Name: percutaneous contrast echocardiography-guided intramyocardial injection and cell delivery in a large preclinical model
Uploaded: 2018-01-21T15:00:00
Description: Watch this Scientific Journal Video about Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model at JoVE.com

The authors thank Sheila Monfort, Brenda Mart&#237;nez, Carlos Mic&#243;, Alberto Mu&#241;oz, and Manuel Molina for excellent support provided during the collection of data, and Carlos Bueno for providing the EGFP(+) HEK-293 cells. This work was supported in part by: Fundaci&#243;n S&#233;neca, Agencia de Ciencia y Tecnolog&#237;a, Regi&#243;n de Murcia, Spain (JT) (Grant number: 11935/PI/09); Red de Terapia Celular, ISCIII-Sub. Gral. Redes, VI PN de I+D+I 2008-2011 (Grant no. RD12/0019/0001) (JMM), Co-financed with Structural funding of the European Union (FEDER) (JMM); and, the University of Reading, United Kingdom (AG, GB) (Central Funding). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The experiments described herein were approved by the Ethical Research Committee of the University of Murcia, Spain, and were performed in accordance with Directive 2010/63/EU of the European Commission. The steps described were performed under standard operating protocols that were part of the plan of work and have not been performed solely for the purpose of filming the accompanying video to this paper.
1. Preparation of Cells and Mammalian Expression Vector
NOTE: H...

The primary goal was to develop a minimally-invasive technique that could be used for the delivery of stem cells into the myocardium of rabbits (a large sized preclinical animal model)17,18, whilst taking advantage of the use of a relatively inexpensive imaging system readily available in many clinical and research centers. Here, we show that, using a clinical echocardiography system, and aided by InI, a widely available agent, with both in situ tracing ...

Cell and gene therapies are exciting and ever developing strategies for regenerating/repairing the injured myocardium in HFrEF. A few studies have compared the effectiveness (e.g., cell retention rate) of the different routes of cell delivery, which have consistently demonstrated the superiority of IMI over intracoronary or intravenous routes1,2,3,4,5. Thus, it is not surprising that a large proportion of studies on translational models of stem cell therapy of the injured myocardium, deliver the injectate via IMI performed under direct view in an open chest procedure6,7. However, this approach has several limitations, including the invasive nature of the procedure, which carries the risk of peri-procedural mortality (often under-reported)8. In addition, an IMI under direct view does not eliminate the possibility for inadvertent injection into the ventricular cavity. In clinical practice an IMI during open chest surgery could be an appropriate method for therapeutic cell delivery, e.g., during coronary artery bypass graft surgery (CABG); however, this approach may not be appropriate for cell delivery in global cardiomyopathy of non-ischemic origin (e.g., HFrEF secondary to anthracycline-induced cardiomyopathy (AICM)).
There is no doubt that ischemic heart disease (IHD) is the most common cause of HFrEF (~ 66%)9,10; however, non-ischemic cardiomyopathy, including AICM, still affects a significant proportion of patients with HFrEF (33%)9. Indeed, recent advances in clinical oncology have resulted in more than 10 million survivors of cancer in the USA alone11, with estimates of a similar number in Europe, consistent with an overall trend towards improved survival of cancer patients12,13. Thus, exploring the benefits of novel therapies such as stem cell transplant for non-ischemic cardiomyopathy, as well as the trialing of an effective and minimally invasive route of stem cell delivery is of utmost importance, given the increasing number of patients affected by cardiotoxicity secondary to anticancer drugs.
Of note, hypothesis testing studies using stem cell therapy aiming to repair/regenerate the injured myocardium frequently involves the use of small rodents (e.g., mice and rats). These models often require expensive high frequency ultrasound systems for evaluation of myocardial function, usually equipped with linear array transducers which have some inherent associated limitations (e.g., reverberation)14. However, other models such as rabbits, representing a large preclinical model, have some advantages for hypothesis testing of stem cell therapies in HFrEF. Thus, in contrast to rats and mice, rabbits maintain a Ca+2 transport system and cellular electrophysiology that resembles that of humans and other large animals (e.g., dogs and pigs)15,16,17,18,19. Another advantage, is their amenability for cardiac ultrasound imaging using relatively inexpensive and widely available clinical echocardiography systems equipped with relatively high frequency phase array transducers, e.g., 12 MHz, such as those frequently used in neonatal and pediatric cardiology. These systems allow excellent echocardiographic imaging with state of the art technology, and they take advantage of the superiority of harmonic imaging20. Furthermore, extensive hypothesis testing of the potential of cardiac regenerative therapies (e.g., stem cell therapy), their safety, efficacy, cardiomyogenic potential, as well as evaluation of the fate of the injectate once delivered into the myocardium, is mandatory before they can be considered for human use, and they require the use of large preclinical animal models, such as the rabbit17,19. Here, we describe a minimally invasive technique for cell delivery via percutaneous contrast-echocardiography guided IMI using a clinical echocardiography system, which is aimed at stem cell transplant-based therapy for non-ischemic cardiomyopathy20. We also describe the benefits of India Ink (InI, also known as China Ink) as an ultrasound contrast agent and in situ tracer of the injectate in the rabbit heart.

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			<td height="21" style="height:21px;width:319px;">HD11 XE Ultrasound System</td>
			<td style="width:131px;">Philips</td>
			<td style="width:183px;">10670267</td>
			<td style="width:259px;">Echocardiography system.</td>
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			<td height="21" style="height:21px;width:319px;">S12-4</td>
			<td style="width:131px;">Philips</td>
			<td style="width:183px;">B01YgG</td>
			<td>4-12 MHz phase array transducer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ultrasound Transmision Gel (Aquasone)</td>
			<td style="width:131px;">Parket laboratories Inc</td>
			<td style="width:183px;">N 01-08</td>
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			<td height="21" style="height:21px;">Vasovet 24G</td>
			<td style="width:131px;">Braun</td>
			<td style="width:183px;">REF 381212</td>
			<td>&#160;over-the-needle catheter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Omnifix-F 1 ml syringe</td>
			<td style="width:131px;">Braun</td>
			<td style="width:183px;">9161406V</td>
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			<td height="21" style="height:21px;">Imalgene (Ketamine)</td>
			<td style="width:131px;">Merial</td>
			<td style="width:183px;">RN 9767</td>
			<td>Veterinary prescription is necessary</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Domtor (Medetomidine)</td>
			<td style="width:131px;">Esteve</td>
			<td style="width:183px;">CN 570686.3</td>
			<td>Veterinary prescription is necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating Pad</td>
			<td style="width:131px;"></td>
			<td style="width:183px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Faber-Castel TG1</td>
			<td style="width:131px;">Faber-Castel</td>
			<td style="width:183px;">16 33 99</td>
			<td style="width:259px;">India (China) Ink</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Holter Syneflash</td>
			<td style="width:131px;">Ela medical</td>
			<td style="width:183px;">SF0003044S</td>
			<td style="width:259px;">24 h Holter ECG system.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Electrodes Blue Sensor&#174;</td>
			<td style="width:131px;">Ambu (NUMED)</td>
			<td style="width:183px;">VLC-00-S</td>
			<td style="width:259px;">Holter ECG electrodes.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Microtome</td>
			<td style="width:131px;">Leica Biosystems</td>
			<td style="width:183px;">RM2155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microscope</td>
			<td style="width:131px;">Olimpus</td>
			<td style="width:183px;">CO11</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">ABC Vector Elite</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:183px;">PK-6200</td>
			<td>Avidin Biotin Complex Kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Chicken anti-GFP antibody</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:183px;">A10262</td>
			<td>Primary antibody.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">Biotinylated goat-anti-chicken IgG Antibody</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:183px;">BA-9010</td>
			<td>Secondary Antibody.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">3,30-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:131px;">DAKO (Agilent)</td>
			<td style="width:183px;">S3000</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">Fluorescence Microscope</td>
			<td style="width:131px;">Carl Zeiss 
			MicroImaging</td>
			<td style="width:183px;">Zeiss AX10 Axioskop</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Holter ECG</td>
			<td>Elamedical</td>
			<td>Syneflash SF0003044S</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">Dulbecco&#8217;s modified Eagle medium (DMEM)&#160;</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10% fetal calf serum (FCS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">11573397</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">25300054</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">Lipofectamine 2000 (Lipid transfection reagent)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Reduced serum medium (Opti-MEM)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">31985070</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:319px;">Hygromycin B</td>
			<td style="width:131px;">Calbiochem (MERCK)</td>
			<td style="width:183px;">400051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Xylene (histological)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">X3S-4</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hydrogen Peroxide Solution (H2O2)</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:183px;">H1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Pronase</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:183px;">53-702-250KU</td>
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</table>

percutaneous contrast echocardiography-guided intramyocardial injection and cell delivery in a large preclinical model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Percutaneous Contrast Echocardiography-guided IMI with InI:
Using the protocol described above, and once the optimal positioning of the tip of the needle was confirmed by echocardiography and the injection initiated, transmural hyperechogenicity was observed during the delivery of InI (10% v/v in PBS) (Figure 2E), as well as shortly after the IMI to the target region (<strong class=...

Watch this Scientific Journal Video about Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model at JoVE.com

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

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