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

Summary

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.

Abstract

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.

Introduction

Myocardial infarction (MI) is the most prevalent form of cardiovascular disease. After MI, the myocardium undergoes serial morphological and functional changes, including the healing of the MI infarct zone, ventricular remodeling (VR), and myocardial dysfunction¹. The healing of MI is a dynamic and well-orchestrated process associated with profound inflammatory infiltration that ends in the formation of a fibrotic scar^2,3. The experimental model of MI in mice is currently used for studying cardiac remodeling under pathological conditions^4,5, and awareness of the precise surgical protocol is essential to develop a reproducible and effective procedure for inducing a permanent coronary ligature. This method is needed to study the healing of MI and its relevance in the temporal evolution of left ventricular remodeling (LVR) and the cardiac dysfunction associated with MI.

Galectins are a group of lectins that recognize specific carbohydrates in intracellular ligands, membrane receptors, and extracellular glycoproteins. Galectin 3 (Gal-3) is a member of this family that acts through the recognition and cross-linking of N- and O-glycans in glycoconjugates on the cell surface, and it is widely expressed in the immune system⁶. Previous studies have investigated the role of Gal-3 as a regulator of inflammation and fibrosis in cardiovascular diseases^{7,8,9,10,11,12}. As targeting the regulatory factors of inflammation during healing is highly relevant because inflammation can notably affect the evolution of remodeling, we aimed to describe a protocol for studying the temporal evolution of post-MI ventricular remodeling and the steps and methods for determining how the genetic mutation of Gal-3 modifies the temporal evolution of healing in MI and affects cardiac remodeling and function in mice.

Protocol

NOTE: All the experiments described in this protocol were approved by the Animal Care and Research Committee of the University of Buenos Aires (CICUAL), in line with the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals¹³. For the experiments, use male, age-matched C57BL/6J and Gal-3 KO mice (8-10 weeks old) weighing 30-35 g, which allow for better manipulation for surgery. Allow the animals access to water and food ad libitum. The Gal-3 KO mice were bred on a C57BL/6J background at the same bioresource facilities as the control C57BL/6J mice.

1. Surgical area and instruments

1. Before starting the surgery, verify that the ventilator is plugged into a power source and working correctly. Confirm that there is enough anesthetic solution. Prepare the solution on the day of the surgery by calculating the number of animals to be used.
2. Confirm that all the surgical instruments are sterilized, including stainless scissors, micro scissors, needle holders for big and small needles, retractors, extra sharp, curved forceps, serrated forceps, and tissue forceps. Clean the working area with 70% ethanol.
3. Ensure the availability of small cotton balls, hyssops, and gauze for immediate cauterization of any potential hemorrhage.
4. Keep ready-prepared 10-0 to 7-0 silicone-coated, braided silk sutures for ligature of the left descending coronary artery (LDA), nylon suture for thorax closing, and linen thread for closing the skin.

2. Anesthesia and intubation

1. Weigh the mice to determine the dose of anesthesia.
2. Prewarm a heating pad surrounded by a Styrofoam base to 40 °C.
3. Anesthetize the mice with intramuscular administration of 0.1 mL/10 g of body weight of a solution containing ketamine (65 mg/kg), xylazine (13 mg/kg), and acepromazine (1.5 mg/kg).
4. Once the mouse is anesthetized and before starting the surgical procedure, check the anesthetic depth by inducing a slightly painful stimulus, such as by pressing the feet with the fingers. If the animal responds to the stimulus, adjust the anesthetic depth.
5. Place the mouse in dorsal decubitus, and tape its legs to the working base above the heating pad, placing it under a binocular stereo microscope. Hyperextend the neck with a thread holding the maxillary teeth attached to the working base.
6. Then, for the intubation part, expose the trachea rings through a minimal incision in the neck, and channel through it with a 20 G intravenous catheter connected to a rodent ventilator (tidal volume: 250 mL/stroke) at a respiratory frequency of 34-38 cycles/min, as described previously¹⁴.
7. Place the animal in lateral decubitus to perform the left lateral thoracotomy at the fourth or fifth intercostal space. Make an incision in the animal's skin; observe the muscles below, and spread them carefully to separate them from the thoracic wall to clearly see the intercostal space. At this point, ensure that the animal is properly connected to the ventilator; then, open the intercostal space by making a hole with the extra sharp forceps.
   NOTE: The LDA should be identifiable along the free left ventricular (LV) wall in the intercostal spaces mentioned above.
8. Perform the pericardiectomy, and identify the LDA by contrasting the coronary arteries with the coronary veins and the LDA ramification below the left auricle. Then, perform the ligature of the LDA using the 8-0 silk thread ~2 mm from the edge of the auricle. Finally, close the thorax by layers using the 6-0 silk thread-close the ribs, ensuring that there is no pneumothorax inside (do this carefully by forcing the lungs to expand with the ventilator), and approach the muscles or suture them before closing the skin.
9. Once the skin is closed, slowly disconnect the mouse from the ventilator in ventral decubitus, and remove the endotracheal tube when the respiratory frequency has recovered. Let the animal recover from the anesthesia in a quiet environment and, preferably, with a stable room temperature at 27 °C.
10. Wait for the animals to recover from the anesthesia, begin moving their limbs, and for their normal respiratory frequency to be restored. Then, house them in individual cages until the end of the protocol.
11. Perform the same procedure on the control or sham-operated animals but without the ligature of the LDA.

3. Study design

1. For testing the temporal evolution of healing and ventricular remodeling after MI, randomize the mice into the following groups:
   1. To study the early phase of ventricular remodeling after 1 week of MI evolution, assign the mice to the following groups: 1) C57 Sham (1 week); 2) Gal-3 KO Sham (1 week); 3) C57 MI (1 week); 4) Gal-3 KO MI (1 week).
   2. To study the late phase of ventricular remodeling at 4 weeks of MI, assign the mice to the following groups: 5) C57 Sham (4 weeks); 6) Gal-3 KO Sham (4 weeks); 7) C57 MI (4 weeks); 8) Gal-3 KO MI (4 weeks).

4. Echocardiography

NOTE: For mouse echocardiograms, linear transducers over 10 MHz must be used for proper visualization of the wall diameters and cavity sizes. This procedure can be performed under anesthesia with intraperitoneal (IP) avertin at 1.15 mL/kg or in conscious animals. However, the latter can lead to confounding results in mice with MI due to the stress and anxiety caused by manipulation.

1. To anesthetize a mouse, pick it up, hold it with its back toward the palm, and turn it over to reach the surface of the abdomen. In that position, inject the IP anesthesia at a 45° angle between the animal and the subcutaneous needle.
2. Once the mouse is anesthetized, shave its chest, and place the mouse over a prewarmed heating pad in the dorsal decubitus position. To obtain parasternal long-axis and short-axis views, move the transducer 90°. Once the correct axis view is obtained, place the cursor at the papillary muscle level, press the 2-D M-mode key to capture the images, and use image analysis software to measure the following parameters:
   1. Measure the LV dimensions, including the wall thickness (LVWT), the LV areas both in systole (S) and diastole (D), and the left ventricular diastolic area (LVDA) and left ventricular systolic area (LVSA) in at least three beats.
   2. In addition, calculate the ventricular function by the ejection fraction (EF, %), the shortening fraction (SF, %), and the cardiac mass (assume an uncorrected cube) using equation (1), equation (2), and equation (3), as previously described¹⁵.
      SF (%) = ([LVEDD - LVESD]/LVEDD) × 100   (1)
      EF (%) = ([LVDA - LVSA]/LVDA) × 100   (2)
      LV mass = 1.055 × ([IVST + LVEDD + PWT]³− [LVEDD]³)   (3)
      Where LVEDD is the left ventricular end-diastolic diameter, LVESD is the left ventricular end-systolic diameter, IVST is the intraventricular thickness, and PWT is the posterior wall thickness.

5. Histology evaluation

1. During necropsy, extract the heart from the animal by opening the thorax from side to side and cutting all the structures that surround it. Clean the blood clot that is inside the cavities by applying gentle pressure using tissue paper.
2. Harvest and weigh the heart on a laboratory precision scale. Immerse it in 10% formaldehyde for at least 72 h at room temperature. Cut the heart manually from the apex to the base in 1 mm thick transverse slices using a blade, and process the slices by embedding them in paraffin. Make serial cuts to the paraffin-embedded sections of 5 µm thickness with a microtome.
3. Place each section between slides, and stain them with hematoxylin and eosin (H&E), Masson's trichrome, rhodamine-conjugated lectin-stained sections, or Picrosirius red^15,16.
   1. By using an appropriate photomicroscope, take digitalized images at 400x for morphometry, fibrosis, and MCSA quantification. Verify that the microscope is attached to a digital camera and connected to a computer with image analysis software. For each morphometric analysis, ensure that the images are in the same areas, with a minimum of 10 high power fields at 400x per section (septum, infarcted zone, and remote zone) and without overlapping the fields.
      1. To measure the MCSA, be aware of the positions of the cardiac myocytes, and count only those myocytes that are transversally sliced and surrounded by at least three nearby capillaries.
      2. In the Picrosirius red-stained slices, identify the scar and septum areas, and image the interstitial collagen in both zones. Upload the images onto the analysis software, and open the threshold tab to highlight all the positive and negative collagen areas To obtain the data, press the measure tab, and save the results. For the calculation of the percentage of collagen per region, use equation (4), and add the collagen-positive zones and divide them by the total tissue, including the collagen-positive areas, as described elsewhere^15,16.
         ​Collagen (%) = Picrosirius red area/Total tissue area   (4)
      3. Quantify the MCSA from the digitalized images obtained from the rhodamine-conjugated lectin-stained sections of the paraffin-embedded samples. To obtain the correct image, use image analysis software to trace the red outlines of the myocytes surrounding the cell membranes. Select the area tab, trace the outlines, and press the measure tab function. Finally, save the results from the cell areas¹⁶.

6. Quantitative determination of infarct size and planimetry to evaluate cardiac remodeling

1. Measure the myocardial infarct size, the wall thickness, and the length of the endocardial and epicardial circumferences using planimetry from the histological images of the Masson's trichrome-stained sections obtained with a light microscope (4x) and the appropriate software.
   1. For the quantification of the infarct size, identify the infarct zone (blue) and the remote zone (red). Trace and measure the total length of the infarct zone and the remote zone at the endocardial and epicardial sides. Calculate the average of the endocardial and epicardial tracings as a percentage of the total LV circumference¹⁷.
   2. Similarly, measure the scar thickness (the average of five equidistant measurements) and the septum thickness (the average of three equidistant measurements) in a middle section of the heart, and use these measurements to determine the scar thickness ratio (equation [5]) and the expansion index (equation [6]¹⁸).
      NOTE: All the values can be recorded in a spreadsheet.
      Scar thickness ratio = Scar thickness/Septum thickness   (5)
      Expansion index = (LV cavity area/total LV area) × (septum thickness/scar thickness)   (6)
      NOTE: Since the remodeling may modify the expansion, resulting in underestimation or overestimation of the infarct size, some time may be needed to perform a pilot experiment for measuring the infarct size at 24 h, followed by euthanasia. In this case, anesthetize the animal with the same IP anesthetic solution used for the MI procedure.
2. Place the animal in dorsal decubitus, and intubate it as described earlier. Once the animal is anesthetized, make a deep diagonal incision that reaches the skin, muscles, and costal bones from the xiphoid apophysis to the axillary hollow.
3. Isolate the aortic arch, make a small hole in ascending aorta, and introduce a catheter to perfuse the heart with Evan's blue. Then, manually remove the stained heart from the animal, and cut it from the apex to the base with a sharp blade. Place the heart slices in 1% triphenyl tetrazolium chloride (TTC) in isotonic phosphate buffer (pH 7.4) and incubate at 37 °C for 30 min ⁴ to confirm that the animals are comparable in terms of infarct size.

Results

Post-MI survival and necropsy
Over 4 weeks of follow-up, 17% (4/23) of the C57 mice versus 40% (8/20) of the Gal-3 KO mice were found dead. The necropsy was performed; the dead Gal-3 KO mice showed larger hearts than the C57 mice (Figure 1), and 38% of the C57 mice compared with 32% of the Gal-3 KO mice had macroscopic chest clots that were directly associated with cardiac rupture. The latter demonstrated that the cause of death was not related...

Discussion

The experimental model of MI by permanent coronary artery ligature is used for studying a wide variety of pathophysiological mechanisms of cardiac repair and remodeling^5,14,17. This article summarizes different methods currently used in this laboratory for studying the temporal evolution of cardiac repair and its effects on post-MI ventricular remodeling^14,17. Two models...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8-0 silk suture	Ethicon
C57BL/6J mice	Department of Bioresources of the Faculty of Veterinary of the University of Buenos Aires, Argentina
Forceps
Hardvard 386 respirator	Hardvard company
Heating pad			maintain animal's temperature during surgery
Image Pro-Plus 6.0	Media Cybernetics		Image Analysis Software
Ketamine	Holiday
Masson Trichrome	BIOPUR
Picrosirius red	BIOPUR
Retractors
Rodent Ventilator Model 683	Harvard Apparatus		Mechanical ventilator
Scissors
Stereoscopic magnifying glass	Arcano
Vivid 7 machine (General Electric Medical Systems, Horten, Norway)	General Electric		Any tracking software can be utilized with this protocol
WGA no. RL-1022, Vector Laboratories, Burlingame	Vector Laboratories
Xylazine	Pro-Ser