This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.
Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.
Acute myocardial infarction (AMI) and its long-term sequelae such as chronic heart failure (CHF) profoundly impact patient prognosis and quality of life, let alone the high cost restraints imposed on our available healthcare resources1. The prevalence of CHF in the western world is estimated at 1-2%, of which ~60% of cases are the consequence of AMI as primary cause2. In the USA alone, about 5.7 million patients suffer from CHF accounting for approximately $30 billion in annual healthcare costs in 2008, with a predicted triplicate in costs rising to $97 billion annually in 20301. Taken together, these numbers make a strong argument for the development of new cardioregenerative treatments that, for swift translation, rely on a reproducible and reliable large animal myocardial infarction model that accurately mimics the human scenario.
Pigs (Sus scrofa) are increasingly being used in cardiovascular research for pharmacological and toxicological testing3. One of the traits responsible for this success as a translational research tool is their similarity in cardiac function and anatomy with the human heart4,5. For instance, pig heart-to-body weight ratio, cardiac size and coronary artery anatomy distribution have all shown to be remarkably similar to man4. Moreover, cardiomyocyte metabolism, electrophysiological properties and response to an ischemic insult such as AMI have been reported to show high levels of agreement with the human situation6,7. Ultimately, to fulfill the above described criteria, a standardized MI-protocol that produces robust and sustainable MI for testing of investigational new drugs (IND) is needed. Here, we present such a standardized model that uses a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD) followed by reperfusion, thereby creating reproducible myocardial infarction covering the anteroapical, septal and inferoseptal walls of the left ventricle.
Todos in vivo experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. Experiments were approved by the local Animal Experimentation Committee.
1. Medication, Anesthesia, Venous Access, and Intubation
2. Transthoracic Echocardiography
3. Surgical Preparation and Vascular Access
4. Invasive Pressure Volume Loop Analysis
5. Intracoronary Pressure and Flow Measurement
6. Induction of MI
Finishing the Surgical Procedure (for Long Term Follow Up)
7. Cardiac Magnetic Resonance Imaging
8. End of Study and Infarct Size
Mortality and Infarct Size
In our center, out of 32 pigs (Female Dalland Landrace, 6 months old, ~70 kg) that were subjected to this MI protocol, five (15.6%) died due to refractory ventricular fibrillation during ischemia. This protocol creates an infarct covering approximately 10-15% of the left ventricle, located in the anteroseptal, septal and inferoseptal walls (Figure 2A). If serial noninvasive assessment of infarct size is warranted, late gadolinium enhancement (LGE) on CMR can be used to follow the nonviable infarct area over time (Figure 2B).
Cardiac Function and Remodeling
Four weeks after MI, global and regional parameters reflecting cardiac function should be decreased compared to healthy baseline values. Specifically, LV ejection fraction (LVEF) should decrease to approximately ~35-45% four weeks post-MI. Besides global systolic function, several parameters reflecting post-MI adverse remodeling can also be measured, such as LV morphology and diameters using CMR and echocardiography (Figures 3A and 3B). Four weeks after MI, an increase in end diastolic volume (EDV) as a sign of adverse remodeling can be expected (Figures 3A and 3B).
Coronary flow and pressure parameters
Angiogenesis and formation of new capillaries are often regarded as important treatment goals in ischemic heart disease8. Assessment of microvascular resistance can be indirectly based on the combined measurement of intracoronary pressure and flow velocity. Representative pressure and flow velocity measurement under normal conditions and maximal hyperemia is shown in Figure 4. Four weeks after MI, the hyperemic microvascular resistance should be increased in the infarct related coronary artery (LAD) compared to the baseline situation8.
Figure 1. MI model based on LAD balloon occlusion. (A) Standard surgical equipment with: 1) towel clamps; 2) mosquitos; 3) dissecting forceps; 4) round container; 5) needle holders (fine and rough); 6) Klinkenberg scissor; 7) dissecting scissors (straight and curved); 9) forceps (De-Bakey, fine and rough); 10) hose clamp; 11) anastomosis clamp; 12) gauzes; 13) electrosurgical pencil; 14) scalpel holder; 15) Dreesman (suction); 16) retractor; 17) lamp holders. (B) Left anterior oblique fluoroscopic view of the LAD and the LCX. (C) After visualizing the second diagonal branch, position the two radiopaque markers (see inset, black arrowheads) of the balloon just distally of the D2. Inflate and ensure that coronary blood flow is successfully blocked by contrast injection (see asterisk). Intracardiac defibrillator lead can be seen in the right ventricle (see white arrowhead). LAD denotes Left anterior descending artery; LCX denotes left circumflex artery; LAO denotes left anterior oblique view; AP denotes anterior posterior view; D1 denotes first diagonal branch; D2 denotes second diagonal branch. Click here to view larger image.
Figure 2. Infarct size after MI. (A) The 90 min balloon occlusion of the LAD leads to extensive myocardial damage and scar formation (white color), visualized by TTC staining at 1 month follow up. (B) Schematic infarct distribution shows that the infarction is located in the anterior, anteroseptal and inferoseptal segments of the heart. (C,D) Short and long axis late gadolinium enhanced CMR images show the extensive infarct scar (white signal, see black arrowheads) localized in the anterior, anteroseptal and inferoseptal segments of the heart. LGE-CMR denotes late gadolinium enhanced cardiac magnetic resonance. Scale bar denotes 3 cm. Click here to view larger image.
Figure 3. Assessment of cardiac function in ischemic MI models. (A) Representative CMR cine-loop images at end diastole and end systole showing functional impairment of the infarct scar segments. (B) M-Mode image of 2D parasternal long axis by echocardiography, showing LV dilatation (increase in LVIDd) 1 month after MI, as well as functional impairment (absence of septal thickening). EDV denotes end diastolic volume; ESV denotes end systolic volume; LVIDd denotes left ventricular internal diameter at diastole and LVIDs denotes left ventricular internal diameter at systole. Click here to view larger image.
Figure 4. Intracoronary pressure and flow velocity derived parameters. Intracoronary pressure and flow velocity recordings using the Combowire showing (A) reference values prior to MI with high response to hyperemia (black arrowhead). (B) 1 month after MI, the infarct related artery (LAD) has a decreased hyperemic response in coronary flow velocity (black arrowheads). As a result, pressure and flow velocity derived parameters (HMR) or flow velocity reserve (CFR) are decreased compared to the baseline. bAPV denotes basal average peak velocity; pAPV denotes peak average peak velocity; CFR denotes coronary flow reserve; HMR denotes hyperemic microvascular resistance. Click here to view larger image.
Figure 5. Overview of different study designs. (A) Schematic of multiple possible study designs to validate investigational new drugs (INDs) in various stages of MI using this LAD MI pig model. Dependent on the chosen phase of MI that is under investigation, functional analysis can be performed just prior to the treatment allocation as the baseline value and assessment of the area at risk. Click here to view larger image.
Intracoronary balloon occlusion of the LAD provides a reproducible and consistent preclinical MI model in pigs that can be used to investigate safety and the efficacy of new cardiovascular therapies that closely mimics the human situation. As shown in Figure 5, the presented ischemia/reperfusion infarction model provides the platform that can be further tailored to investigate different phases of MI and post-MI remodeling whilst the initial ischemia/reperfusion injury is identical for both.
The success of the described protocol outlined here is dependent on the myocardial ischemia as the most critical phase of the protocol. Correct placement of the balloon distal to the second diagonal branch of the LAD is crucial for reaching adequate infarct size whilst ensuring a high survival rate. Based on this MI model, a ~15% mortality rate was observed, while extensive mid and apical segments of the anterior, septal and inferior walls were infarcted as seen on CMR and TTC staining (Figures 2A and 2B). The duration of ischemia can be tailored according to the desired infarct size. Although we have used Landrace pigs in this protocol, minipigs (i.e. Göttingen minipigs) usually require longer durations of myocardial ischemia (e.g. 150 min occlusion).
Outcome analysis in preclinical and clinical MI studies is often based on LVEF. Although lower LVEF has been firmly associated with increased risk for cardiovascular mortality, it remains dependent on hemodynamical parameters such as preload9. Arguably, given that on average only 10-15% of the LV is infarcted, several conceptual and practical limitations are related to LVEF being a global measure of LV systolic function rather than reflecting local improvement10. Therefore, the proposed measures of outcome used in this model shed light on different aspects of MI and post-MI remodeling thereby providing the investigators the means to accurately assess the efficacy of new therapies on multiple levels.
To optimize translation from preclinical models to clinical practice, we choose using large pigs instead of minipigs. Hemodynamic measurements, medication dosages and surgical devices can easily be exchanged with clinical practice. Compared to minipigs, large pigs gain relatively much weight. This may cause a problem in long-term follow up, with regard to comparability of serial results. Female Dalland Landrace pigs weigh approximately 70 kg at an age of 6 months. To prevent abundant weight gain during the follow up period, animals are kept on a restricted diet. Pigs receive 750 g of custom made low calorie food (containing: proteins 15.6%, fat 2.0%, fibers 14.8%, ashes 8.8%, calcium 0.9%, phosphorous 0.57%, magnesium 0.29%, and potassium 0.18%) twice a day and gain about 10 kg of weight in 4 weeks.
McCall and coworkers have previously published a similar protocol for myocardial infarction in pigs11. Considerable overlap exists between this protocol and theirs, emphasizing the preference for the LAD rather than the left circumflex artery (LCX) or the right coronary artery (RCA). In our experience, there is a lesser extent of infarct size of the total left ventricle using the LCX while the RCA infarction is accompanied with higher chance of unwanted conduction disturbances (i.e. sinus node dysfunction, AV-node dysfunction). One difference between the two protocols pertains to the use of increased pharmacological platelet inhibition in this protocol, as we have observed higher rates of no-reflow based on thrombus formation as the result of 90 min of hemostasis in the occluded coronary artery. This observation is in line with known hypercoagulability observed in pigs12. Although McCall proposed using a single, high-dose, bolus of heparin, this protocol relies on the use of heparin in multiple lower doses spread throughout the surgery to minimize thrombotic complications.
In summary, we present a porcine MI model that enables researchers to make use of an effective, reproducible and above all practical large animal model of human disease to study new therapeutics as an essential step towards a first-in-man clinical trial.
The authors have nothing to disclose.
Cees Verlaan, Joyce Visser, Merel Schurink, and Grace Croft are kindly acknowledged for their excellent technical assistance with the animal experiments.
0.9% Saline | Braun | ||
6-0 Prolene | Ethicon | ||
Acetylsalicylic acid | 80 mg (Ratiopharm) | ||
Adenosine | 3mg/ml apotheek UMCU | ||
AdVantage PV loop system | Transonic Scisence | ||
Amiodarone | Cordarone I.V. (Sanofi) | ||
Amiodaron HCl (PCH) | |||
Amoxicilline/ clavulaanzuur | 500/50mg (Sandoz) | ||
Atropinesulfaat | 0,5 mg/ml (PCH) | ||
Bone Marrow Wax | Syneture | ||
Cardiac Defibrillator | Philips | ||
Clopidogrel | Clopidrogel 75A (Apothecon B.V.) | ||
Contrast agent | Telebrix | ||
Endotracheal tube | Covidien | ||
Fentanyl patch | Durogesic® 25 mcg/h (Janssen-Cilag) | ||
Fogarty catheter | Edwards Lifesciences | ||
Gadolinium | Gadovist | ||
Guidewire | Abbott | ||
Heparin | Leo | ||
I.V. cannula | Abbocath® (Hospira venisystems) | ||
Iodine | Jodiumtictuur 2% (Eurovet) | ||
Ketamine | Narketan® 10 Vétoquinol | ||
Midazolam | 5mg/ml (Actavis) | ||
Nitroglycerin | 1mg/ml Pohl Boskamp | ||
Pancuronium bromide | 2mg/ml | ||
Seldinger vascular sheath 8F | Arrow | ||
Sufentanil | 50 mcg/ml (Sufentanil-hameln) | ||
Swann-Ganz catheter | Criticath ref 680078 (Argon) | ||
Synolux | 250 mg (Pfizer) | ||
Tetra-polar catheter | Transonic Scisence | ||
Thiopental | 0,5 g (Inresa) | ||
Triphenyl-tetrazolium chloride | Merck | ||
Venofundin | Braun | ||
Vicryl 2-0 | Ethicon | ||
Volcano ComboMap system | Volcano |