The study was supported by the Swiss National Foundation [SNF 310030-149986 to MNG], the University of Fribourg, and Fribourg Hospital.

All animals received humane care in compliance with the European Convention on Animal Care. Surgical procedures were performed in accordance with the Swiss Animal Protection Law after obtaining permission of the State Veterinary Office, Fribourg, approved by the Swiss Federal Veterinary Office, Switzerland.
1. Heart Harvesting
NOTE: All surgical interventions were performed under isoflurane anesthesia. Efforts were made to diminish animal suffering. In particular, all...

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G., 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=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, 2996-3006 (2014).</li><li>Landa, N., 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=Effect+of+injectable+alginate+implant+on+cardiac+remodeling+and+function+after+recent+and+old+infarcts+in+rat.">Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat.</a> Circulation. 117, 1388-1396 (2008).</li><li>Valente, M., 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=Optimized+heart+sampling+and+systematic+evaluation+of+cardiac+therapies+inmouse+models+of+ischemic+injury:+Assessment+of+cardiacremodeling+and+semi-automated+quantification+of+myocardial+infarctsize.">Optimized heart sampling and systematic evaluation of cardiac therapies inmouse models of ischemic injury: Assessment of cardiacremodeling and semi-automated quantification of myocardial infarctsize.</a> Curr. Protoc. Mouse Biol. 5, 359-391 (2015).</li><li>Csonka, C., 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=Measurement+of+myocardial+infarct+size+in+preclinical+studies.">Measurement of myocardial infarct size in preclinical studies.</a> J. Pharmacol. Toxicol. Methods. 61, 163-170 (2010).</li><li>Zornoff, L. A., Paiva, S. A., Minicucci, M. F., Spadaro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+myocardium+infarction+in+rats:+Analysis+of+the+model.">Experimental myocardium infarction in rats: Analysis of the model.</a> Arq. 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Critical Steps within the Protocol
Fibrotic tissue can be accurately assessed in a chronic MI rat model using systematic sampling of the harvested heart and image analyses of trichromatic-stained histological sections obtained from base to apex. Two steps are particularly important for successful protocol implementation. First, the use of KCl for heart harvesting allows the cardiac muscle to be maintained in a relaxed state. This step is important for comparisons of infarct di...

Myocardial infarction (MI) is a leading cause of death and disability worldwide. Coronary heart disease is the main cause; MI results from ischemia consecutive to coronary events such as occlusion. When reperfusion is not performed within the first 6 hr, ischemia induces irreversible myocardial necrosis. In patients, the characterization of MI relies on different diagnostic tools, including clinical signs, electrocardiography, assessment of plasma levels of biomarkers, echocardiography, MRI imaging, and histological analyses1. Acute and chronic MI are classified as two different phases of injury according to the timing of the myocardial necrosis relative to the time of the coronary occlusion. The acute phase, occurring during the first 7 days, is associated with the loss of cardiomyocytes, extensive inflammation, and the recruitment of fibroblasts. The sub-acute phase, characterized by healing of the cardiac tissue and the formation of a scar, occurs between 1 and 4 - 6 weeks. Expansion of the infarct, ventricle wall thinning, and ventricle dilatation characterize the chronic phase. Extensive remodeling of the left ventricle progressively results in severe heart failure2.
MI induced by permanent left anterior descending artery (LAD) ligation represents the standard rodent model of chronic myocardial infarction. The coronary ligature mimics the coronary occlusion. The size of the infarct depends on the site of the ligature. Characterization of myocardial ischemic injury in a rodent model is classically performed using biomarker plasma levels, such as troponin I and T3, echocardiography, MRI, and histology4,5. Biomarker levels are correlated with the extent of cardiomyocyte death. Echocardiography evaluates the left ventricular function impairment resulting from regional wall motion abnormalities. In addition, non-invasive imaging techniques, such as MRI or high-resolution echocardiography, allow the assessment of the reduction in wall motion, the volume of the scar area with reduced perfusion and viable myocardium, and the wall thinning. LV dimensions permit the accurate evaluation of infarct size. Finally, the quantification of viable and dead myocardium can be performed postmortem using specific stains of histological sections of harvested hearts and allows verification of the infarct size (IS). Another important feature is the evaluation of the infarct expansion index (EI)6. The EI is associated with the transmural infarct and starts within the first 3 days. The EI is characterized by a progressive reduction in wall thickness, an increase in the LV cavity size, and consequent changes in LV shape.
In order to evaluate the therapeutic efficacy of novel treatments--in particular, the regenerative strategies based on cells, matrices, and gene delivery-accurate assessment of MI in rodents is of paramount importance. When measured on a single cross section obtained at the papillary muscle level, the IS size may be biased due to the large variability that exists in infarct development following LAD ligation; the apex infarct might be then occulted. Importantly, more accurate methodologies to determined MI size have been described for mice7-9 or rats10. Nevertheless, IS is insufficient to accurately quantify LV remodeling or therapeutically induced reductions (or preventions) of the remodeling. Indeed, IS is commonly expressed as a percentage of total LV volume assessed on cross sections of the heart. Although this method is valid for acute MI, the thinning of the LV wall occurring during remodeling remains under-evaluated11,12. A complete morphometric quantification of infarct size and structural changes should quantify several parameters, such as endocardial and epicardial lengths and diameters, as well as infarct and healthy areas. We describe a methodological approach to accurately assess MI and remodeling in a chronic rat model.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrylic rat heart matrix 2mm</td>
			<td style="width:119px;">72-5015</td>
			<td style="width:136px;">Harvard Appartus</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:119px;">HARVARD APPARATUS</td>
			<td style="width:136px;">557058</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CATHETER INSYTE 14G</td>
			<td style="width:119px;">BD</td>
			<td style="width:136px;">381267</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">O.C.T</td>
			<td style="width:119px;">BDHA361603E</td>
			<td style="width:136px;">VWR</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TTC</td>
			<td style="width:119px;">T8877-10G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mayer hematoxylin</td>
			<td style="width:119px;">MHS32-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acid Fuchsin 
			CI 42685</td>
			<td style="width:119px;">F8129-50G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ponceau Xylidin 
			CI 16150</td>
			<td style="width:119px;">P2395-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Orange G 
			CI 16230</td>
			<td style="width:119px;">O3756-100G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Light green 
			CI 42095</td>
			<td style="width:119px;">L5382-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">KCl</td>
			<td style="width:119px;">P9333-500G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Xylol</td>
			<td style="width:119px;">10315083</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol absolute</td>
			<td style="width:119px;">10303990</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-methylbutane</td>
			<td style="width:119px;">M32631-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Stereogical microscope</td>
			<td style="width:119px;">SM2800</td>
			<td style="width:136px;">Nikon</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Formaldehyde</td>
			<td style="width:119px;">99340</td>
			<td style="width:136px;">Reactolab</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Embedding cassette</td>
			<td style="width:119px;">K113.1</td>
			<td style="width:136px;">Carl Roth</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Bersoft Image measurement Software</td>
			<td style="width:119px;"></td>
			<td style="width:136px;">Bersoft.com</td>
			<td style="width:273px;">Licensed software</td>
		</tr>
	</tbody>
</table>

histological quantification of chronic myocardial infarct in rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

Six weeks post-LAD ligation, hearts were harvested from Lewis rats. 2-mm tissue sections were obtained from apex to base. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red (Figure 2). Depending on the site of ligation of the LAD, the infarct size varies. For large MI, transmural infarcts were observed from apex to base (Figure 2A). Smaller infarcts presented white...

Watch this Scientific Journal Video about Histological Quantification of Chronic Myocardial Infarct in Rats at JoVE.com

Histological Quantification of Chronic Myocardial Infarct in Rats

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

The overall goal of this histological procedure is to perform morphometric quantification of infarct expansion index and heart structural parameters using a myocardial infarction rat model in order to assess infarct size and left ventricle remodeling. This method can help answer key question in the cardiac field such as the effect of regenerative therapy following myocardial infarction or novel strategies aiming at reducing infarct expansion. The main advantage of this technique is the use of systematic sampling of harvested rat heart to accurately assess the infarct size and take into account the left ventricle changes throughout remodeling.Following this method can provide insight into infarct size in chronic rat model of myocardial infarction. It can also be applied to mice or other ischemic model, such as acute myocardial infarction or ischemia reperfusion. To begin this procedure, perform a sternotomy on a myocardialy infarcted animal under anesthesia.Make an incision into the skin and the muscles along the sternum with surgical scissors. Then, cut the ribs on the left and the right to remove the chest. After that, remove the adhesions remaining after LAD ligation.Cut the aorta and remove the heart then place the tissue in PBS with one molar potassium chloride for less than one minute before washing it with PBS. In order to preserve the tissue, PBS inside the ventricles should be carefully removed. Subsequently, transfer the infarcted heart to an acrylic rat heart matrix and position it longitudinally.Then keep it at minus 20 degrees celsius for one hour. For systematic sampling after an hour, cut the heart directly in the matrix using a razor blade. Ensure each section is two millimeters thick.Next, perform TTC staining by incubating the sections in 1%TTC for 15 minutes at 37 degrees celsius, then incubate them in 4%PFA for 20 to 60 minutes at room temperature. Afterward, place the sections between two glass plates with two millimeter spacers and take pictures with a stereological microscope coupled with a camera at 15x magnification. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red.For a large myocardial infarction, transmural infarcts were observed from apex to base. Smaller infarcts presented white infarcted tissue visible from the apex to the midsection of the heart. For small, non-transmural infarcts, fibrotic tissue was observed on only one or two sections from the apex.To freeze the sections, place each section in a plastic mold with mounting medium for cryotomy and position the apex downward in the mold. Freeze the blocks with 2-Methylbutane vapors under liquid nitrogen cooling for 10 to 15 minutes. After that, store the tissues at minus 80 degrees celsius.For paraffinization, place each section in an embedding cassette with the apex pointing at the bottom, then keep the cassettes in 4%PFA for 24 hours. Afterward, place them in a tissue processor overnight. Once processing is complete, make blocks by embedding each heart section in paraffin.In this procedure, prepare 5-micron-thick tissue sections from the paraffin blocks with a manual microtome. Next, stain one slide from each heart section for each rat with Masson-Goldner trichrome staining. For the paraffin sections, melt the tissues at 60 degrees celsius in an oven.Under a fume hood, deparaffinize them in xylol twice for ten minutes each. Then, rehydrate them in 100%ethanol, 95%ethanol, 70%ethanol, and then distilled water for three minutes each. For the deparaffinized sections and for the cryo sections, fix them in Bouin solution overnight.The following morning, rinse them in running tap water for 10 to 15 minutes, then rinse again using distilled water. Fixation with Bouin is the most important step to ensure a splendid Goldner staining. Incubate them in Mayer's hematoxylin for three minutes.After three minutes, remove the slides and leave them in distilled water for five minutes. Subsequently, incubate them in ponceau acid fuchsin for five minutes before rinsing with 1%acetic acid for one minute. Next, incubate the sections in phosphomolybdic acid orange g for one minute and then rinse them in 1%acetic acid for one minute.Incubate in light green dye for ten minutes and then rinse in 1%acetic acid for one minute. Finally, dehydrate the sections in 70%ethanol for 30 seconds, 95%ethanol for 30 seconds, and 100%ethanol for five minutes sequentially. Apply one drop of resinous mounting medium on to the tissue sections, cover them with cover slips, and place them to dry.In this step, acquire one image from each slide using a stereo microscope coupled with a camera at 15x magnification. Photograph a ruler under the same settings. Thin five-micron sections from each heart slice were cut and stained with Masson-Goldner trichrome.Healthy tissues appeared in red and the connective tissue in green. Next, use the image analysis software to measure the scar thickness in the middle of the infarct, the septum thickness, the left ventricle cavity area, the infarct area, and the LV tissue area. To do so, set the scale with the ruler picture.Click on measurements and select set conversion factor. Draw a line on the calibration bar. Right click on the picture and click on end calibration.Note the bar value and then click OK.Use the multiple segment tool to select the LV point by point and then right click copy paste anywhere. After that, measure the scar and septum thickness using the single segment measurement tool. Then, automatically detect the LV cavity, infarct, and LV areas using the automatic area measurement tool in RGB mode.Activate only borders and contiguous in the RGB mode. Finally, click inside the area of interest to detect it. After that, calculate the infarct size as the ratio of the infarct area to the LV area.Then, calculate the expansion index using this formula. Finally, calculate an average for each heart using the formula shown. The expansion index calculated from Goldner staining is a good index to evaluate the infarct development.As shown in the figure, it correlates with the size of infarct expressed as the percentage of the left ventricle and with the ejection fraction measured by echocardiography. On the graphs are represented the linear regression and the 95%confidence intervals. The infarct expansion index was calculated as described in the formula here.The whole LV area was measured as the combined LV cavity and LV tissue area. The percentage of the infarct was calculated as shown. The function was assessed at six weeks post LAD ligation using a high-resolution echocardiography.After watching this video, you should have a good understanding of how to perform a systematic sampling of the heart and how to assess infarct size based on an image analysis of stain sections.

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14.3K vistas.  University of Fribourg. The overall goal of this histological procedure is to perform morphometric quantification of infarct expansion index and heart structural parameters using a myocardial infarction rat model in order to assess infarct size and left ventricle remodeling. This method can help answer key question in the cardiac field such as the effect of regenerative therapy following myocardial infarction or novel strategies aiming at reducing infarct expansion. The main advantage of this technique is the use of...