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, 
			05.1071.001 
			and 
			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&#38;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&#38;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&#228;ger, L&#252;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&#39; 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&#252;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&#38;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&#38;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, 
			62.6616, 
			62.5607 
			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&#252;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 &#956;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 ...

Watch this Scientific Journal Video about Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures at JoVE.com

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

10.7K Views.  University of Bern. 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.

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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

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 ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

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 ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

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 ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...