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 animals received a subcutaneous injection of 0.1 mg/kg buprenorphine pre-anesthesia. The surgical protocol for inducting&#160;myocardial&#160;infarction has been previously described elsewhere13.
<ol>
	<li>Perform a sternotomy on a myocardially infarcted animal under anesthesia (intubated animal, 2.5% isoflurane, proper anesthesia confirmed by paw pinch reflex).
	<ol>
		<li>Open the animal by cutting the skin and then the muscles with surgical scissors. Cut the ribs on the left and right, and then remove the chest.</li>
		<li>Remove the adhesions remaining after the previous surgery (LAD ligation). Cut the aorta and remove the heart. Put the tissue in 1 M KCl (in PBS), and then wash it with PBS.</li>
	</ol>
	</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Put the infarcted heart longitudinally in an acrylic rat heart matrix. Keep it at -20 &#176;C for 1 hr.</li>
	<li>Cut the heart directly in the matrix using a razor blade with a transversal. Ensure each slice is 2 mm thick. Cut approximately 5 - 7 slides for each heart (based on the remodeling level); this is called systematic sampling.</li>
	<li>At this step, performing the 2,3,5-triphenyltetrazolium chloride (TTC) staining is optional (keep the orientation of the heart slices base-to-apex).
	<ol>
		<li>Incubate in 1% TTC in PBS for 50 min at 37 &#176;C. Incubate it in 4% paraformaldehyde (PFA) for 20 min to 1 hr. Put the sections between two glass plates with 2-mm spacers and take pictures with a stereological microscope coupled with a camera at 15X magnification. 
		CAUTION! Paraformaldehyde is toxic.</li>
	</ol>
	</li>
	<li>Slide freezing (1st option)
	<ol>
		<li>Put each slide in a plastic mold (10 x 10 x 5 mm) with mounting medium for cryotomy (optimal cutting temperature, OCT), maintain the orientation (place the apex-oriented side of the section down on the bottom of the mold). Freeze the blocks with 2-methylbutane vapors under liquid nitrogen cooling for 10 - 15 min. Finally, store the tissue at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Paraffinization (2nd option)
	<ol>
		<li>Place each slide in embedding cassettes (maintain the orientation by placing the apex-oriented side of the section down on the bottom of the cassette) and then in 4% PFA for 24 hr. Put it in a tissue processor machine overnight.
		<ol>
			<li>Incubate it in ethanol 70% for 2 hr. Incubate it in ethanol 95% for 2 hr. Incubate it in ethanol 100% for 3 hr. Incubate it in xylol for 4 hr. Incubate it in paraffin (molten at 60 &#176;C in an oven) for 5 hr. Finally, make blocks by embedding each heart slide in paraffin.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Masson-Goldner Trichrome Staining
<ol>
	<li>Make tissue sections from paraffin blocks with a manual microtome (thickness: 5 &#181;m) or from OCT blocks with a cryostat set to -18 &#176;C (thickness: 7 &#181;m).</li>
	<li>Stain one slide from each heart part for each rat (3 - 4 transversal sections per slide) with Masson-Goldner trichrome staining (see annex). 
	NOTE: Start from this step for the paraffin sections.
	<ol>
		<li>Melt the slides at 60 &#176;C in an oven. Under a fume hood, deparaffinize them in xylol twice for 10 min each. Rehydrate them in 100% ethanol, 95% ethanol, 70% ethanol, and distilled water for 3 min each. 
		NOTE: Start from this step for the cryosections.</li>
		<li>Fix them in Bouin solution overnight. Then, rinse them in running tap water for 10 - 15 min. Rinse them in distilled water. Incubate them in Mayer&#39;s hematoxylin for 3 min.</li>
		<li>Remove the slides and leave them in distilled water for 5 min. Then, incubate them in acid Fuchsin-Ponceau for 5 min. Rinse them in 1% acetic acid for 1 min.</li>
		<li>Next, incubate them in phosphomolybdic acid Orange G for 1 min. Rinse them in 1% acetic acid for 1 min, incubate them in light green dye for 10 min, and rinse them in 1% acetic acid for 1 min.</li>
		<li>Dehydrate them in 70% ethanol (30 sec), 95% ethanol (30 sec), and 100% ethanol (5 min). Put one drop of a resinous mounting medium on the tissue sections, cover them with coverslips, and let them dry.</li>
	</ol>
	</li>
</ol>
4. Infarct Size Analysis
<ol>
	<li>Acquire one image from each slide on a stereomicroscope (15X magnification) coupled with a camera. Photograph a ruler with the same settings.</li>
	<li>Use image analysis software to measure the scar thickness in the middle of the infarct, the septum thickness, the left ventricle (LV) cavity area, the infarct area, and the LV tissue area using.
	<ol>
		<li>Set the scale with the ruler picture (Figure 1A).
		<ol>
			<li>Click on Measurements then 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.</li>
		</ol>
		</li>
		<li>Select the LV from the stained heart picture (Figure 1B). Use the Multiple segment tool. Select the LV point-by-point, and then right click Copy/Paste anywhere.</li>
		<li>Measure the scar and septum thickness using the single segment measurement tool (Figure 1C).</li>
		<li>Automatically detect the LV cavity, infarct, and LV areas using the automatic area measurement tool in RGB mode (Figure 1D).
		<ol>
			<li>Click on the tool. Activate Only Borders and Contiguous in RGB mode. Finally, click inside the area of interest to detect it. If necessary, use precision tools.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate the infarct size as the ratio of the infarct area to the LV area.</li>
	<li>Calculate the expansion index as follows: [LV cavity area / whole LV area] / [infarct thickness / septum thickness], with the whole LV area including both the LV cavity and LV tissue areas.</li>
	<li>Finally, calculate an &#34;average&#34; for each heart as follows: [average of expansion index from the infarcted slides] * [Number of infarcted slides / Total number of slides].</li>
</ol>

<ol>
	<li>Amsterdam, E. A., 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=AHA/ACC+Guideline+for+the+Management+of+Patients+with+Non-ST-Elevation+Acute+Coronary+Syndromes:+a+report+of+the+American+College+of+Cardiology/American+Heart+Association+Task+Force+on+Practice+Guidelines.">AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.</a> J Am Coll Cardiol. 64, e139-e228 (2014).</li><li>Konstam, M. A., Kramer, D. G., Patel, A. R., Maron, M. S., Udelson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+remodeling+in+heart+failure:+current+concepts+in+clinical+significance+and+assessment.">Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment.</a> JACC Cardiovasc Imaging. 4, 98-108 (2011).</li><li>Frobert, A., 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=Prognostic+Value+of+Troponin+I+for+Infarct+Size+to+Improve+Preclinical+Myocardial+Infarction+Small+Animal+Models.">Prognostic Value of Troponin I for Infarct Size to Improve Preclinical Myocardial Infarction Small Animal Models.</a> Front Physiol. 6, 353 (2015).</li><li>Redfors, B., Shao, Y., Omerovic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarct+size+and+area+at+risk+assessment+in+mice.">Myocardial infarct size and area at risk assessment in mice.</a> Exp Clin Cardiol. 17, 268-272 (2012).</li><li>Guex, A. 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. Bras Cardiol. 93, 434-440 (2009).</li><li>Lichtenauer, 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=Myocardial+infarct+size+measurement+using+geometric+angle+calculation.">Myocardial infarct size measurement using geometric angle calculation.</a> Eur J Clin Invest. 44, 160-167 (2014).</li><li>Lutgens, E., 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=Chronic+myocardial+infarction+in+the+mouse:+cardiac+structural+and+functional+changes.">Chronic myocardial infarction in the mouse: cardiac structural and functional changes.</a> Cardiovasc Res. 41, 586-593 (1999).</li><li>Frobert, A., Valentin, J., Cook, S., Lopes-Vicente, L., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+for+heart+failure+in+rat:+double+thoracotomy+for+myocardial+infarction+and+epicardial+implantation+of+cells+and+biomatrix.">Cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix.</a> J Vis Exp. , e51390 (2014).</li><li>Takagawa, J., 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> J Appl Physiol (1985). 102, 2104-2111 (2007).</li><li>Fishbein, M. 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=Early+phase+acute+myocardial+infarct+size+quantification:+validation+of+the+triphenyl+tetrazolium+chloride+tissue+enzyme+staining+technique.">Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.</a> Am Heart J. 101, 593-600 (1981).</li><li>Weisman, H. F., Bush, D. E., Mannisi, J. A., Weisfeldt, M. L., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+myocardial+infarct+expansion.">Cellular mechanisms of myocardial infarct expansion.</a> Circulation. 78, 186-201 (1988).</li><li>Mannisi, J. A., Weisman, H. F., Bush, D. E., Dudeck, P., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+administration+after+myocardial+infarction+promotes+early+infarct+expansion.+A+study+in+the+rat.">Steroid administration after myocardial infarction promotes early infarct expansion. A study in the rat.</a> J Clin Invest. 79, 1431-1439 (1987).</li></ol>

The authors have nothing to disclose.

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 dimensions from different hearts. Second, the overnight fixation of the section in Bouin solution is critical to obtain bright trichrome staining.
Modifications and Troubleshooting
Absence of staining may be due to difficulties during the fixation of the section in Bouin solution, such as reduced incubation time or expired Bouin solutions. The TTC staining is optional and could be used to visualize the infarct size in a rapid but non-quantitative way. It is important to note that TTC can be performed for the same heart prior to the inclusion in either OCT or paraffin.
Limitations of the Technique
The present approach permits one to choose between paraffin-embedded and cryo-preserved heart sections and can be used for immunostaining. Nevertheless, the full heart has to be sectioned, and the tissue cannot be used for further analysis, such as western blot or RT-PCR for protein and gene expression analyses.
Significance of the Technique with Respect to Existing/Alternative Methods
Because the quantification procedures and, in particular, the number of analyzed sections vary widely between investigations, defining the minimal number of sections necessary to obtain a reliable quantification is paramount. Takagawa et al.9 demonstrated that reliability is maximized with a minimal heart slice number of 6 - 8 sections of the whole heart of a mouse using 1-mm intervals. In the present protocol, 5 - 7 sections of the entire rat heart were obtained when performing 2-mm thick sectioning. Furthermore, 2-mm sections are a standard and frequently used size for TTC staining. In addition, the systemic sampling is simple to apply and presents a periodic aspect that allows characterization of the entire heart.
From each of the 2-mm slices, 5-&#181;m sections were cut and stained. For each 5-&#181;m section, quantification of the septum thickness, scar thickness and length, total scar area, total LV area, and LV cavity area were performed, and the average of each parameter was calculated per animal. We used Masson-Goldner staining of thin sections rather than TTC staining of 2-mm sections to improve the accuracy of both the automatic color detection and the assessment of lengths. Indeed, TTC staining is mostly interesting for early-phase or ischemic reperfusion models for the detection of the area at risk8,14, rather than for the chronic stage.
MI induced from LAD ligation may vary depending on the ligature site. In the presented chronic infarction model, the ligation size was intentionally modified for each animal, and results for various conditions of infarct size and remodeling were present after 6 weeks. Accordingly, the calculated EI revealed a wide range of myocardial infarcts and supported the difference observed in heart function correlated with EF. When extensive thinning of the infarcted segment occurred, the area of the infarct was greatly reduced in the case of transmural fibrosis. In this condition, defining the infarct size by calculating the area of the infarct tissue in relation to the entire LV (expressed as a percentage of the LV) would weakly evaluate the extent of the infarct. The lack of consideration for remodeling-induced LV dilation and extreme thinning of the infarct LV wall may underestimate the infarct size, while an infarct present in the absence of wall thinning would over-estimate the infarct size, as represented by the values outside of the 95% confidence interval. This shortcoming could be eliminated through the calculation of the EI.
It has been shown that the LV shape change is positively correlated with the EI and wall thinning15, 16. Although EI can be a predictor of LV function, it is important to emphasize that echocardiography and histological analyses are complementary methods to assess the myocardial infarct at the functional and tissue level, respectively. Echocardiography allows longitudinal study, and histological analyses provide fundamental end-point assays that allow additional quantification of LV morphology, such as wall thickness.
Future Applications or Directions after Mastering This Technique
The systemic sampling of the entire heart and the calculation of the EI are of particular interest when assessing chronic MI. In addition, this method will be suitable for the evaluation of treatment efficacy, in particular for novel treatments, such as cell- and matrix-based treatments that aim at reducing infarct size and remodeling. Reliable quantification of infarct expansion is of paramount importance for comparisons between non-treated and treated animals, as end-point histological analyses preclude the comparison of infarct size pre- and post-treatment.

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><tbody><tr><td>Acrylic rat heart matrix 2 mm</td><td>72-5015</td><td>Harvard Appartus</td><td></td></tr><tr><td>Inspira advanced volume controlled ventilator</td><td>Harvard Apparatus</td><td>557058</td><td></td></tr><tr><td>Catheter Insyte 14 G</td><td>BD</td><td>381267</td><td></td></tr><tr><td>O.C.T</td><td>BDHA361603E</td><td>VWR</td><td></td></tr><tr><td>TTC</td><td>T8877-10G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Mayer hematoxylin</td><td>MHS32-1L</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Acid Fuchsin&lt;br/&gt; CI 42685</td><td>F8129-50G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Ponceau Xylidin&lt;br/&gt; CI 16150</td><td>P2395-25G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Orange G&lt;br/&gt; CI 16230</td><td>O3756-100G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Light green&lt;br/&gt; CI 42095</td><td>L5382-25G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>KCl</td><td>P9333-500G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Xylol</td><td>10315083</td><td>HoneyWell</td><td></td></tr><tr><td>Ethanol absolute</td><td>10303990</td><td>HoneyWell</td><td></td></tr><tr><td>2-methylbutane</td><td>M32631-1L</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Stereogical microscope</td><td>SM2800</td><td>Nikon</td><td></td></tr><tr><td>Formaldehyde</td><td>99340</td><td>Reactolab</td><td></td></tr><tr><td>Embedding cassette</td><td>K113.1</td><td>Carl Roth</td><td></td></tr><tr><td>Bersoft Image measurement Software</td><td></td><td>Bersoft.com</td><td>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 infarcted tissue visible from the apex to the mid-section of the heart (Figure 2B). For small non-transmural infarcts, fibrotic tissue was observed on only one or two sections from the apex (Figure 2C).
Thin 5-&#181;m sections from each heart slice were cut and stained with Masson-Goldner trichrome. Pictures of the whole cross section of the heart were acquired under a stereomicroscope (Figure 3). Healthy tissues appeared in red and the connective tissue in green. Discrimination between each color can be easily performed with an image analysis software.
The thicknesses of the infarct and the septum, the LV cavity area and the LV total area were measured and computed to calculate the EI (Table 1). The EI was calculated for each section. Depending on the heart size, the number of sections varied from 5 to 7. For each heart, the EI is an average of the EI from each section. The EI varied from 0 to 0.278 and revealed a wide range of myocardial infarcts, with a CV of 58%. In comparison, the average of the percentage of infarct to the LV varied from 0 to 0.241, with a CV of 54% (Table 1). The EI and the percentage of infarct were significantly correlated; a non-parametric Spearman analysis provided a correlation coefficient r-value of 0.491 (p = 0.005) (Figure 4A). For close EI values, such as 0.11 and 0.13, the percentage of infarct varied from 5.8 to 24.1%.
Heart function was assessed by high-resolution echocardiography at 6 weeks post-LAD ligation on anesthetized animals and was performed once prior to heart harvesting. The EI calculated from histological quantification correlated significantly with the EF. A non-parametric Spearman analysis provided an r-value of -0.709 (p = 0.005) (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/54914/54914fig1.jpg" /> 
Figure 1. Step-by-step Illustration of the Use of the Image Analysis Software. Automatic color delimitation and length measurements included the several steps. A. Calibration: The scale of the image was set up. A picture of a ruler was taken in the same condition as the heart section. The length of the ruler was marked to define the scale conversion factor. Conversion from pixel to millimeters was performed. B. LV selection: The right ventricle was cut out of the picture using the selection tool of the software. C. LV wall and septum thickness measurement: The single measurement tool was used to quantify distances. The scale bars indicate 3 mm. D. Automatic area quantification: Automatic color delimitation was performed in RGB mode. Auto-selection was performed after selecting the auto-selection mode. Color-based selection can be modified; the investigator performed visual control of the selected green tissue and increased or decreased the selected region as necessary. The scale bars indicate 3 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54914/54914fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54914/54914fig2.jpg" /> 
Figure 2. Heart Sections Stained with TTC. 2-mm sections of the full heart were stained with TTC. Healthy myocardium appeared in red and fibrotic tissues in white. Three hearts with different infarct sizes are presented. A: large transmural infarct, B: medium infarct, and C: small infarct. The scale bars indicate 3 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54914/54914fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54914/54914fig3.jpg" /> 
Figure 3. Heart Sections Stained with Masson-Goldner Trichrome. 5-&#181;m sections were cut from each heart section obtained 6 weeks post-LAD ligation. Stained sections were placed under a stereomicroscope. Pictures of the full sections were obtained with 15X magnification. The representative picture, obtained at the level of the papillary muscles (back arrow), shows healthy myocardium in red and fibrotic tissues in green. The picture illustrates the automatic color delimitation with the image analysis software, as well as the site at which the infarct and septum thickness were measured (black lines). The scale bars indicate 3 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54914/54914fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54914/54914fig4.jpg" /> 
Figure 4. Correlation Between the Expansion Index (EI) and the Percentage of the Infarct Related to LV (A) or the Ejection Fraction (EF) (B). The linear regression (black line) and 95% confidence intervals (dotted lines) are represented. The non-parametric Spearman r-value is reported. A: The percentage of infarct and EI significantly correlated (r = 0.567; p = 0.003). The infarct expansion index (EI) was calculated as [LV cavity area/whole LV area]/[infarct thickness/septum thickness]. The whole LV area was measured as the combined LV cavity and LV tissue area. The percentage of the infarct was calculated as LV tissue area/infarct tissue area. B: The function assessed at 6 weeks post-LAD ligation using a high-resolution echocardiography significantly correlated with EI. Both parameters were significantly correlated (r = -0.709; p = 0.005). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54914/54914fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54914/54914table1.jpg" /> 
Table 1: Parameters Measured on Masson Goldner-stained Heart Sections.

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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 ...

histological-quantification-of-chronic-myocardial-infarct-in-rats

Nanyang Technological University

Research

JoVE Journal

Medicine

14.5K Views.  University of Fribourg. 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. 

Video: Histological Quantification of Chronic Myocardial Infarct in Rats

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioengineering

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Histological Quantification of Chronic Myocardial Infarct in Rats

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