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.
	<ol>
		<li>Prepare 1 mL of ureido-pyrimidinone (UPy) gel in accordance to previously-described protocols12,<sup class="xref"...

<ol>
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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.

<table border="0" cellpadding="0" cellspacing="0" width="610">
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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>
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		<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 ...

Narrator}The overall goal of this protocol is to facilitate a structured and reproducible method for 3D whole-heart tissue processing that can be used to assess the targeting accuracy of intramyocardial injections into the infarct border zone. This method can help to answer key questions in the cardioregenerative medicine field, such as practice development and optimization of injection techniques. The main advantage of this technique is that it offers a standardized and reproducible method for 3D whole-heart tissue processing.The implications of this technique extend towards development of cardiac regenerative therapies and validating injection accuracy of normal delivery techniques. The aim of this cardiac stem cell therapy is to inject cells into the infarct border zone for stimulation of local vasculogenesis and cardiomyocytes protection. Capturing the heart in end diastolic phase requires practice and patience.A visual demonstration of these steps is critical. Begin by separating the pericardium from the microbead injected heart, keeping the atria and ventricles intact. When all of the tissue has been removed, use Klinkenberg scissors to dissect the ascending aorta about one centimeter above the aortic valve, and cut the inferior caval and pulmonary veins about one centimeter from the atrium.After fixing the apex of the heart to the bottom of the plastic embedding container to prevent flotation, place 2-0 sutures through the remaining part of the aorta to the rims of the container, and fixate the heart. Take care that the heart is centered and is not touching the walls of the container. When the organ has been secured in an end diastolic like geometry, use mosquito clamps to grip the inferior caval vein and use a 50 milliliter syringe to slowly inject freshly prepared 50 to 60 degree Celsius agar into the right atrium via the superior caval vein until both the right atrium and ventricle are completely filled.To obtain an embedded heart resembling the end diastolic geometry as closely as possible, fix the left ventricle apex to the bottom of the container and fill the right ventricle with agar while the pulmonary veins are clamped. Next, place a mosquito clamp on the pulmonary veins and gently pass a new agar filled 50 milliliter syringe in the retrograde direction through the aortic valves. Slowly inject the solution into the left ventricle until the ventricle and left atrium are completely full, clamping the aorta once the left ventricle has been filled.Now, pour the remaining agar into the container until the heart is fully covered and place two rigid plastic tubes within the embedding container to serve as reference structures during the image registration. Then place the heart at two to seven degrees Celsius for at least four hours to solidify the agar. After magnetic resonance imaging, turn the container upside down and allow air between the agar and the sides of the container to remove the solid agar solution and the heart from the container.Remove the plastic rods. Then use a meat slicer to section the agar block and the heart in five millimeter slices from the apex to the base of the heart, cutting parallel to the bottom of the agar block to keep the angulation of the slices the same as in the acquired magnetic resonance images. When all of the slices have been obtained, stain the tissue samples in TTC.After 15 minutes, photograph the slices on both sides at a perpendicular angle, then carefully rinse the sections in 0.9%saline. To image the samples by fluorescence microscopy, select fluorescence mode imaging on the variable mode scanner and set the pixel size to 100 by 100 microns. For the first filter block, select a band pass filter that overlaps with the emission wavelength of the bead fluorescence in channel one, then to band pass filter for the second filter block outside the emission wavelength in channel two.Next, select the appropriate filter block, set the photomultiplier tube to the appropriate voltage, and the excitation laser to the excitation wavelength closest to that of the injected microbeads. After all of the images have been obtained, in the appropriate post processing software, segment the myocardium and endo-and epicardial left ventricle borders in the magnetic resonance images followed by a similar segmentation of the myocardium and injection sites in the fluorescence images. Then linearly interpolate the image registered segments from both sides of the slices to reconstruct the original sample thickness and to create a 3D model of the data.With the heart secured in an end diastolic like geometry as just demonstrated, in this representative experiment, the agar successfully adhered to the heart tissue enabling the tissue to be sliced at the desired angulation with equal slice thicknesses. In both the 2D fluorescence and magnetic resonance images, the scar and injection sites are clearly distinct. TTC stained tissue and late gadolinium enhanced magnetic resonance images provide a control for scar assessment in fluorescence imaging.Post processing imaging enables reconstruction of the 3D geometry of the ex vivo heart based on the segmentations and fluorescence images, allowing assessment of the 3D injection accuracy. In this study, the injection depositions and the infarct border zone were protected onto the endocardial wall, and the distances between the projections on the endocardial surface were measured. Following this procedure, other methods like a surgical assessment, infarct size quantification, or validation of other three dimensional imaging modalities can be performed.We have used this technique to assess the accuracy of injection into the infarct border zone. This research was conducted to improve cardioregenerative therapy for heart failure. After watching this video, you should have a good understanding of how to prepare the heart, how to embed and process cardiac tissue, and how to perform fluorescence imaging of cardiac tissue samples.

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