Name: combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli
Uploaded: 2016-09-25T14:00:00
Description: Watch this Scientific Journal Video about Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli at JoVE.com

This work was supported by the Korea Healthcare Technology R&#38;D Project, Ministry of Health and Welfare (HI12C1847, HI12C0066), the Bio &#38; Medical Technology Development Program (2010-0019862) and Global Research Lab (GRL) program (NRF-2015K1A1A2028228) of the National Research Foundation, funded by the Korean government.

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)
<ol>
	<li>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...

<ol>
	<li>Saver, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+is+brain--quantified.">Time is brain--quantified.</a> Stroke. 37 (1), 263-266 (2006).</li><li>Latchaw, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+imaging+of+acute+ischemic+stroke:+a+scientific+statement+from+the+American+Heart+Association.">Recommendations for imaging of acute ischemic stroke: a scientific statement from the American Heart Association.</a> Stroke. 40 (11), 3646-3678 (2009).</li><li>Kim, D. 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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...

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 &#34;Time is Brain&#34;. 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 hyperacute ischemic stroke. CT is particularly valuable for informing the critical early decisions: administering tissue plasminogen activator (tPA) for thrombolysis and/or triaging to endovascular clot-retrieval2. Current CT-based thrombus imaging, however, cannot serially track cerebral thromboemboli in vivo, because it uses indirect methods to demonstrate thrombi: after opacification of the blood pool by iodinated contrast, the thrombi are demonstrated as filling defects in the vessels. There are dose limits and risks associated with the repeated administration of iodinated contrast, which preclude repeated imaging of thrombi in this manner.
Thus, there is a critical need for a direct imaging methodology for cerebral thrombi in stroke patients, to allow faster and better treatment decisions to be made. We propose to accomplish this by enhancing the value of CT, the currently used frontline imaging modality for stroke, with the use of a thrombus-seeking nanoparticular molecular imaging agent.
We have demonstrated the use of this agent using micro-computed tomography (microCT), a high-resolution ex vivo or in vivo (small animal) imaging version of CT that allows rapid data acquisition3,4. Even with the relatively poor soft tissue contrast available for small animal microCT (much worse than available from human sized scanners), the imaging agent was able to seek and mark thrombi by making them hyperdense on CT, a &#39;dense vessel sign&#39; enhanced by molecular imaging.
Complementing the CT technique, our group has previously developed an optical direct thrombus imaging technique using Cy5.5 near-infrared fluorescent (NIRF) probe to visualize cerebral thrombus burden5. This is an ex vivo technique on post mortem brains, but is highly sensitive, and serves to confirm in vivo data in the research setting.
Having both CT and NIRF based thrombus-seeking imaging techniques allows us to compare and contrast these techniques to achieve highly informative data on the role of thrombus and thrombus imaging in the process of ischemic stroke development.
Here, we describe a detailed protocol of a combined technique of in vivo microCT and ex vivo NIRF imaging to directly visualize thromboemboli in a mouse model of embolic stroke. These simple and robust methods are useful to advance our understanding of thrombotic diseases by enabling the accurate in vivo assessment of thrombus burden / distribution and characterization of dynamic thrombus evolution in a prompt and quantitative manner in vivo during therapy, followed by ex vivo data that serves as a control and reference standard for the confirmation of in vivo imaging findings.

<table border="0" cellpadding="0" cellspacing="0" width="588">
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		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Machines</td>
			<td style="width:219px;"></td>
			<td style="width:119px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microCT</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIRF imaging system</td>
			<td>Roper-scientific,Tucson, AZ</td>
			<td>coolsnap-Ez</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Doppler flowmeter</td>
			<td>Perimed, Stockholm, Sweden</td>
			<td>PeriFlux System 5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical microscope</td>
			<td>Leica Microsystems, Seoul, Korea</td>
			<td>EZ4HD</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Inhalation anesthesia machine</td>
			<td>PerkinElmer, Massachusetts, USA</td>
			<td>XGI-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">NFR control</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td style="width:96px;">microCT control software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Lucion</td>
			<td>Infinitt, Seoul, Korea</td>
			<td>Lucion</td>
			<td style="width:96px;">3D render imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab chart 7</td>
			<td>ADInstruments, Colorado, USA</td>
			<td>Lab chart 7</td>
			<td>rCBF</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Image J software</td>
			<td>Wanye Rasband, NIH, USA</td>
			<td>1.49d</td>
			<td>imaging analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Devices/Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Infusion pump</td>
			<td>Harvard, Massachusetts, USA</td>
			<td>pump 22(55-2226)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homeothermic blanket</td>
			<td>Panlab, Barcelona, Spain</td>
			<td>HB101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pocket cautery</td>
			<td>Daejong, Seoul, Korea</td>
			<td>DJE-39</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain matrice</td>
			<td>Ted pella, CA, USA</td>
			<td>15003</td>
			<td style="width:96px;">coronal section</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-50 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-45(PE-50)</td>
			<td style="width:96px;">I.D. 0.58 mm O.D. 0.96 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-10 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-10(PE-10)</td>
			<td style="width:96px;">I.D. 0.28mm O.D. 0.61 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">30 gauge needle</td>
			<td>sungshim-medical, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>CPL-medical, Ansan, Korea</td>
			<td></td>
			<td>1 &#38; 3 cc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gauze</td>
			<td>Panamedic, Cheonan, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tape</td>
			<td>Scotch, Seoul, Korea</td>
			<td>3M-810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro forceps</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td>&#160;11253-27</td>
			<td>Dumont #L5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">15000-03</td>
			<td>Vannas spring</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">14084-08</td>
			<td>8.5 cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black silk suture</td>
			<td>Ailee, Busan, Korea</td>
			<td>SK6071, SK728</td>
			<td>6-0 and 7-0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">meloxicam</td>
			<td>Yuhan, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vet ointment</td>
			<td>Novartis, Basel, Swiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% Povidone-iodine (betadine)</td>
			<td>Firson, Cheon-an, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FeCl3</td>
			<td>Sigma, Missouri, United States</td>
			<td>157740-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TTC</td>
			<td>Amresco, Ohio, USA</td>
			<td>0765-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Hana-Pham, Gyeonggi, Korea</td>
			<td>Ifran</td>
			<td>100 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Welgene, Daegu, Korea</td>
			<td style="width:119px;">LB001-02</td>
			<td>500 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold nanoparticles</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C15 optical agent</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue plasminogen activator</td>
			<td style="width:219px;">Boehringer Ingelheim, Biberach, Germany</td>
			<td style="width:119px;">rtPA(actilyse)</td>
			<td>20 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Daihan Pham, Seoul, Korea</td>
			<td>48N3AF3</td>
			<td>20 mL</td>
		</tr>
	</tbody>
</table>

combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli

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&#160;&#8212; 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.

Baseline microCT images, obtained in vivo after administering fib-GC-AuNP (10 mg/ml, 300 &#181;l) at 1 hr after embolic stroke, clearly visualized cerebral thrombus in the MCA &#8211; 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...

Watch this Scientific Journal Video about Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli at JoVE.com

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

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 ...

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Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

Near Infrared Optical Projection Tomography for Assessments of ÃƒÅ½Ã‚Â²-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...