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Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Published: September 25th, 2016



1Molecular Imaging and Neurovascular Research Laboratory, Dongguk University College of Medicine, 2Biomedical Research Center, Korea Institute of Science and Technology, 3Research Institute of Advanced Materials, Department of Materials Science and Engineering, Seoul National University, 4Departments of Radiology and Cancer Systems Imaging, University of Texas M.D. Anderson Cancer Center

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker — a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

One in 6 people will have a stroke at some point in their lifetime. Ischemic stroke is by far the most common stroke type, and accounts for about 80 percent of all stroke cases. Because thromboemboli cause the majority of these ischemic strokes, there is an increasing interest in direct thrombus imaging.

It was estimated that about 2 million brain cells die during every minute of middle cerebral artery occlusion1, leading to the slogan "Time is Brain". Computed tomography (CT) studies can be done rapidly, and are widely available; for this reason, CT remains the imaging of choice for the initial diagnosis and treatment of....

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All animal procedures demonstrated in this protocol have been reviewed and approved by the Dongguk University Ilsan Hospital Animal Care and Use Committee and conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals.

1. Preparation of Exogenously Formed Clot Labeled with Fluorescence Marker (Figure 1)

  1. Anesthetize a mouse in an induction chamber using 3% isoflurane mixed with 30% oxygen (1.5 L/min 100% oxygen). Ensure adequate depth of anesthes.......

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Baseline microCT images, obtained in vivo after administering fib-GC-AuNP (10 mg/ml, 300 µl) at 1 hr after embolic stroke, clearly visualized cerebral thrombus in the MCA – ACA bifurcation area of the distal internal carotid artery (Figure 6). Follow-up microCT imaging showed no change in the COW thrombus with saline treatment. However, treatment with tPA showed a gradual dissolution of the COW thrombus (blue arrowheads in Figure 6). T.......

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We demonstrated the use of two complementary molecular imaging techniques for direct thrombus imaging in experimental models of embolic stroke: a fibrin targeted gold nanoparticle (fib-GC-AuNP) for in vivo microCT-based imaging, and a FXIIIa targeted optical imaging probe for ex vivo fluorescent imaging.

After intravenous administration of fib-GC-AuNPs, thrombi became visible to CT as dense structures, caused by the particles becoming entrapped in the thrombi by the action of.......

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This work was supported by the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare (HI12C1847, HI12C0066), the Bio & Medical Technology Development Program (2010-0019862) and Global Research Lab (GRL) program (NRF-2015K1A1A2028228) of the National Research Foundation, funded by the Korean government.


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Name Company Catalog Number Comments
microCT NanoFocusRay, JeonJu, Korea NFR Polaris-G90
NIRF imaging system Roper-scientific,Tucson, AZ coolsnap-Ez
Laser Doppler flowmeter Perimed, Stockholm, Sweden PeriFlux System 5000
Surgical microscope Leica Microsystems, Seoul, Korea EZ4HD
Inhalation anesthesia machine PerkinElmer, Massachusetts, USA XGI-8
NFR control NanoFocusRay, JeonJu, Korea NFR Polaris-G90 microCT control software
Lucion Infinitt, Seoul, Korea Lucion 3D render imaging software
Lab chart 7 ADInstruments, Colorado, USA Lab chart 7 rCBF
Image J software Wanye Rasband, NIH, USA 1.49d imaging analysis
Infusion pump Harvard, Massachusetts, USA pump 22(55-2226)
Homeothermic blanket Panlab, Barcelona, Spain HB101
Pocket cautery Daejong, Seoul, Korea DJE-39
Brain matrice Ted pella, CA, USA 15003 coronal section
PE-50 tubing Natsume, Tokyo, Japan SP-45(PE-50) I.D. 0.58 mm O.D. 0.96 mm
PE-10 tubing Natsume, Tokyo, Japan SP-10(PE-10) I.D. 0.28mm O.D. 0.61 mm
30 gauge needle sungshim-medical, Seoul, Korea
Syringe CPL-medical, Ansan, Korea 1 & 3 cc
Gauze Panamedic, Cheonan, Korea
Tape Scotch, Seoul, Korea 3M-810
Micro forceps Fine Science Tools, Vancouver, Canada  11253-27 Dumont #L5
Micro scissor Fine Science Tools, Vancouver, Canada 15000-03 Vannas spring
Scissor Fine Science Tools, Vancouver, Canada 14084-08 8.5 cm
Black silk suture Ailee, Busan, Korea SK6071, SK728 6-0 and 7-0
meloxicam Yuhan, Seoul, Korea
vet ointment Novartis, Basel, Swiss
10% Povidone-iodine (betadine) Firson, Cheon-an, Korea
FeCl3 Sigma, Missouri, United States 157740-5G
TTC Amresco, Ohio, USA 0765-100g
Isoflurane Hana-Pham, Gyeonggi, Korea Ifran 100 mL
PBS Welgene, Daegu, Korea LB001-02 500 mL
Gold nanoparticles Synthesis
C15 optical agent Synthesis
Tissue plasminogen activator Boehringer Ingelheim, Biberach, Germany rtPA(actilyse) 20 mg
Normal saline Daihan Pham, Seoul, Korea 48N3AF3 20 mL

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