Name: 3d whole-heart myocardial tissue analysis
Uploaded: 2017-04-12T13:00:00
Description: Watch this Scientific Journal Video about 3D Whole-heart Myocardial Tissue Analysis at JoVE.com

The authors would like to thank Marlijn Jansen, Joyce Visser, and Martijn van Nieuwburg for their assistance with the animal experiments. We greatly acknowledge Martijn Froeling and Anke Wassink for their assistance with the MRI imaging.

The in vivo experiment was conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Research. The experiment was approved by the local Animal Experiments Committee.
1. Preparation of Injectable and Embedding Solution
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	<li>Prepare the injectable gel.
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		<li>Prepare 1 mL of ureido-pyrimidinone (UPy) gel in accordance to previously-described protocols12,<sup class="xref"...

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Whole-heart 3D myocardial tissue processing according to this protocol provides a structured method that enables the 3D analysis of the infarct, the IBZ, and the performed injections with respect to the cardiac anatomy. The filling volume of the heart depends on the desired analysis. In this study, to assess the injection accuracy, we aimed to fill the heart to resemble the end-diastolic geometry as closely as possible. To enforce this, the LV apex is fixed to the bottom of the container and the LV is filled with agar wh...

Ischemic heart disease has been the world&#39;s leading cause of death for the past decades1. Acute treatment after myocardial infarction aims to restore blood flow to the myocardium via percutaneous coronary intervention or coronary artery bypass grafting. In severe infarctions, a large area of the myocardium is scarred, and these cases often result in ischemic heart failure (HF)2. Current treatment options for HF focus on prevention and the preservation of cardiac function for the HF patients, but not on regeneration.
In the last decade, cardiac regenerative therapies have been investigated as a treatment option for HF3. This therapy aims to deliver biologicals, such as stem cells or growth factors, directly to the injured myocardium to induce revascularization, cardiomyocyte protection, differentiation, and growth4. For optimal therapeutic effect, it is hypothesized that the biological must be injected in the infarct border zone (IBZ) to facilitate good tissue perfusion for the survival of the biological and for optimal effect to the target zone5,6. Multiple techniques have been developed to perform identification and visualization of the IBZ to guide intramyocardial injections7,8,9,10,11. Besides identification and visualization of the IBZ, the delivery also relies on the biomaterials and injection catheters used. To validate the injection accuracy of the delivery techniques, an accurate and reproducible quantification method is required.
We have developed a protocol for whole-heart myocardial tissue processing that offers two-dimensional (2D) and three-dimensional (3D) imaging, which can be used for qualitative and quantitative study aims. The protocol covers the embedding process and the digital image analysis. In this paper, we demonstrate a protocol for the assessment of the targeting accuracy of intramyocardial injections in the IBZ in a large porcine model of chronic myocardial infarction.

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			<td height="20" style="height:20px;width:216px;">0.9% Saline</td>
			<td style="width:108px;">Braun</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Agarose</td>
			<td style="width:108px;">Roche Diagnostics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Scientific grade multipurpose agar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biomolecular fluorescence scanner Typhoon 9410&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Embedding container</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Plastic, dimensions 17 x 14,5 x 14 cm</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">FluoSpheres Polystyrene Microspheres</td>
			<td style="width:108px;">Invitrogen</td>
			<td>F8834</td>
			<td>red, 10 &#181;m</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gadolinium</td>
			<td style="width:108px;">Gadovist</td>
			<td style="width:119px;"></td>
			<td>1,0 mmol/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">dS 32 channel head coil</td>
			<td style="width:108px;">Philips</td>
			<td style="width:119px;"></td>
			<td>Or similar</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Matlab</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To insure compatability 2015a or newer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Meat slicer</td>
			<td style="width:108px;">Berkel</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Myostar injection catheter</td>
			<td style="width:108px;">Biosense Webster</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Super paramagnetic iron oxide particles</td>
			<td style="width:108px;">Sinerem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Triphenyl-tetrazolium chloride</td>
			<td style="width:108px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UPy-PEG10k</td>
			<td style="width:108px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Vicryl 2-0</td>
			<td style="width:108px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

3d whole-heart myocardial tissue analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

Tissue Embedding
Through the embedding process, an end-diastolic-like geometry was established. The agar successfully adhered to the heart tissue, enabling the tissue to be sliced at the desired angulation with equal slice thicknesses (Figure 2A and 2C).
Scar- and Injection-site Assessment
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Watch this Scientific Journal Video about 3D Whole-heart Myocardial Tissue Analysis at JoVE.com

3D Whole-heart Myocardial Tissue Analysis

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

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