Histological Quantification of Chronic Myocardial Infarct in Rats

Summary

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.

Abstract

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.

Introduction

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 histology^4,5. 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 mice^7-9 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-evaluated^11,12. 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.

Protocol

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 myocardial infarction has been previously described elsewhere¹³.

1. Perform a sternotomy on a myocardially infarcted animal under anesthesia (intubated animal, 2.5% isoflurane, proper anesthesia confirmed by paw pinch reflex).
   1. 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.
   2. 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.

2. Tissue Preparation

1. Put the infarcted heart longitudinally in an acrylic rat heart matrix. Keep it at -20 °C for 1 hr.
2. 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.
3. At this step, performing the 2,3,5-triphenyltetrazolium chloride (TTC) staining is optional (keep the orientation of the heart slices base-to-apex).
   1. Incubate in 1% TTC in PBS for 50 min at 37 °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.
4. Slide freezing (1^st option)
   1. 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 °C.
5. Paraffinization (2^nd option)
   1. 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.
      1. 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 °C in an oven) for 5 hr. Finally, make blocks by embedding each heart slide in paraffin.

3. Masson-Goldner Trichrome Staining

1. Make tissue sections from paraffin blocks with a manual microtome (thickness: 5 µm) or from OCT blocks with a cryostat set to -18 °C (thickness: 7 µm).
2. 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.
   1. Melt the slides at 60 °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.
   2. 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's hematoxylin for 3 min.
   3. 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.
   4. 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.
   5. 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.

4. Infarct Size Analysis

1. Acquire one image from each slide on a stereomicroscope (15X magnification) coupled with a camera. Photograph a ruler with the same settings.
2. 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.
   1. Set the scale with the ruler picture (Figure 1A).
      1. 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.
   2. 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.
   3. Measure the scar and septum thickness using the single segment measurement tool (Figure 1C).
   4. Automatically detect the LV cavity, infarct, and LV areas using the automatic area measurement tool in RGB mode (Figure 1D).
      1. 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.
3. Calculate the infarct size as the ratio of the infarct area to the LV area.
4. 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.
5. Finally, calculate an "average" for each heart as follows: [average of expansion index from the infarcted slides] * [Number of infarcted slides / Total number of slides].

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Acrylic rat heart matrix 2mm	72-5015	Harvard Appartus
INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR	HARVARD APPARATUS	557058
CATHETER INSYTE 14G	BD	381267
O.C.T	BDHA361603E	VWR
TTC	T8877-10G	Sigma Aldrich
Mayer hematoxylin	MHS32-1L	Sigma Aldrich
Acid Fuchsin CI 42685	F8129-50G	Sigma Aldrich
Ponceau Xylidin CI 16150	P2395-25G	Sigma Aldrich
Orange G CI 16230	O3756-100G	Sigma Aldrich
Light green CI 42095	L5382-25G	Sigma Aldrich
KCl	P9333-500G	Sigma Aldrich
Xylol	10315083	HoneyWell
Ethanol absolute	10303990	HoneyWell
2-methylbutane	M32631-1L	Sigma Aldrich
Stereogical microscope	SM2800	Nikon
Formaldehyde	99340	Reactolab
Embedding cassette	K113.1	Carl Roth
Bersoft Image measurement Software		Bersoft.com	Licensed software