Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.
Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson’s trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.
Cardiovascular disease is a major public health concern that claims 17.9 million lives each year, accounting for 31 percent of global mortality1. The most prevalent type of cardiovascular anomaly is coronary heart disease, and myocardial infarction (MI) is one of the major manifestations of coronary heart disease2. MI is usually caused by thrombotic occlusion of a coronary artery due to the rupture of a vulnerable plaque3. The resulting ischemia causes profound ionic and metabolic changes in the affected myocardium, as well as a rapid decrease in systolic function. MI results in the death of cardiomyocytes, which can further lead to ventricular dysfunction and heart failure4.
Research on MI in patients is limited due to the scarcity of tissues obtained from patients with MI5. As such, murine models of MI are useful in both studying disease mechanisms as well as developing potential therapeutic targets. Currently available murine models of MI includeirreversible ischemia models (LCA and ablation methods) and reperfusion models (ischemia/reperfusion, I/R)6. Permanent ligation of the left coronary artery (LCA) in mice is the most used method, and it imitates the pathophysiology and immunology of MI in patients7,8,9. Permanent MI can also be induced by ablation methods, which involve electrical damage or cryoinjury. Ablation methods are able to generate uniform-sized infarction at the precise location10. On the other hand, scar formation, infarct morphology, and molecular signaling mechanisms may vary among the ablation methods10,11. The murine I/R method is another important MI model as it represents the clinical scenario of reperfusion therapy12. The I/R model is associated with challenges such as a variable infarct size, difficulty in distinguishing responses of initial injury, and reperfusion6.
Although widely used, LCA ligation methods are associated with low survival rates and post-operative pain13. This protocol demonstrates the murine surgical MI model of LCA ligation that involves the preparation and intubation of mice, LCA ligation, post-operative care, and validation of MI. Rather than using an invasive tracheotomy14, this method employs endotracheal intubation. The animal is intubated by illuminating the oropharynx using a laryngoscope, making the procedure easier, safer, and less traumatic15. The mouse is kept on ventilator support and under isoflurane anesthesia throughout the procedure. Further, echocardiography and Masson's trichrome staining are performed to evaluate heart function and cardiac fibrosis after MI, respectively. Overall, this method provides a reliable and reproducible surgical murine model of MI that can be used to study pathophysiology and inflammation after MI.
The present study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh. Eight (sham n = 4 and MI n = 4) 1-year-old female C57BL/6J mice weighing 24-30 g were used for these experiments. Approximately 100% and at least 80% of mice survived in the first 24 h and 28 days, respectively.
1. Preparation and endotracheal intubation of the mice
2. Permanent ligation of the left coronary artery
3. Post-operative care
4. Echocardiographic Evaluation
NOTE: Echocardiography was performed to evaluate the parameters of heart failure on day 28 after MI.
Figure 1 demonstrates the representative active ECG and respiration signals during the echocardiographic evaluation of sham (Figure 1A) and MI (Figure 1B) mice. Verification of active ECG and respiration signals are important before acquiring the echocardiographic data. Figure 2 shows echocardiographic measurement of cardiac functional parameters following 28 days after LCA ligation. Figure 2 shows M-mode images of the para sternal short axis view of sham (Figure 2A) and MI (Figure 2B) hearts. Figure 2B shows defective heart wall movement following LCA ligation. Indicators of heart failure, such as increased LV mass (Figure 2C), decreased ejection fraction (Figure 2D), and decreased cardiac output (Figure 2E), were observed in the MI group as compared to the sham group.
All the animals were euthanized according to standard protocols using an excessive dose of CO2 gas. The hearts were fixed and frozen in optimal cutting temperature (OCT) compound. Masson's trichrome staining18 was performed for three different ventricle sections (lower, middle, and upper) and images were taken using a research slide scanner under 10x magnification to examine the degree of cardiac fibrosis. Figure 3 shows increased collagen staining (blue) in the infarcted heart, indicating augmented fibrosis.
Figure 1: Active ECG and respiration signals during the echocardiographic evaluation. Representative active ECG and respiration signals during the echocardiographic evaluation of (A) sham and (B) MI mice. Green = ECG signals, yellow = respiration signals. Please click here to view a larger version of this figure.
Figure 2: Echocardiographic evaluation of cardiac functional parameters following LCA ligation on day 28 after the surgery. Representative parasternal short axis (PSAX) M-mode echocardiographic images of (A) sham and (B) MI mice. Assessment of (C) left ventricular mass (mg), (D) ejection fraction (%), and (E) cardiac output (mL/min) of sham and mice with MI. LVAW;d = left ventricular anterior wall thickness in diastole; LVAW;s = left ventricular anterior wall thickness in systole; LVPW;d = left ventricular posterior wall thickness in diastole; LVPW;s = left ventricular posterior wall thickness in systole; LVID;d = left ventricular internal diameter in diastole; LVID;s = left ventricular internal diameter in systole. Data are shown as Mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Please click here to view a larger version of this figure.
Figure 3: Assessment of fibrosis after LCA ligation on day 28 after the surgery. Representative images showing Masson's trichrome staining of (A) sham and (B) MI hearts 28 days after the surgery. The fibrotic regions in the infarcted heart are characterized by collagen deposition and stained blue after Masson's trichrome staining. Scale bar = 500 μm. Please click here to view a larger version of this figure.
The murine model of MI is gaining popularity in cardiovascular research laboratories, and this study describes a reproducible and clinically relevant MI model. This protocol improves the LCA ligation process in several ways. To begin with, the use of injectable pre-operative anesthetics such as xylazine/ketamine or sodium pentobarbital14,15 is avoided. Only isoflurane anesthesia was used, which helps enhance animal survival rates (>80% survival 28 days after surgery), minimize drug-induced complications, and has minimal cardiac effects as compared to other agents19. However, isoflurane does also slow the heart, albeit at a lower degree compared to other anesthetic agents20. This protocol involves less invasive endotracheal intubation avoiding tracheostomy21, which reduces post-operative pain and discomfort. Previous murine LCA ligation studies have recommended making a mid-neck incision to improve the visualization of endotracheal intubation; however, the current protocol uses a laryngoscope instead to illuminate the oropharynx15. Lugrin et al. recently demonstrated a murine LCA MI model without thoracentesis14; however, the current protocol includes an effective thoracentesis, which will help remove excess blood and air from the chest cavity, preventing pneumothorax19. In addition, this method uses sterile gauze for bleeding management in place of a cauterizer, as using a cauterizer to reduce bleeding can result in iatrogenic burns and may alter inflammatory readings21.
One of the critical steps in this surgical model is identification and ligation of the LCA. The location of the coronary artery may vary depending on mouse strains and genotypes9. In most cases, the artery is not visible under a microscope. From experience, ligating the myocardial tissue 2-4 mm below the edge of the left atrium results in efficient blanching of the left ventricular wall. Furthermore, the procedure can be simply modified to induce temporary myocardial ischemia followed by reperfusion (I/R) by removing the ligation22. This animal model mimics the restoration of coronary blood flow in MI patients following percutaneous coronary intervention23,24. Since the permanent LCA occlusion model differs from the I/R model in several aspects, such as the size of the infarcted area, location of the infarct, and infiltration of inflammatory cells, researchers must be cautious while selecting the relevant model depending on the study7,14,25.
There are multiple approaches to ensure successful ligation of the LCA and subsequent development of MI. Observing immediate blanching of the lower left ventricle is the earliest confirmation of successful LCA ligation. Apart from this, the extent and location of the myocardial infarct can be visualized by staining the whole heart with Evan's blue or 2,3,5-triphenyl tetrazolium chloride (TTC)26. Measurement of circulating cardiac troponin can further validate the myocardial tissue injury21. Electrocardiography can be used as a non-invasive method of confirming elevation of the ST segment after MI17. The degree of cardiac fibrosis associated with MI can be evaluated by Masson's trichrome staining and cardiac MRI27,28,29. Echocardiography can be used to evaluate the parameters of heart failure on days 1 and 28 after MI. To examine cardiac remodeling following the MI, Masson's trichome staining and echocardiography can be utilized17. It is also possible to use qPCR and immunoblot to further investigate and confirm the expression of the genes and proteins implicated in fibrosis, inflammation, and heart failure after MI14.
The major limitation of LCA ligation is the high incidence of mortality, which could be due to post-operative cardiac arrhythmias, ventricular rupture, hemorrhage, pneumothorax, and post-operative discomfort19,30. However, a successful thoracentesis, minimizing the non-target tissue damage, and proper post-operative pain and temperature management may help reduce the death of the animal. As with any other surgical model, exact reproducibility is another limitation of this surgical model. Researchers can, however, reproduce MI, control infarct size, and improve post-surgery survival by rigorous practice and experience.
The authors have nothing to disclose.
This work was supported by National Institute of Health grants (R01HL143967, R01HL142629, R01AG069399, and R01DK129339), AHA Transformational Project Award (19TPA34910142), AHA Innovative Project Award (19IPLOI34760566), and ALA Innovation Project Award (IA-629694) (to PD).
22 G catheter needle | Exel INT | 26741 | Thoracentesis |
24 G catheter needle | Exel INT | 26746 | Endotracheal intubation |
4-0 nylon suture | Covetrus | 29263 | Suturing of muscles and skin |
8-0 nylon suture | S&T | 3192 | Ligation of LAD |
Anesthetic Vaporizers | Vet equip | VE-6047 | Anesthetic support |
Animal physiology monitor | Fujifilm | VEVO 3100 | Monitor heart rate,respiration rate and body temperature |
Betadine solution | PBS animal health | 11205 | Antispetic |
Buprenorphine | Covetrus | 55175 | Analgesic |
Disecting microscope | OMANO | OM2300S-V7 | Binocular |
Electric razor | Wahl | 79300-1001M | Shaving |
Electrode gel | Parker Laboratories | W60698L | Electrically conductive gel |
Ethanol | Decon Laboratories | 22-032-601 | Disinfectant |
Forceps | FST | 11065-07 | Stainless Steel |
Gauze | Curity | CAR-6339-PK | Sterile |
Heat lamp | Satco | S4998 | Post surgery care |
Heating pad | Kent scientific | Surgi-M | Temperature control |
Hot Bead sterilizer | Germinator 500 | 11503 | Sterilization of surgical instrument |
Isoflurane | Covetrus | 29405 | Anesthesia |
Masson’s trichrome staining kit | Thermoscientific | 87019 | Measurement of cardiac Fibrosis |
Micro Needle Holder | FST | 12500-12 | Stainless Steel |
Micro scissors | FST | 15000-02 | Stainless Steel |
Ophthalmic ointment | Dechra | Puralube Vet | Sterile occular lubricant |
Scanning Gel | Parker Laboratories | Aquasonic 100 | Aqueous ultrasound transmission gel |
Scissors | FST | 14060-11 | Stainless Steel |
Small Animal Laryngoscope | Penn-Century | Model LS-2-M | Illuminating the oropharynx |
Small animal ventilator | Harvard apparatus | 557058 | Ventilator support |
Surgical light | Cole parmer | 41723 | Illuminator Width (in): 7 |
Vevo 3100 preclinical imaging platform | Fujifilm | VEVO 3100 | Echocardiography |
VevoLAB software | Fujifilm | VevoLAB 3.2.6 | Echocardiography data analysis |