The authors wish to acknowledge Professor Christian Heinis for providing the FXIIa inhibitor and the respective control. We also gratefully acknowledge Olgica Beslac, Dr. Daniel Mettler and Kay Nettelbeck from the Experimental Surgery Unit, Department for Biomedical Research, University of Bern for technical support. Celine Guillod and Matthias Rausch from the Department for Diagnostic, Interventional and Pediatric Radiology, Bern University Hospital, Inselspital provided support with the X-ray equipment and techniques.This project was funded by the Swiss National Science Foundation, project no. 320030_156193.&#160;We also would like to thank Mr. Reto Haenni from communication and marketing, Bern University Hospital, Inselspital for video recording our experiment.

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 &#177;&#160;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)
<ol>
	<li>Anesthesia and preparation of the animal:
	<ol>
		<li>Fast the animals for 12 h before starting the experiment.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Depth of anesthesia is&#160;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.&#160;Core temperature was continuously monitored and maintenance of normothermia (38-38.5 &#176;C) was ensured with passive warming (isolating blankets and warm bottles).</li>
		<li>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.</li>
		<li>Administer a bolus dose of 250 &#181;g Fentanyl analgesic through the central venous line followed by 250 &#181;g/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.&#160;</li>
		<li>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 &#176;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.</li>
		<li>Aliquot 200 &#181;L of plasma or serum into 500 &#181;L tubes and store all samples at -80 &#176;C for further analysis.</li>
		<li>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&#39;s instructions.</li>
		<li>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&#39;s instructions. See Figure 2 for time points.</li>
		<li>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.</li>
		<li>Monitor ACT every 30-45 min as mentioned in 2.1.11. Inject 2500 IU unfractionated heparin intravenously if ACT is &#60; 180 s.</li>
	</ol>
	</li>
	<li>Myocardial infarction experiment
	<ol>
		<li>Use a standard C arm fluoroscopy equipment (coronary angiography program, angle 0&#176;, 12 frames per second, ~70 kV) - or alternatively a dedicated angiography system - to perform the coronary intervention.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Monitor the animal closely to immediately detect and treat (2.2.10) cardiac arrhythmias. Extrasystoles usually occur and increase in frequency (&#62;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.</li>
		<li>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).</li>
		<li>Repeat if necessary and increase the energy to 175 J after the 3rd shock. Use higher energy settings for heavier animals.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.&#160;Death was confirmed by the absence of ECG signals and pulse waves.</li>
	</ol>
	</li>
</ol>
3. Sampling techniques
<ol>
	<li>Extracting, dissecting and sampling the heart (figure 4)
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Rinse the heart inside and out with room temperature saline. Weigh the whole heart.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Photograph the slices of the heart using a digital single lens reflex camera.</li>
		<li>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.</li>
		<li>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.</li>
		<li>First analyze the Evans Blue negative sections (the ischemic area at risk, AAR). Weigh them all and put them all into a plastic container.</li>
		<li>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 &#176;C while gently shaking.</li>
		<li>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 &#176;C for further analysis. Transfer the rest into 4% formaldehyde solution and store at room temperature for histology sections.</li>
		<li>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 &#176;C for further analysis. These samples should have the same weight.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Calculation of the AAR and the infarct size
	<ol>
		<li>%AAR of left ventricle = (weight of AAR in g/ weight of left ventricle in g)* 100.</li>
		<li>Use ImageJ software to calculate the total surface area of both the AAR and NIT (both sides of each piece) based on the photographs.</li>
		<li>Adjust the scale bar by selecting the scale bar length using the straight line from the angle tool. Choose from the menu Analyze &#62; Set Scale and insert the known distance and unit of the scale bar. Choose &#34;global&#34; so the same scale will be applied to all pictures.</li>
		<li>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).</li>
		<li>Set the measurement by choosing &#34;area&#34; and &#34;display label&#34; from Analyze &#62; Set Measurements menu. Measure the surface area from Analyze &#62; Measure.</li>
		<li>Repeat step 3.2.5 to measure NIT (mark only the non-stained tissue). Note: Don&#39;t include the fatty tissue (figure 6-D) in the NIT calculation. Repeat the step on the other side of the tissue.</li>
		<li>Calculate the average AAR and NIT for each piece of tissue.</li>
		<li>Use the values obtained from 3.2.7 to calculate the NIT as a percentage of the AAR:%NIT of AAR = (&#931; average surface area of NIT in cm2/ &#931; average surface area of AAR in cm2)* 100.</li>
		<li>Two different investigators should repeat the above method. The acceptable margin of difference is &#60; 10%.</li>
	</ol>
	</li>
	<li>Ischemia markers
	<ol>
		<li>Use the EDTA plasma samples, which have been previously stored at -80 &#176;C (2.1.9) to measure the level of cardiac troponin-I using a single-plex Luminex-type assay as previously described 19.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

Myocardial I/R injury has a significant effect on the final infarct size which is directly translated into the patient&#39;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...

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.

<table><tbody><tr><td>ABL 90 Flex, blood gas analyser</td><td>Radiometer</td><td>-</td><td>Blood gas analysis (BGA)</td></tr><tr><td>ACT Plus</td><td>Medtronic</td><td>-</td><td>Activated clotting time</td></tr><tr><td>Atropin</td><td>Sintetica</td><td>-</td><td>Atropinum Sulfas, 0,5mg/ml</td></tr><tr><td>Balance (20-500 Kg)</td><td>NAGATA Scale, Tiwan</td><td>HTB/HTR</td><td>Alternative products can be used</td></tr><tr><td>Balance (21-4200 g)</td><td>Mettler toledo, Switzerland</td><td>MS4002SDR</td><td>Alternative products can be used</td></tr><tr><td>Blood collection tubes: EDTA, citrate and serum</td><td>S-Monovette, Nuembrecht, Germany</td><td>05.1167.001,&lt;br/&gt; 05.1071.001&lt;br/&gt; and&lt;br/&gt; 05.1557.001 respectively</td><td>Alternative products can be used</td></tr><tr><td>BV Pulsera mobile C-arm</td><td>Philips</td><td>-</td><td>Alternative products can be used</td></tr><tr><td>Centrifuge</td><td>Labcare, UK</td><td>ALC PK120R</td><td>Alternative products can be used</td></tr><tr><td>Defibrillator Lifepak 12</td><td>Medtronic</td><td>-</td><td>Alternative products can be used</td></tr><tr><td>Dextran from Leuconostoc mesenteroides</td><td>Sigma-Aldrich, Germany</td><td>D3759</td><td>Average M.wt 48000-90000</td></tr><tr><td>Digital single lens reflex camera</td><td>Sony, Thailand</td><td>DSLR-A500/A550</td><td>Alternative products can be used</td></tr><tr><td>Dissecting forceps</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>EMPIRA RX PCI dilatation catheter</td><td>Cordis, Johnson&amp;amp;Johnson, USA</td><td>85R15300S</td><td>Diameter 3 mm, length 15 mm, Alternative products can be used</td></tr><tr><td>EMPIRA RX PCI dilatation catheter</td><td>Cordis, Johnson&amp;amp;Johnson, USA</td><td>85R15350S</td><td>Diameter 3.5 mm, length 15 mm, Alternative products can be used</td></tr><tr><td>Evans Blue</td><td>Sigma-Aldrich, Germany</td><td>E2129</td><td>Toxic</td></tr><tr><td>Fabius GS premium respirator</td><td>Dr&amp;auml;ger, L&amp;uuml;beck, Germany</td><td>-</td><td>Anesthesia work station, Alternative products can be used</td></tr><tr><td>Fentanyl</td><td>Inselspital ISPI</td><td>-</td><td>Fentanyl 2500mcg/50ml</td></tr><tr><td>Formaldehyde</td><td>Pathology Institute, Bern University</td><td>SI148701</td><td>Alternative products can be used</td></tr><tr><td>FXIIa inhibitor</td><td>Provided by Prof. Christian Heinis&#039; laboratory in EPFL</td><td></td><td>Novel bicyclic peptide</td></tr><tr><td>Galeo, coronary guidewire</td><td>Biotronik, Germany</td><td>125497</td><td>Alternative products can be used</td></tr><tr><td>Guidance catheter</td><td>Boston Scientific, Florida, USA</td><td>34356-06</td><td>6F (100 cm, EB3.75). Alternative products can be used</td></tr><tr><td>Heparin Sodium</td><td>Drossapharm, Basel, Switzerland</td><td>-</td><td>Liquemin, 25000 U.I./5 ml</td></tr><tr><td>High end electrosurgery</td><td>BOWA, Germany</td><td>ARC 400</td><td>Electrical source for blood suction. Alternative products can be used</td></tr><tr><td>Hydro-Guardmini breathing filter</td><td>Intersurgical, Lithuania</td><td>1745000</td><td>Filters</td></tr><tr><td>Image J</td><td>National Institute of Health, USA</td><td>1.47v</td><td>Alternative products can be used</td></tr><tr><td>Inflation device, Atrion QL2530</td><td>Atrion medical product, Alabama, USA</td><td>96402</td><td>Alternative products can be used</td></tr><tr><td>IntelliVue MP 70</td><td>Philips, Boeblingen, Germany</td><td>-</td><td>Monitor (ECG, heart rate, blood presure and body temperature). Alternative products can be used</td></tr><tr><td>KCl</td><td>Sintetica SA</td><td>-</td><td>Potassium chloride 15%</td></tr><tr><td>Ketamine</td><td>Vetoquinol</td><td>-</td><td>Narketan, 1ml/100mg</td></tr><tr><td>LR-ACT</td><td>Medtronic</td><td>402-01</td><td>ACT special syringes</td></tr><tr><td>Midazolam</td><td>Roche</td><td>-</td><td>Dormicum, 5mg/ml</td></tr><tr><td>Monopolar scalpel</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>Needle holder</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>High resolution camera, PathStand Macro Imaging Stand for Grossing</td><td>Spotimaging, USA</td><td></td><td>1080 p HD resolution. Alternative products can be used</td></tr><tr><td>PBS</td><td>In-house preparation</td><td>-</td><td>Alternative products can be used</td></tr><tr><td>Peripheral venous cannula, 18 G</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>PowerLab 4/35 data acquisition system</td><td>Adinstruments, Spechbach, Germany</td><td>-</td><td></td></tr><tr><td>Rotamax120T</td><td>Heidolph, Germany</td><td>544-41200-00</td><td>Shaker. Alternative can be used</td></tr><tr><td>R&amp;uuml;schelit-Super Safety Clear Tube</td><td>Teleflex, Dublin, Irland</td><td>112480</td><td>Air way tubing, Alternative products can be used</td></tr><tr><td>Safe Pico Aspirator Syringes</td><td>Radiometer</td><td>956-622</td><td>BGA special syringes</td></tr><tr><td>Saline</td><td>Sintetica Bioren</td><td>-</td><td>NaCl 0,9%. Alternative products can be used</td></tr><tr><td>Sevorane 1.5%</td><td>AbbVie AG</td><td>-</td><td>Sevorane 250ml 100%</td></tr><tr><td>Sheath</td><td>Cordis, Johnson&amp;amp;Johnson, USA</td><td>504-607 A</td><td>AVANTI + / 7 F, Alternative products can be used</td></tr><tr><td>Space infusion pump</td><td>B.Braun Medical AG, Germany</td><td>-</td><td>For infusion of fentanyl. Alternative products can be used</td></tr><tr><td>SPR-350 (Millar catheter)</td><td>Adinstruments, Texas, USA</td><td>840-8166</td><td>MIKRO-TIP, 5F, 120 cm</td></tr><tr><td>Sternotomy saw</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>Sutures</td><td>ETHICON, Johnson&amp;amp;Johnson, USA</td><td>Y3110H</td><td>Monocryl 3-0 SH-1 Plus. Alternative products can be used</td></tr><tr><td>Syringes (20, 10 and 5 mL)</td><td>CODAN,Baar, Switzerland</td><td>62.7602,&lt;br/&gt; 62.6616,&lt;br/&gt; 62.5607&lt;br/&gt; respectively</td><td>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</td></tr><tr><td>Thorax spreader</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>Tissue-tek</td><td>SAKURA, Netherlands</td><td>4583</td><td>O.C.T. compound</td></tr><tr><td>Vascular forceps</td><td></td><td></td><td>Alternative products can be used</td></tr><tr><td>Xenetix 300 contrast media</td><td>Guerbet, Z&amp;uuml;rich, Switzerland</td><td>-</td><td>Lobitridol, 300 mg iodide/ml</td></tr><tr><td>Xylazine</td><td>Vetoquinol</td><td>-</td><td>Xylapan, 20mg/1 mL</td></tr><tr><td>2,3,5-Triphenyltetrazolium choride</td><td>Sigma-Aldrich, Austria</td><td>T8877</td><td></td></tr><tr><td>500 &amp;mu;L tubes (eppendorf)</td><td>Trefflab, Switzerland</td><td>96.08185.9.03</td><td>Alternative products can be used</td></tr></tbody></table>

improvement of a closed chest porcine myocardial infarction model by standardization of tissue and blood sampling procedures

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. &#176;&#176;&#176;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.

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 ...

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

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.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

improvement-closed-chest-porcine-myocardial-infarction-model

Research

JoVE Journal

Medicine

10.8K Views.  University of Bern. Hier tonen we een protocol om te standaardiseren bemonsteringsprocedures van een gevestigde varkens model van acuut myocardinfarct teneinde haar translationeel waarde in de kennis van de pathofysiologie van myocardiale ischemie/reperfusie letsel en het testen van nieuwe drugkandidaten.

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

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

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.

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 ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

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

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