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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 anesthesia by observing muscle tone and confirming the absence of the toe pinch reflex.
2. Place the animal on a sterile drape in prone position, and k....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Machines
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
Software
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
Devices/Instruments
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
Reagents
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