koudelka S., Kulich P., Kratochvílová I., Miller A.D., Ledvina M., Raška M., et al. Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure. Anal. Biochem. 2011;408:95–104. doi: 10.1016/j.ab.2010.08.023. PubMed DOI

Overgaard K., Sereghy T., Boysen G., Pedersen H., Høyer S., Diemer N.H. A rat model of reproducible cerebral infarction using thrombotic blood clot emboli. J. Cereb. Blood Flow Metab. 1992;12:484–490. doi: 10.1038/jcbfm.1992.66. PubMed DOI

Chameettachal S., Pati F. 6–3D printed in vitro disease models. In: Kalaskar D.M., editor. 3D Printing in Medicine. Woodhead Publishing; Sawston, UK: 2017. pp. 115–138. DOI

Kaneko N., Mashiko T., Ohnishi T., Ohta M., Namba K., Watanabe E., Kawai K. Manufacture of patient-specific vascular replicas for endovascular simulation using fast, low-cost method. Sci. Rep. 2016;6:39168. doi: 10.1038/srep39168. PubMed DOI PMC

Nagassa R.G., McMenamin P.G., Adams J.W., Quayle M.R., Rosenfeld J.V. Advanced 3D printed model of middle cerebral artery aneurysms for neurosurgery simulation. 3D Print. Med. 2019;5:11. doi: 10.1186/s41205-019-0048-9. PubMed DOI PMC

De Long W.B. Anatomy of the middle cerebral artery: The temporal branches. Stroke. 1973;4:412–418. doi: 10.1161/01.STR.4.3.412. PubMed DOI

Elnager A., Abdullah W.Z., Hassan R., Idris Z., Arfah N.W., Sulaiman S.A., Mustafa Z. In Vitro Whole Blood Clot Lysis for Fibrinolytic Activity Study Using D-Dimer and Confocal Microscopy. Adv. Hematol. 2014;2014:814684. doi: 10.1155/2014/814684. PubMed DOI PMC

Leenaerts D., Loyau S., Mertens J.C., Boisseau W., Michel J.B., Lambeir A.M., Jandrot-Perrus M., Hendriks D. Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models. J. Thromb. Haemost. JTH. 2018;16:2057–2069. doi: 10.1111/jth.14249. PubMed DOI

Meunier J.M., Wenker E., Lindsell C.J., Shaw G.J. Individual lytic efficacy of recombinant tissue plasminogen activator in an in vitro human clot model: Rate of “nonresponse”. Acad. Emerg. Med. Off. J. Soc. Acad. Emerg. Med. 2013;20:449–455. doi: 10.1111/acem.12133. PubMed DOI PMC

Walvick R.P., Bratane B.T., Henninger N., Sicard K.M., Bouley J., Yu Z., Lo E., Wang X., Fisher M. Visualization of clot lysis in a rat embolic stroke model: Application to comparative lytic efficacy. Stroke. 2011;42:1110–1115. doi: 10.1161/STROKEAHA.110.602102. PubMed DOI PMC

Schellinger P.D., Fiebach J.B., Hacke W. Imaging-based decision making in thrombolytic therapy for ischemic stroke: Present status. Stroke. 2003;34:575–583. doi: 10.1161/01.STR.0000051504.10095.9C. PubMed DOI

Petroková H., Mašek J., Kuchař M., Wünschová A.V., Štikarová J., Bartheldyová E., Kulich P., Hubatka F., Kotouček J., Knotigová P.T., et al. Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders. Pharmaceutics. 2019;11:642. doi: 10.3390/pharmaceutics11120642. PubMed DOI PMC

Spero R.C., Sircar R.K., Schubert R., Taylor R.M., Wolberg A.S., Superfine R. Nanoparticle Diffusion Measures Bulk Clot Permeability. Biophys. J. 2011;101:943–950. doi: 10.1016/j.bpj.2011.06.052. PubMed DOI PMC

Benson J.C., Fitzgerald S.T., Kadirvel R., Johnson C., Dai D., Karen D., Kallmes D.F., Brinjikji W. Clot permeability and histopathology: Is a clot’s perviousness on CT imaging correlated with its histologic composition? J. Neurointerv. Surg. 2020;12:38–42. doi: 10.1136/neurintsurg-2019-014979. PubMed DOI PMC

Botnar R.M., Perez A.S., Witte S., Wiethoff A.J., Laredo J., Hamilton J., Quist W., Parsons E.C., Vaidya A., Kolodziej A., et al. In Vivo Molecular Imaging of Acute and Subacute Thrombosis Using a Fibrin-Binding Magnetic Resonance Imaging Contrast Agent. Circulation. 2004;109:2023–2029. doi: 10.1161/01.CIR.0000127034.50006.C0. PubMed DOI PMC

Sirol M., Fuster V., Badimon J.J., Fallon J.T., Toussaint J.-F., Fayad Z.A. Chronic thrombus detection with in vivo magnetic resonance imaging and a fibrin-targeted contrast agent. Circulation. 2005;112:1594–1600. doi: 10.1161/CIRCULATIONAHA.104.522110. PubMed DOI

Kotouček J., Hubatka F., Mašek J., Kulich P., Velínská K., Bezděková J., Fojtíková M., Bartheldyová E., Tomečková A., Stráská J., et al. Preparation of nanoliposomes by microfluidic mixing in herring-bone channel and the role of membrane fluidity in liposomes formation. Sci. Rep. 2020;10:5595. doi: 10.1038/s41598-020-62500-2. PubMed DOI PMC

Korninger C., Collen D. Studies on the Specific Fibrinolytic Effect of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood and in Various Animal Species in Vitro. Thromb. Haemost. 1981;46:561–565. doi: 10.1055/s-0038-1653411. PubMed DOI

Tomkins A.J., Hood R.J., Levi C.R., Spratt N.J. Tissue Plasminogen Activator for preclinical stroke research: Neither “rat” nor “human” dose mimics clinical recanalization in a carotid occlusion model. Sci. Rep. 2015;5:16026. doi: 10.1038/srep16026. PubMed DOI PMC