Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Summary

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Abstract

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. °°°This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Introduction

Ischemic heart disease, in particular acute myocardial infarction (MI), is the main cause of death in developed countries ¹. Today, the standard treatment of MI is percutaneous coronary intervention (PCI), the balloon catheter treatment. One of the critical factors that affects quality of life and prognosis of patients after PCI-treated acute MI is the infarction size. The reduction of the size can have a great impact on patient survival and prognosis ². Myocardial ischemia/reperfusion (I/R) injury has a significant influence on the infarction size so one of the main aims in cardiovascular research is to prevent or reduce myocardial I/R injury ³. The exact mechanisms of I/R injury are still under investigation ⁴. Activation of the plasma cascades and endothelial cells are hallmarks of I/R injury ⁵. Activation of the coagulation system is clearly involved ^6,7. Recently, the role of FXII, as an early upstream peptide involved in contact phase activation of the coagulation cascade, has been shown in a FXII knock out rat model of cerebral I/R injury ⁸. Validation of these results in a porcine model is an important step into clinical translation. Therefore, we are testing a novel bicyclic (80 kDa protease) FXIIa inhibitor in the context of myocardial I/R injury in a pilot study.

Animal models, which mimic the clinical situation of acute MI and PCI treatments, are essential to improve our understanding of the pathophysiology of myocardial I/R injury and to test novel treatment options. Pigs represent a good animal model for clinical myocardial I/R injury. This is not only because their hearts are very similar to human hearts with respect to anatomy and coronary circulation, but they also show similar pathophysiological responses to myocardial ischemia and reperfusion ^9,10. Other models such as rats and mice do not fulfill these criteria and show considerable differences when compared to human hearts ^11,12, whereas dogs for example, have many more collateral coronary vessels as compared with humans ¹³.

The porcine acute myocardial infarction model has been widely used in cardiovascular research to investigate ischemic heart disease including myocardial I/R injury ^14,15,16,17. The latter is an inflammatory condition, because of which minimizing the inflammatory reaction related to sternotomy or thoracotomy used in open-chest surgery is essential. The closed chest model using a clinical C-arm angiography setting overcomes this problem. Furthermore, one of the most important points is that our protocol provides an accurate distinction between ischemic (area at risk, AAR) and non-ischemic areas of the left ventricle (area not at risk, ANR) so that the infarct size (necrotic ischemic tissue, NIT) can be accurately determined. Our aim for this paper is to clearly define a reproducible methodology of a porcine myocardial I/R injury model, in particular with respect to myocardial tissue sampling, which will allow for a more precise analysis of the molecular mechanisms of I/R injury and a clearer picture of the effects of novel drug treatments.

Protocol

1. Animals:

All animals were treated according to the guidelines of the Swiss national laws. The study was approved by the local animal experimentation committee of the Canton of Bern (permission no. BE 25/16).

Use large white pigs of both sexes (~30 ± 5 kg). Divide the animals blindly into two groups, one group receiving a bicyclic (80 kDa protease) inhibitor of FXIIa or treatment of choice and the other an inactive control.

2. Surgical procedure (Figure 1)

1. Anesthesia and preparation of the animal:
   1. Fast the animals for 12 h before starting the experiment.
   2. Pre-medicate the animal with 20 mg/kg Ketamine and 2 mg/kg Xylazine via an intramuscular injection into the neck, using a 10 mL syringe. Record the animal weight and sex.
   3. Induce anesthesia by injecting 0.5 mg/kg Midazolam and 0.05 mg/kg Atropin into the auricular vein. Intubate the animal with an endotracheal tube.
   4. Maintain the anesthesia by mechanical ventilation using a respirator (O₂/air 1:3, Sevorane 1.5%), a 7 - 8 mm airway tube and a filter. Adjust the fraction of inspired oxygen (FiO₂) to 35% and the tidal volume to 6-10 mL/kg.
   5. Depth of anesthesia is assessed as adequate in absence of either motor or autonomic responses to nose pinching. Palpebral reflexes and jaw tone were monitored continuously as well targeting relaxed jaw tone and absence of palpebral reflexes. Core temperature was continuously monitored and maintenance of normothermia (38-38.5 °C) was ensured with passive warming (isolating blankets and warm bottles).
   6. Dissect free, as previously described by Koudstaal and his colleagues steps 3-1 to 3-3¹⁸, the carotid arteries on both sides and cannulate them with a 7F sheath. Cannulate the left jugular vein with a 7F sheath for venous blood sampling.
   7. Administer a bolus dose of 250 µg Fentanyl analgesic through the central venous line followed by 250 µg/h as a continuous intravenous infusion using an infusion pump. Monitor body temperature, heart rate and rhythm with a 3-lead electrocardiogram (ECG), arterial and central venous pressure during the whole experiment. 
   8. Using standard blood collection tubes, withdraw the following baseline blood samples from the venous line: 5 mL citrated plasma and 2.9 mL EDTA plasma into the respective tubes. Centrifuge immediately at 2,000 x g for 15 min at 4 °C. Take 2.9 mL blood into a serum tube and allow to coagulate for 30 min at room temperature before centrifuging as described above.
   9. Aliquot 200 µL of plasma or serum into 500 µL tubes and store all samples at -80 °C for further analysis.
   10. Withdraw 0.5 mL of blood from the arterial line using the special blood gas analysis (BGA) syringes to measure BGA using the BGA machine according to the manufacturer's instructions.
   11. Withdraw 0.5 mL of blood from the venous line using a standard 2 mL syringe and immediately transfer it into the ACT cartridge using a 30 G needle. Insert the filled cartridge into the ACT machine to measure clotting time according to the manufacturer's instructions. See Figure 2 for time points.
   12. Administer 5000 IU unfractionated heparin into the venous line using a 2 mL syringe and allow the animal to stabilize for 20 min before starting the MI experiment.
   13. Monitor ACT every 30-45 min as mentioned in 2.1.11. Inject 2500 IU unfractionated heparin intravenously if ACT is < 180 s.
2. Myocardial infarction experiment
   1. Use a standard C arm fluoroscopy equipment (coronary angiography program, angle 0°, 12 frames per second, ~70 kV) - or alternatively a dedicated angiography system - to perform the coronary intervention.
   2. Use fluoroscopic guidance to insert a pressure catheter (5F, 120 cm) via the previously placed sheath in the left carotid artery. Advance it into the left ventricle. Connect the pressure catheter to an acquisition system to record left ventricular pressure. The acquisition system needs to continuously calculate and record heart rate, developed pressure, dP/dt maximum (contractility of left ventricle) and dP/dt minimum (relaxation of left ventricle) during the entire experiment. Record baseline values for 10 min.
   3. Insert a 6F (100 cm, EB3.75) guiding catheter via the previously placed sheath into the right carotid artery. Advance it to the left coronary artery to reach the left anterior descending coronary artery (LAD) under X-ray guidance. Inject contrast medium using a 20 mL syringe via the guiding catheter to perform a baseline coronary angiography.
      NOTE: Injection of contrast medium was done by manual pressure but a dedicated power injector system could be used as well.
   4. Assess the size of the LAD on the X-ray monitor to select the appropriate size of the percutaneous coronary intervention (PCI) catheter. Use PCI balloons with a length of 15-25 mm and diameters between 2 and 3.5 mm depending on the LAD size. Assemble PCI catheter and coronary guidewire (F 014/J, 175 cm). Connect the PCI catheter with the inflation device pre-filled with contrast medium.
      NOTE: The LAD diameter can be measured directly on a calibrated monitor using a ruler and the size of the PCI balloon is then selected accordingly.
   5. Insert the assembled system from 2.2.4 into the lumen of the guiding catheter. Advance the guidewire into the LAD until it reaches beyond the second diagonal branch of the LAD.
   6. Use fluoroscopic guidance to advance the PCI catheter until it reaches about the middle of the LAD. Choose the LAD blocking site depending on the anatomy of the coronaries, usually after the second, sometimes after the first diagonal branch (Figure 3) in order to have similar percentages of the AAR of the left ventricle (LV).
      NOTE: The choice of the blocking site depends on the length of the diagonal branches and thus the area of tissue which is supplied by blood via the respective branch. In case of a long, bifurcated first diagonal branch the blocking site will be just after this. In case of a shorter first diagonal branch, the blocking is done after the second diagonal.
   7. Remove the guidewire and then increase the pressure in the inflation device to 7-10 bar to inflate the balloon of the PCI catheter and induce myocardial ischemia for 1 h. Gradually increase the FiO₂ to 50-60% between 15 and 40 min of ischemia. Keep tidal volume at 6-10 mL/kg.
      NOTE: This procedure will reduce the occurrence of extrasystoles and decrease the frequency of ventricular fibrillations.
   8. Record a 5-10 s video sequence while injecting contrast medium through the guiding catheter to have an angiogram of the balloon of the PCI catheter in place just after starting ischemia; repeat after 10 min of ischemia to verify complete occlusion of the LAD distal to the balloon of the PCI catheter.
   9. Monitor the animal closely to immediately detect and treat (2.2.10) cardiac arrhythmias. Extrasystoles usually occur and increase in frequency (>3 per min) between 20 and 40 min after induction of myocardial ischemia. If this occurs, gently massage the neck on both sides just below the cheek. In most cases this will be sufficient to re-establish a regular heartbeat, probably by stimulation of the vagal nerve baroreceptors located on the common carotid artery.
   10. If cardiac arrhythmias progress into ventricular fibrillation, use an external, biphasic defibrillator to re-establish a sinus rhythm. Apply 5-10 chest compressions using the defibrillator pads immediately before applying the shock in order to fill the coronaries with oxygenated blood and then shock with 150 J (for 30 kg animals).
   11. Repeat if necessary and increase the energy to 175 J after the 3^rd shock. Use higher energy settings for heavier animals.
   12. Five min before the end of the ischemia time repeat the blood sampling mentioned in 2.1.8-2.1.13. Inject the test substance (either the bicyclic FXIIa inhibitor or control 4 mg/kg, do this this blindly) intravenously through the central venous line and flush the line with 20 mL saline.
   13. Withdraw one plasma citrate sample (as mentioned in 2.1.8 and 2.1.9) 4 min after injecting the substance in 2.2.12.
   14. Perform an angiogram (2.2.3) to confirm LAD occlusion, then deflate the balloon of the PCI catheter and remove this catheter from the guiding catheter. Confirm perfusion of the LAD distal to the occlusion site by angiogram immediately after deflation and removal of the balloon of the PCI catheter, 10 min thereafter, whenever signs of myocardial ischemia were visible by ECG for more than 5 min, and immediately before re-occlusion of the LAD.
   15. Allow reperfusion of the ischemic myocardium for 2 h. Take plasma and serum samples at 10, 30, 60 and 115 min of reperfusion (2.1.8 and 2.1.9). Monitor BGA at 60 and 115 min (2.1.10).
   16. Reinsert the PCI catheter together with the guidewire to exactly the same position as used for the ischemia. Inflate the balloon of the PCI catheter as before and confirm LAD occlusion by angiogram (2.2.3). Remove the pressure catheter from the left ventricle and stop recording.
   17. Inject 100 mL 2% Evans Blue in phosphate buffered saline (PBS, pH 7.4) into the central venous line. About 30 s later, when the whole animal turns blue, inject 40 mL 20% KCl to euthanize the animal. Death was confirmed by the absence of ECG signals and pulse waves.

3. Sampling techniques

1. Extracting, dissecting and sampling the heart (figure 4)
   1. Perform a sternotomy to expose the heart. Follow the protocol previously described by Koudstaal and colleagues, steps 8-2 and 8-3¹⁸. Cut open the pericardium while inspecting for abnormalities, which might stem from earlier pericarditis, and exclude the animal from further evaluation.
   2. Deflate and remove the PCI as well as the guiding catheter. Excise the heart for further analysis. Cut the vena cava and remove blood using a suction pump, then cut all the large vessels connecting the heart with the body.
   3. Rinse the heart inside and out with room temperature saline. Weigh the whole heart.
   4. Within 30-40 min, cut the heart into slices of about 3-5 mm from the apex to the Chordae tendinae of the mitral valve, perpendicular to the long axis using a sharp knife.
   5. Be careful to always place the heart in the same orientation with the ventral side facing up in order to keep the orientation of the cut samples (Figure 5).
   6. Photograph the slices of the heart using a digital single lens reflex camera.
   7. Cut away the right ventricle (discard as not needed). Photograph the left ventricle slices and weigh all the slices for the total weight of the left ventricle.
   8. Differentiate between the Evans Blue positive and Evans Blue negative tissue in all the sections. Dissect the slices to separate the ischemic (Evans Blue negative) from the non-ischemic tissue (Evans Blue positive) using a scalpel.
   9. First analyze the Evans Blue negative sections (the ischemic area at risk, AAR). Weigh them all and put them all into a plastic container.
   10. Cover the slices entirely with 100-150 mL (according to the heart size) triphenyl tetrazolium chloride solution (2 g TTC, 16 g Dextran, molecular weight 48000-90000, in 200 mL PBS, freshly prepared) so that the heart pieces can move freely inside the solution. Cover the container and incubate for 20 min at 37 °C while gently shaking.
   11. During this 20 min incubation time weigh the Evans Blue positive pieces (area not at risk, ANR), take samples for Tissue-Tek embedding (choose the most distal part from the injury) and store at -80 °C for further analysis. Transfer the rest into 4% formaldehyde solution and store at room temperature for histology sections.
   12. Remove the pieces of the AAR from the TTC solution. The red stained tissue is viable ischemic tissue (VIT) and the non-stained tissue is necrotic ischemic tissue (NIT). Cut 2 small pieces (blocks of 2-3 mm) from both the NIT and VIT, embed them into Tissue -Tek and store at -80 °C for further analysis. These samples should have the same weight.
   13. Fix the rest of the pieces (all slices derived from the AAR) by pinning them down in a Styrofoam container and covering completely with 4% formaldehyde solution for 24 h at room temperature in a fume hood. The pieces should stay flat for the photographic documentation in the next step.
   14. The next day photograph both sides of the pieces with a high-resolution camera with the same zoom setting and distance from the tissue (same magnification). Add automatic scale bars to all pictures. All bars need to have the same length.
       NOTE: The high-resolution camera should be connected to software that automatically adds the scale bar to each photo.
2. Calculation of the AAR and the infarct size
   1. %AAR of left ventricle = (weight of AAR in g/ weight of left ventricle in g)* 100.
   2. Use ImageJ software to calculate the total surface area of both the AAR and NIT (both sides of each piece) based on the photographs.
   3. Adjust the scale bar by selecting the scale bar length using the straight line from the angle tool. Choose from the menu Analyze > Set Scale and insert the known distance and unit of the scale bar. Choose "global" so the same scale will be applied to all pictures.
   4. Mark the whole surface area of the tissue using the free hand selection tool to calculate AAR. Be careful not to include the side (height) of the tissue and/or the fatty tissue (figure 6-C).
   5. Set the measurement by choosing "area" and "display label" from Analyze > Set Measurements menu. Measure the surface area from Analyze > Measure.
   6. Repeat step 3.2.5 to measure NIT (mark only the non-stained tissue). Note: Don't include the fatty tissue (figure 6-D) in the NIT calculation. Repeat the step on the other side of the tissue.
   7. Calculate the average AAR and NIT for each piece of tissue.
   8. Use the values obtained from 3.2.7 to calculate the NIT as a percentage of the AAR:%NIT of AAR = (Σ average surface area of NIT in cm²/ Σ average surface area of AAR in cm²)* 100.
   9. Two different investigators should repeat the above method. The acceptable margin of difference is < 10%.
3. Ischemia markers
   1. Use the EDTA plasma samples, which have been previously stored at -80 °C (2.1.9) to measure the level of cardiac troponin-I using a single-plex Luminex-type assay as previously described ¹⁹.

Results

One animal died prematurely before administration of FXIIa inhibitor or control peptide due to a technical error (sudden drop of blood pressure during ischemia time, before addition of test substance). One animal was excluded from the FXIIa inhibitor group because no ischemia/reperfusion injury was observed due to abnormal anatomy of the left anterior descending artery (LAD). A large part of the left ventricle, including the apex, was perfused by the circumflex artery in this animal. The ...

Discussion

Myocardial I/R injury has a significant effect on the final infarct size which is directly translated into the patient's prognosis after acute myocardial infarction³. Understanding the pathophysiology of myocardial I/R injury is the first step to reduce or prevent it. Myocardial I/R injury is an acute condition that occurs directly after reperfusion of the occluded vessels. I/R injury leads to activation of the innate immune response and cellular damage occurs at the site of reperfusion and th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ABL 90 Flex, blood gas analyser	Radiometer	-	Blood gas analysis (BGA)
ACT Plus	Medtronic	-	Activated clotting time
Atropin	Sintetica	-	Atropinum Sulfas, 0,5mg/ml
Balance (20-500 Kg)	NAGATA Scale, Tiwan	HTB/HTR	Alternative products can be used
Balance (21-4200 g)	Mettler toledo, Switzerland	MS4002SDR	Alternative products can be used
Blood collection tubes: EDTA, citrate and serum	S-Monovette, Nuembrecht, Germany	05.1167.001, 05.1071.001 and 05.1557.001 respectively	Alternative products can be used
BV Pulsera mobile C-arm	Philips	-	Alternative products can be used
Centrifuge	Labcare, UK	ALC PK120R	Alternative products can be used
Defibrillator Lifepak 12	Medtronic	-	Alternative products can be used
Dextran from Leuconostoc mesenteroides	Sigma-Aldrich, Germany	D3759	Average M.wt 48000-90000
Digital single lens reflex camera	Sony, Thailand	DSLR-A500/A550	Alternative products can be used
Dissecting forceps			Alternative products can be used
EMPIRA RX PCI dilatation catheter	Cordis, Johnson&Johnson, USA	85R15300S	Diameter 3 mm, length 15 mm, Alternative products can be used
EMPIRA RX PCI dilatation catheter	Cordis, Johnson&Johnson, USA	85R15350S	Diameter 3.5 mm, length 15 mm, Alternative products can be used
Evans Blue	Sigma-Aldrich, Germany	E2129	Toxic
Fabius GS premium respirator	Dräger, Lübeck, Germany	-	Anesthesia work station, Alternative products can be used
Fentanyl	Inselspital ISPI	-	Fentanyl 2500mcg/50ml
Formaldehyde	Pathology Institute, Bern University	SI148701	Alternative products can be used
FXIIa inhibitor	Provided by Prof. Christian Heinis' laboratory in EPFL		Novel bicyclic peptide
Galeo, coronary guidewire	Biotronik, Germany	125497	Alternative products can be used
Guidance catheter	Boston Scientific, Florida, USA	34356-06	6F (100 cm, EB3.75). Alternative products can be used
Heparin Sodium	Drossapharm, Basel, Switzerland	-	Liquemin, 25000 U.I./5 ml
High end electrosurgery	BOWA, Germany	ARC 400	Electrical source for blood suction. Alternative products can be used
Hydro-Guardmini breathing filter	Intersurgical, Lithuania	1745000	Filters
Image J	National Institute of Health, USA	1.47v	Alternative products can be used
Inflation device, Atrion QL2530	Atrion medical product, Alabama, USA	96402	Alternative products can be used
IntelliVue MP 70	Philips, Boeblingen, Germany	-	Monitor (ECG, heart rate, blood presure and body temperature). Alternative products can be used
KCl	Sintetica SA	-	Potassium chloride 15%
Ketamine	Vetoquinol	-	Narketan, 1ml/100mg
LR-ACT	Medtronic	402-01	ACT special syringes
Midazolam	Roche	-	Dormicum, 5mg/ml
Monopolar scalpel			Alternative products can be used
Needle holder			Alternative products can be used
High resolution camera, PathStand Macro Imaging Stand for Grossing	Spotimaging, USA		1080 p HD resolution. Alternative products can be used
PBS	In-house preparation	-	Alternative products can be used
Peripheral venous cannula, 18 G			Alternative products can be used
PowerLab 4/35 data acquisition system	Adinstruments, Spechbach, Germany	-
Rotamax120T	Heidolph, Germany	544-41200-00	Shaker. Alternative can be used
Rüschelit-Super Safety Clear Tube	Teleflex, Dublin, Irland	112480	Air way tubing, Alternative products can be used
Safe Pico Aspirator Syringes	Radiometer	956-622	BGA special syringes
Saline	Sintetica Bioren	-	NaCl 0,9%. Alternative products can be used
Sevorane 1.5%	AbbVie AG	-	Sevorane 250ml 100%
Sheath	Cordis, Johnson&Johnson, USA	504-607 A	AVANTI + / 7 F, Alternative products can be used
Space infusion pump	B.Braun Medical AG, Germany	-	For infusion of fentanyl. Alternative products can be used
SPR-350 (Millar catheter)	Adinstruments, Texas, USA	840-8166	MIKRO-TIP, 5F, 120 cm
Sternotomy saw			Alternative products can be used
Sutures	ETHICON, Johnson&Johnson, USA	Y3110H	Monocryl 3-0 SH-1 Plus. Alternative products can be used
Syringes (20, 10 and 5 mL)	CODAN,Baar, Switzerland	62.7602, 62.6616, 62.5607 respectively	Used to inject anesthetic materials intramuscularly or directly into the central venous line. Also to inject heparin or FXIIa or the respective control. Alternative products can be used
Thorax spreader			Alternative products can be used
Tissue-tek	SAKURA, Netherlands	4583	O.C.T. compound
Vascular forceps			Alternative products can be used
Xenetix 300 contrast media	Guerbet, Zürich, Switzerland	-	Lobitridol, 300 mg iodide/ml
Xylazine	Vetoquinol	-	Xylapan, 20mg/1 mL
2,3,5-Triphenyltetrazolium choride	Sigma-Aldrich, Austria	T8877
500 μL tubes (eppendorf)	Trefflab, Switzerland	96.08185.9.03	Alternative products can be used