Ligation of Left Anterior Descending Coronary Artery in Rats for Developing Occlusive Myocardial Infarction Model

Summary

The protocol shows the development of a myocardial infarction (MI) model by permanently ligating the left anterior descending coronary artery in rats. Animal survival post-MI is essential to salvage cardiomyocytes in the ischemic zone, prevent infarct expansion, and reduce post-myocardial inflammation and fibrosis.

Abstract

Cardiovascular disease (CVD), including ischemic heart disease and stroke, is a major cause of mortality and morbidity throughout the world. Myocardial infarction is a clinical syndrome of ischemic heart disease that occurs due to prolonged ischemia. Ischemic myocardium undergoes functional, metabolic, and structural alterations, leading to irreversible damage to a portion of the myocardium due to necrosis. Experimental models of myocardial infarction are crucial for understanding the acute and chronic cellular, molecular, and morphological changes occurring during and post-MI and for developing and optimizing novel targets or strategies for treatment. The surgical model of MI in small animals employing permanent closure of the left anterior descending coronary artery (LAD) nearly resembles human MI. The goal of this study is to establish a surgically induced MI model involving the closure of the LAD coronary artery indelibly in rats. The experimental protocol includes an initial induction of anesthesia with ketamine 80 mg/kg and xylazine 12 mg/kg i.p., intubation of an endotracheal cannula using an otoscope without performing tracheotomy under ventilator-assisted ventilation with the use of extrinsic positive end-expiratory pressure (PEEP) to prevent the collapse of alveoli. A thoracotomy procedure was adopted that limits the lesions caused to skeletal muscles during surgery. This approach is minimally invasive, repeatable, and lowers mortality post-surgery. In this model, animals survived 4 days post-MI to understand the short-term pathobiological changes, mainly post-MI inflammation, angiogenesis as a natural process of infarct healing, and ultimately, fibrosis in infarcted myocardium.

Introduction

Cardiovascular disease (CVD), mainly comprising ischemic heart disease (IHD) and stroke, is a major cause of mortality and morbidity across the world. According to the World Heart Federation, 20.5 million people died from cardiovascular disease in 2021^1,2. Myocardial infarction (MI), also known as a heart attack, is an acute clinical syndrome of IHD. During MI, myocardial tissue suffers irreversible damage as a result of prolonged ischemia, which is usually caused by an occlusive thrombus formed over a ruptured atherosclerotic plaque in a coronary artery³.

Timely reperfusion using percutaneous coronary intervention (PCI) or thrombolytic agents is the mainstay therapy to salvage ischemic myocardium from necrosis^3,4. In developed countries, due to greater implementation of PCI, mortality following MI has declined, but the incidence of MI is increasing in developing countries like South Asia⁵. After MI, a portion of the myocardium that undergoes necrosis is surrounded by a zone of reversibly damaged myocardial cells and an ischemic zone. The ischemic myocardium undergoes post-MI ventricular remodeling that can lead to ventricular dilation and dysfunction, ultimately progressing to heart failure⁶.

Currently, extensive research is being carried out to promote the regeneration of damaged cardiomyocytes by means of stem cell therapy⁷ and to facilitate myocardial repair by stimulating angiogenesis within the ischemic zone⁸. However, considerable improvements in strategies are required for the clinical success of both approaches. Thus, experimental models mimicking myocardial infarction or ischemia are crucial for understanding the pathological, cellular, molecular, and morphological changes occurring during and post-MI so that novel therapeutic targets or strategies for treatment can be identified and optimized for clinical translation. In this study, an experimental model of MI is developed by performing closure of the left anterior descending (LAD) coronary artery irreversibly in rats. Firstly, endotracheal intubation was performed in anesthetized animals without tracheotomy under ventilator-assisted ventilation by applying extrinsic positive end-expiratory pressure (PEEP) to prevent atelectasis (deflation/collapse of air sacs in lungs). A minimally invasive procedure was adopted to open the chest cavity, avoiding any injury to skeletal muscles during surgery. The location of ligation involves the origin of the first diagonal branch of the LAD, which produces sufficient infarction and mimics the clinical condition of Occlusive Myocardial Infarction (OMI). As the location of ligation is the mid-LAD region, the mortality rate after surgery, as well as post-surgical complications, is lower⁹. In this model, animals survived 4 days post-MI to understand the short-term modifications occurring in myocardial tissue post-MI and identification of novel targets and strategies to treat MI.

Protocol

The experimental procedures were performed as per protocol after approval from Institutional Animal Ethics Committee (IAEC) [Protocol No. (IAEC/KCP/2023/009)]. The guidelines of the Committee for the purpose of control and supervision (CCSEA) and the Ministry of Environment, Forest and Climate Change, Government of India, were followed in handling and taking care of experimental animals.

1. Anesthesia and intubation of endotracheal cannula (Duration: 10-15 min)

1. Use Wistar rats of either sex, weighing 200 - 240 g, aged more than 6 months. Record the weight of the rats. Anesthetize the rat by administering Ketamine (80 mg/kg) and Xylazine (12 mg/kg) according to body weight through the intraperitoneal route.
   NOTE: Ketamine, a dissociative anesthetic, produces rapid anesthesia but partial muscle immobilization¹⁰. Thus, it is used in combination with Xylazine to produce muscle relaxation and achieve a state of surgical anesthesia¹¹.
2. Pinch the tail to ensure the extent of anesthesia. If the rat shows reflexes, administer 1/3^rd of the dose of Ketamine. Connect a heating pad to the Ventilator to lay over the operating table to provide homeothermic warming.
3. Place the rat in a dorsal decubitus position on the heating pad (Figure 1A). Retain the rat in this position by applying adhesive tapes and set the operation table in an inclined position.
4. Adjust the ventilator respiratory parameters with the tidal volume at 8 mL/kg (2-3 mL/min), respiratory rate at 77 breaths per min (bpm), and peak pressure (peak inspiratory pressure) at 15 cm H₂O. To prevent the collapse of the lungs, adjust positive end-expiratory pressure (PEEP) to 2 cm H₂O.
5. To perform endotracheal intubation, grasp the tongue and raise it with blunt tweezers. Insert an otoscope (Figure 1B) in the mouth to view the vocal cords and introduce the endotracheal cannula (Figure 1B and Figure 2A) through the vocal cords. To clearly see the epiglottis, project a flashlight around the throat from above.
6. Connect the endotracheal cannula with the ventilator tube (Figure 1A and Figure 2B) and monitor the chest movements to confirm correct intubation. Swelling of abdomen indicates a suspicion of esophageal intubation, repeat the intubation as per step 1.8.
7. After all the setup is done, use a surgical stereomicroscope for LAD ligation (Figure 1A), and use a heating lamp during the operation to maintain the rat body temperature at 37 °C.
8. Apply depilation cream to the rat's neck and thoracic region and leave for 10 - 20 min. Gently remove the rat's fur from the neck to the thoracic region.

2. Surgical induction of myocardial infarction (Duration: 20 - 25 min)

1. Use the tweezers to pluck the skin on the xiphoid process and make a slight incision (about 1 cm) with the help of scissors, followed by cutting longitudinally beginning at the xiphoid process, directed towards the sternocleidomastoid muscle.
   1. Avoid harming fascia so as to prevent severe bleeding. If there is bleeding, use wrapped wipes to gently press on the bleeding area.
2. From the base of the sternocleidomastoid muscle to the 5^th- 6^th rib, cut the fascia and pectoral muscles along the left side of the sternum.
3. Use forceps to make a puncture at the 3^rd and 4^th intercostal space towards the left side of the sternum. Do not damage the heart or lungs while creating a hole (about 6-8 mm). Apply the peep at this step.
4. Use a rib-cut nipper to cut the 4^th, 5^th, and 6^th ribs along with intercostal muscles and detach the remaining intercostal muscles with forceps (Figure 2C).
5. Insert the rib retractor gently to hold the ribs apart and expose the heart (Figure 2D). To clearly view the left atrium, separate the pericardium and thymus from the front side of the heart using tweezers and curved scissors.
6. Identify the location of the left anterior descending (LAD) coronary artery for ligation by suture.
   1. After carefully removing the pericardium, identify LAD as a prominent superficial artery running diagonally from the base of the heart to the apex along the interventricular groove. The LAD divides into two sets of branches: first, the septal branches (S1 and S2) that perforate the interventricular septum. The second set of branches, called diagonal branches (D1 and D2), perforate the anterior of the left ventricle.
   2. Identify the three regions of LAD, namely, proximal, mid, and distal regions. Proximal LAD: the region of LAD from its origin to the first branch, which could be either a septal or a diagonal; Mid LAD: the region between the first branch and the second diagonal branch; Distal LAD: the region beyond the second diagonal branch is known as distal LAD (Figure 2E).
   3. LAD appears as a pulsatile reddish artery with thicker walls. To confirm its location, gently press with micro forceps, and a pulsation is felt.
7. Perform permanent closure of LAD by passing 8-0 silk suture in the mid-LAD region near the origin of the first diagonal branch D1 using a needle holder and tugging the suture gently, preventing any harm to the heart (Figure 2E).
8. Using forceps, make two knots of ligature in identical orientation (surgeon's knot). Fasten the ligature by making multiple knots in a two-sided direction with the help of forceps.

3. Sham surgery

1. Perform steps 2.1-2.6. After performing a left-sided thoracotomy, expose the heart, pass a silk suture, and pull underneath the left main coronary artery (LAD) without ligating.

4. Administration of treatment drug (Duration: 2-5 min)

1. After LAD ligation, immediately inject Doxazosin 0.06 mg/kg intravenously through the tail vein in the treatment group (Figure 3)

5. Chest cavity closure after surgical procedure (Duration: 5 min)

1. Remove the retractors so that the ribs and intercostal muscles revert to their original position. Close the chest cavity by suturing the left and right intercostal muscles using a 6-0 silk suture. Make stitches with a 4-0 Prolene suture to close the skin (Figure 2F).

6. Post-surgery care and monitoring (Duration: 40 min - 60 min)

1. Follow the operated rat for recovery from anesthesia for 40 min or more. Assure complete recovery by lifting or pinching the tail.
2. Remove the connection from the ventilator temporarily and ensure the self-regulated breathing of the rat by examining the normal movement of the thoracic region.
3. Switch off the ventilator and remove the endotracheal cannula. Detach the adhesive tapes fastened to the limbs.
4. Inject Tramadol (12.5 mg/kg) through the intraperitoneal route to relieve post-operative pain.
5. Keep the rat on the heating pad to keep the body temperature at 37 °C for at least 30 min. Continuously monitor the rat for any morbid conditions, such as irregular breathing or difficulty, or any dangerous signs of infarction.
6. When the rat is able to move around freely, consider it in a state of complete recovery from anesthesia. Even though the rat is not capable of moving voluntarily, keep it in the cage within 45 min after removal from the ventilator.
7. Observe the rat daily for the first 4 days. On the 4th day, sacrifice the rat using a CO₂ euthanasia chamber.

7. Analysis of biochemical markers of myocardial infarction

1. Assess the troponin level as a biomarker of Myocardial infarction at 6 h and 4 days after surgery in blood samples of animals.
   1. Sample collection and preparation: Collect blood sample (200 µL) from the tail vein after 6 h of infarction in EDTA-coated or heparin-coated tubes and by retro-orbital puncture (1 mL) after 4 days of MI.
      NOTE: An Anticoagulated whole blood sample is preferred for Troponin T testing.
   2. Apply 150 µL of sample to the test strip using cardiac pipettes and disposable dosing syringes supplied in the kit.
   3. Incubate at room temperature for 15-20 minutes, and then interpret the results based on the appearance of a signal line (reddish line) in the detection zone within 15-20 min. Use the following parameters: No detection line, only control line = negative; visible detection line and control line = positive (Figure 6B).
2. Detect Troponin I in a serum sample using the Standard F analyzer.
   1. Sample collection and preparation: Collect blood samples (200 µL) from a tail vein after 6 h of infarction in EDTA-coated or heparin-coated tubes and by retro-orbital puncture (1 mL) after 4 days of MI.
   2. Allow the kit and blood sample to be at room temperature before the test. Check the expiry date on the back of the foil pouch. Open the foil pouch and check the test device (Figure 4).
   3. Insert the test device into the test slot of the analyzer. The analyzer reads the barcode data and checks the test device is valid (Figure 4).
   4. Collect 100 µL of sample with a fixed volume dropper (100 µL). Dispense the collected sample into the extraction buffer tube. Then, discard the used fixed volume dropper (100 µL).
   5. Mix the sample and buffer 2x-3x with the disposable dropper (100 µL). Then, collect 100 µL of the sample mixture.
   6. After applying the sample in the sample well of the test device, immediately press the TEST START button of the analyzer. The analyzer will automatically display the test result within 10 min.
      NOTE: If Troponin I (TnI) is present, it will be bound to the europium microparticle. This complex will be captured by the capture antibody on the test line, and the intensity of fluorescence light will be detected by the analyzer.

8. Analysis of the infarction area

1. After sacrificing the rats 4 days post-infraction, determine the area of infarction using the macroscopic method of 2,3,5-Triphenyltetrazolium chloride (TTC) dye.
   1. Remove the heart from the animal body and keep overnight at 4 °C. Excise both the auricles, the root of the aorta, and the right ventricle. Cut the frozen left ventricle into 2-4 mm-thick slices using a surgical blade. Incubate the ventricular slices at 37 °C in 1% TTC solution (prepared using 0.1 M Tris buffer maintained at pH 7.8) for a duration of 20 min.
   2. Analyze the staining pattern as the viable myocardium, which contains a dehydrogenase enzyme and a cofactor NADH, stained brick red with TTC due to the formation of a formazan, whereas the myocardial area that undergoes infarction remains unstained.
2. Determine the infarcted area by volume method.
   1. Place ventricular slices between two glass slides and a transparent plastic grid with 100 squares in 1 cm placed over it.
   2. Calculate the average area of each ventricular slice by counting the number of squares on either side. Similarly, calculate the number of squares falling on stained and unstained areas on both sides. The non-stained dull yellow area represented the infarcted portion, and the red area represented the viable portion.
   3. Express the infarct size as a percentage of total left ventricular volume (%LVV).

9. Histopathological analysis

1. Hematoxylin and eosin staining
   1. For histopathological findings, sacrifice the animal 4 days post-LAD ligation, remove the heart, wash with phosphate buffer (PBS, pH 7.4), and preserve in formalin solution (10%) immediately.
   2. Perform tissue processing by dehydrating the tissue for 1 h in a series of increasing alcohol concentrations (70%, 80%, 95%, 95% and absolute alcohol) so that water is removed from the tissue and alcohol enters inside the tissue. Perform clearing of alcohol from tissue by immersion in xylene.
   3. After clearing, transfer the tissue using forceps to molten paraffin wax in a stainless steel mold. The clearing agent diffuses out, and the molten wax infiltrates into the tissue. Chill the mold on the cold plate to solidify the paraffin. When the wax is completely cooled and hardened (30 min), remove the paraffin block easily from the mold.
   4. Cut the paraffin blocks containing embedded tissue into sections of 4 µm thickness using a microtome. Place the ribbon of paraffin sections in a water bath at 40-45 ˚C. Mount the sections on a slide and air dry or gently warm the slide. Then, deparaffinize the sections by washing them with xylene and rehydrating them with absolute alcohol and water.
   5. Stain with hematoxylin for 15 min. Wash the stained sections with water and treat them with a 1% acid alcohol mixture for 20 s.
   6. After washing with water, stain the sections with counterstain (1% aqueous solution of eosin) for 2 min. Observe the slides under the microscope to visualize histopathological changes at 10x and 40x magnification.
2. Masson trichrome staining
   1. Prepare the following reagents.
      1. Bouin's Solution: Add 75 mL of saturated Picric acid, 25 mL of 40% Formaldehyde, and 5 mL of Glacial acetic acid. Weigert's Iron Hematoxylin Solution: Prepare stock solution A containing Hematoxylin 1 g in 100 mL of 95% alcohol. Prepare stock solution B containing 29% Ferric chloride in 4 mL of water, 1 mL of concentrated hydrochloric acid, and 95 mL of distilled water. Add equal parts of stock solution A and B for use. The mixture is only stable for no more than 3 months.
      2. Biebrich Scarlet-Acid Fuschin Solution: Add 90 mL of 1% aqueous Biebrich Scarlet, 10 mL of 1% aqueous Acid fuschin, and 1 mL of Glacial acetic acid. Phosphomolybdic-Phosphotungstic Acid Solution: Add 25 mL of 5% Phosphomolybdic acid and 25 mL of 5% Phosphotungstic acid. Aniline Blue Solution: Add 2.5 g of Aniline blue and 2 mL of glacial acetic acid to 100 mL of distilled water. 1% Glacial Acetic Acid Solution.
   2. Deparaffinize the paraffin-embedded heart sections using xylene, followed by rehydration in different concentrations of alcohol.
   3. Incubate the heart sections with Bouin's solution for 30 min at 56 °C to increase the final color intensity. Rinse the sections in running tap water until the yellow color is removed.
   4. Stain the tissue with hematoxylin for 1 min then, wash with running tap water for 2-3 min. Stain the sections with Biebrich scarlet-acid fuchsin solution for 15 min.
   5. Differentiate with 5% phosphomolybdic - phosphotunstic acid for 5 - 8 min. Transfer the section directly (without rinsing) to an aniline blue solution for 6- 8 min.
   6. Wash with distilled water. Then, differentiate with 1% acetic acid solution. Dehydrate with 95% ethanol, clear with xylene, mount on the slide, and examine under a microscope¹².

Results

Endotracheal intubation is necessary to assist ventilation during the LAD ligation procedure involving open-chest thoracic procedures in rats. Successful intubation is confirmed by basal respiratory parameters before surgery and the maintenance of stable respiration, tidal volume, and minute volume during the surgical procedure (Figure 5).

The success of the LAD ligation procedure was confirmed by evaluating cardiac Troponin T and Troponin I as biochemical markers...

Discussion

Our present study represents the induction of Myocardial Infarction through permanent closure of the left anterior descending coronary artery (LAD) in rats. In this surgical protocol, anesthetic Ketamine 80 mg/kg administered by intraperitoneal route induced quick anesthesia, along with intraperitoneal administration of Xylazine 12 mg/kg, produced sufficient muscle relaxation, thereby facilitating the whole surgical procedure effectively as well as smooth post-surgery recovery after a short duration with minimal side eff...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18-Gauge intravenous cannula	Kent scientific, US
20-Gauge intravenous cannula	Kent scientific, US
4-0 Polypropylene suture	Khanna Surgical
6-0 silk suture	Nanak Surgical
8-0 silk suture	Nanak Surgical
Adson forceps	Nanak Surgical
Anaesthesia apparatus	Orchid Scientific
Anaesthesia induction chamber	Orchid Scientific
Curved scissors	Nanak Surgical
Endotracheal intubation tube	Kent Scientific, US
Hair removal cream	Nanak Surgical
Heating pad	Kent scientific, US
Hooked forceps	Nanak Surgical
Ketamine			Gift sample from KVAS, Amritsar
Micro needle holder	Nanak Surgical
Nitrile gloves	Nanak Surgical
Operation table
Otoscope	Nanak Surgical
Pean forceps	Nanak Surgical
Rib retractor	Nanak Surgical
Straight tweezer	Nanak Surgical
Strait nipper	Nanak Surgical
Surgical hair cap	Nanak Surgical
Surgical mask	Nanak Surgical
Surgical Stereomicroscope	Olympus Magnus	24B1187	Stereo zoom MSZ-TR microscope with arm boom stand, zoom ratio: 1:7 objective zoom: 0.65 - 4.5x Eyepiece 10x/22 mm Magnification : 6.5 - 45x with 10x eye piece LED ring illumination
Toothless forceps	Nanak Surgical
Tramadol			Gift sample from Amandeep hospital, ASR
Tweezers	Nanak Surgical
Ventilator	RoVent (Kent Scientific,  US)	RV-04-220	Automatic Ventilator with volume and pressure control and temprature warming and monitoring
Xylazine			Gift sample from KVAS, Amritsar