Name: histological quantification of chronic myocardial infarct in rats
Uploaded: 2016-12-11T14:00:00
Description: Watch this Scientific Journal Video about Histological Quantification of Chronic Myocardial Infarct in Rats at JoVE.com

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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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 ...

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