The authors have no acknowledgements.

The institutional animal care committee approved all experimental protocols and animals were treated following the &#8220;Guide for the Care and Use of Laboratory Animals&#8221; prepared by the Institute of Laboratory Animal Resources, National Research Council, 1996. Male Yorkshire pigs (40&#8211;50 kg) were used to perform the orthotopic heart transplants (animal size can vary according to the investigators discretion and experimental goals).
1. Donor Procedure
<ol>
	<li>Anesthetic induction and animal preparation:
	<ol>
		<li>Premedicate the animal using an intramuscular injection of Ketamine (20 mg/kg), Midazolam (0.3 mg/kg), and Atropine (0.04 mg/kg). Perform anesthetic induction and maintenance using inhalational Isoflurane (end tidal concentration: 1%&#8211;3%) + 3 L/min O2 via face mask.</li>
		<li>Confirm anesthetic adequacy by ensuring a relaxed jaw tone and an absence of pain during toe pinch. Anesthetic adequacy needs to follow institutional guidelines.</li>
		<li>Once anesthetic adequacy is confirmed, perform an orotracheal intubation using an endotracheal tube of size 6.5&#8211;8 mm.</li>
		<li>Place an oxygen saturation monitor on the ear or bottom lip for continuous monitoring. Place the cautery electrode pad on the animal&#8217;s back.</li>
		<li>Insert a peripheral intravenous access via ear vein (e.g. 20 g Angiocath). Start a maintenance infusion (e.g. 0.9% NaCl). Administer 2 g of Magnesium Sulfate to prevent arrhythmias.</li>
		<li>Insert a percutaneous central venous sheath introducer using the Seldinger technique into the right jugular vein if right-sided heart catheterization and cardiac output measurements are to be performed. Place the animal in Trendelenburg position to facilitate venous access. Alternatively, this can be done in the left internal jugular vein. If access cannot be established, this step may be performed following midline sternotomy by dissecting the internal jugular vein (left or right) and inserting the sheath directly.</li>
	</ol>
	</li>
	<li>Donor heart procurement:
	<ol>
		<li>Perform a midline sternotomy from the mid cervical region to below the xiphoid process using a cautery pen. Open the sternum with a bone saw. Ensure adequate hemostasis throughout the entire procedure (e.g. cauterize sternum, and/or application of bone wax).</li>
		<li>In the cervical region, retract the sternocleidomastoid muscle to the center and dissect the right carotid artery (alternatively, this can be done with the left carotid artery). Place an arterial access line (e.g. 20 g Angiocath) into the right carotid artery for invasive arterial pressure monitoring.</li>
		<li>Dissect and remove the thymus from over the pericardium. Gently lift the thymus from the pericardium, using the cautery, and dissect the structure from the pericardium. To prevent bleeding, cauterize the small vessels that originate from the aorta and superior vena cava (SVC) to irrigate the thymus.</li>
		<li>Open the pericardium. Dissect the aortopulmonary space using the cautery. For this, have the assistant retract the right ventricular outflow track inferiorly and the pulmonary artery to the left and have the surgeon retract the aorta to the right. Do this carefully to avoid direct lesions to the pulmonary artery.</li>
		<li>Clear the anterior aspect of the ascending aorta from connective tissue. Carefully retract the right ventricular outflow tract and place a purse-string suture using a 4-0 prolene on the proximal ascending aorta adventitia (avoid full thickness sutures). Secure this suture with a tourniquet.</li>
		<li>Administer 30,000 units of heparin (&#62;300 U/Kg) to achieve systemic anticoagulation. Insert the cardioplegia delivery cannula (e.g. DLP aortic root cannula) into the ascending aorta between the purse-string suture previously placed and secure by tightening the tourniquet. Prepare the cardioplegic solution that will be used and connect to the delivery cannula.</li>
		<li>Open the SVC and inferior vena cava (IVC), and the left inferior pulmonary vein to ensure adequate cardiac venting. Place the aortic cross-clamp on the distal ascending aorta (above the cardioplegia cannula). Alternatively, the left ventricle can be vented by opening the left atrial appendage and placing suction.</li>
		<li>Initiate cardioplegia infusion targeting an aortic root pressure of 80 to 100 mmHg. 
		NOTE: The authors applied a model using 1.5 L of a standard extracellular hyperkalemic cardioplegic solution at 4 &#176;C. Different solutions and volumes can be used according to the experimental set-up.</li>
		<li>Place ice slush (0.9% NaCl) in the thoracic cavity and over the organ for cooling following initiation of cardioplegia. After the cardioplegia infusion is finished, proceed with cardiectomy in a traditional manner. Section the aorta and the pulmonary artery after the innominate artery and at the bifurcation, respectively, to ensure sufficient length for implant.</li>
		<li>After removal, place the organ in an organ bag with at least 500 mL of preservation solution (standard extracellular hyperkalemic solution). Place this on ice and keep at 4 &#176;C. This step can be modified according to the experimental design and objective.</li>
	</ol>
	</li>
</ol>
2. Recipient Procedure:
<ol>
	<li>Anesthetic induction and animal preparation:
	<ol>
		<li>Perform anesthesia and monitoring as described in the donor procedure (steps 1.1.1 to 1.1.6).</li>
		<li>After inserting the percutaneous central venous sheath introducer into the right jugular vein, insert a central venous catheter (e.g. double-lumen) into the left jugular vein using the Seldinger technique. Alternatively, this can be done after the midline sternotomy as described above.</li>
	</ol>
	</li>
	<li>Cardiopulmonary bypass (CPB):
	<ol>
		<li>Perform the midline sternotomy and expose the heart and great vessels as described in the donor procedure (steps 1.2.1 to 1.2.4).</li>
		<li>Dissect between the SVC and the innominate artery, and the IVC and pericardium using the Metzenbaum and &#8220;right-angle&#8221; forceps. Encircle the SVC and IVC using an umbilical tape (alternatively a simple O silk suture can be used). Secure each tape/suture with a tourniquet.</li>
		<li>Place 2 concentric purse-string sutures using a 4-0 prolene suture on the distal ascending aorta adventitia (avoid full thickness sutures). Place purse-string sutures using 4-0 prolene on the IVC and the SVC at the level of the pericardial reflection. Secure these sutures with tourniquets.</li>
		<li>During CPB preparation, have an assistant setup and prime the system according to the investigators&#8217; and experimental needs. The current procedure uses the same setup used as the institution&#8217;s Cardiovascular Surgery Division and employs the help of a trained perfusionist. The bypass system is primed with 2 litres of crystalloid solution (e.g. Plasmalyte) with 500 mg of Solumedrol.</li>
		<li>Administer 30,000 units of heparin (&#62;300 U/Kg) to achieve systemic anticoagulation. Activated clotting time (ACT) should be above 300 s, if the test is available.</li>
		<li>Cannulate the aorta with a 17 to 21 F arterial cannula. Use an extracorporeal membrane oxygenator (ECMO) cannula (e.g. EOPA arterial cannula) inserted using the Seldinger technique to facilitate this step and avoid blood loss. Alternatively, a standard bypass arterial cannula can be used.</li>
		<li>Connect the cannula to the arterial line of the bypass circuit using a 3/8-3/8 connector. Ensure complete deairing to avoid air embolism.</li>
		<li>Perform a bicaval cannulation. For this, cannulate the SVC and then the IVC using 24 to 28 F right angled single-stage venous cannula (e.g. DLP single lumen angled venous cannula).
		<ol>
			<li>First, make a small incision (5 mm) at the center of the purse-string suture. Dilate the incision with a small angled instrument (e.g. right angle or a snap). Insert the cannula directing the angle superiorly in the SVC and inferiorly in the IVC (away from the heart). Secure by tightening the tourniquet holding the purse-string suture.</li>
			<li>Between each step, cover the incision with a finger to avoid excessive bleeding. Connect the cannulas to the venous line of the bypass circuit using a 3/8-3/8-1/2 Y connector. Ensure deairing to avoid an airlock in the system.</li>
		</ol>
		</li>
		<li>Initiate CPB. Adjust flowrates to maintain an arterial pressure above 50 mmHg (approximately 4 L/min). Maintain normothermia throughout the procedure. 
		&#8203;NOTE: These settings can be modified according to the experimental design. Vasoactive medications should only be administered during CPB if needed to aid in pressure regulation (e.g. epinephrine infusion).</li>
	</ol>
	</li>
	<li>Donor heart implantation:
	<ol>
		<li>After initiation of CPB, open the left pleura and retract the native heart to the right. Dissect and encircle the left hemiazygous vein using a sharp dissection instrument (e.g. Metzenbaum) and a right-angle, respectively. Ligate distally with an O Silk tie. Only one ligature is needed as the native heart will be removed.</li>
		<li>Cross-clamp the recipient aorta proximally to the arterial cannula. Snare both vena cava with the tourniquets previously placed using O silk ties. Remove the recipient&#8217;s native heart. Alternatively, the heart can be arrested using standard hypothermic blood-based cardioplegia.</li>
		<li>During cardiectomy, make sure to maintain large cuffs in the recipient to facilitate donor heart implant. For this, section the aorta and pulmonary artery proximally, close to their roots. Similarly, the left and right atriums must be kept with large cuffs. Leave the right and left atrial appendages in the recipient cuffs, which may be needed during the anastomoses.</li>
		<li>Prepare the donor heart for implant.
		<ol>
			<li>Dissect the pulmonary artery off the left atrium and separate the pulmonary artery completely from the aorta. Leave at least 2-3 cm of each vessel to be trimmed during implant as needed. Ligate both vena cava with an O silk tie. Unite all pulmonary veins, creating a single left atrial cuff to be anastomosed.</li>
			<li>Compare left atrial cuff sizes (donor and recipient) and trim each as needed to become similar sizes. The recipient&#8217;s left atrial appendage can be shortened, or the donor&#8217;s left atrial roof and appendage can be opened for this.</li>
		</ol>
		</li>
		<li>Deliver the first cardioplegia dose to the donor heart using the previously placed cardioplegia cannula as described in step 1.2.6. Cardioplegic protective solution consists of 500 mL of a 2:1 mixture of blood:crystalloid containing 24 mEq of potassium and delivered at 10&#176;C. Achieve the desired potassium concentration by adding potassium chloride to the cardioplegic mixture.</li>
		<li>After the completion of each anastomosis, deliver an additional dose of 300 mL of cardioplegia at 10 &#176;C containing 8 mEq of potassium.</li>
		<li>Following all anastomoses and before removal of the aortic cross-clamp, administer an additional dose of 500 mL of warm (37 &#176;C) blood cardioplegia with 8 mEq of potassium.</li>
		<li>Remove the donor heart from storage and implant with the standard biatrial anastomotic technique in the following sequence: left atrium, right atrium, pulmonary artery, and aorta. Use a 4-0 prolene suture with a SH needle for the left and right atrium, and the aorta and a 5-0 prolene suture with a BB needle for the pulmonary artery.
		<ol>
			<li>Left atrium: place a 4-0 prolene suture at the junction between the left atrium and the IVC (right inferior margin) and another at 180&#176; from the first, connecting the donor and recipient cuffs. Complete the posterior wall anastomosis. Complete the anterior wall anastomosis. This is performed from the superior suture to the inferior suture.</li>
			<li>Right atrium: open the donor right atrium from the appendage towards the IVC, creating a donor cuff that matches the recipient cuff size. Starting at the inferior angle (junction between IVC and right atrium), complete the interior wall anastomosis and then the lateral wall.</li>
			<li>Pulmonary artery: trim the edges of both recipient and donor pulmonary arteries to create matching sizes. Place a 5-0 prolene suture at the left lateral wall uniting donor and recipient vessels and another at the right lateral edge. Complete the inferior wall anastomosis and then the anterior wall anastomosis.</li>
			<li>Aorta: trim as described with the pulmonary artery. Place a suture on the left lateral wall connecting donor and recipient vessels. Complete the inferior wall and then the anterior wall anastomosis.</li>
			<li>Perform single layered anastomoses, except for the pulmonary artery, where a double layered anastomosis is required. The porcine tissue is extremely fragile and should be handled carefully to avoid tears. Importantly, the pulmonary artery anastomosis is the most delicate step of the implant and must be done with extreme care. The implant technique can be modified according to the surgeons&#8217; preference and the experimental design.</li>
		</ol>
		</li>
		<li>Following completion of all anastomoses and warm cardioplegia dose delivery as described above, remove the aortic cross-clamp. Inspect all anastomoses for sites of bleeding, they should be corrected at this point.</li>
		<li>Reperfuse the donor heart for 60 min on CPB. Ventricular arrhythmias can be treated with internal defibrillation (20-50 J). If required, ventricular pacing can be used to maintain a heart rate of 100 beats per min. Antiarrhythmic drugs (e.g. Amiodarone, Lidocaine, or Magnesium Sulphate) can be used at the investigators discretion, if required.</li>
		<li>After 60 min of reperfusion, administer 1 g of calcium chloride. Initiate weaning from CPB by decreasing the flow to half, then one-quarter and then off. The central venous line can be used to monitor central venous pressure, targeting 10 mmHg. Initiate infusion of vasoactive and inotropic medications (e.g. dobutamine, epinephrine, norepinephrine, and vasopressin) according to the experimental design or the investigators&#8217; discretion.</li>
		<li>Weaning is deemed successful if the animal maintains a systolic arterial pressure above 60 mmHg for over 30 min after discontinuation of CPB. As this is not a survival model, do not reverse the heparin; continuous bleeding can occur from needle holes and dissected structures (e.g sternum). Donor hearts respond well to small and repeated doses of volume replacement using the CPB system. Additionally, the porcine model responds well to dobutamine. 
		NOTE: The recipient management should be tailored to the investigators&#8217; experience and the experimental design. A cardiac anesthesiologist may aid in this manner.</li>
	</ol>
	</li>
</ol>
3. Graft Assessment:
<ol>
	<li>Functional assessment:
	<ol>
		<li>This large animal model has the advantage of having an open chest approach at all times, which facilitates direct functional assessment. To measure cardiac contractility, use pressure-volume (PV) loop analyses, echocardiography, and/or right-sided catheterization.</li>
		<li>Pressure-volume loops10: Place an umbilical tape around the IVC, and insert a PV conductance catheter into the left ventricle through a small apical ventriculotomy to permit continuous measurements of left ventricular PV relations. Obtain steady-state recordings to generate volume-dependent parameters (e.g. developed pressure and stroke work) and then obtain occlusion recordings in triplicate by occluding the IVC to generate volume-independent parameters (e.g. preload recruitable stroke work).</li>
		<li>Echocardiography: have cardiac anesthesiologists obtain epicardial images using a standard transesophageal probe.</li>
		<li>Right-sided catheterization: Insert a Swans-Ganz catheter through the venous sheath placed at the beginning of the procedure and guid towards the pulmonary artery. This enables the measurement of central venous pressure, right ventricular pressure, pulmonary artery pressures, pulmonary capillary wedge pressure, and cardiac output using the thermodilution technique.</li>
		<li>Perform contractile evaluation at baseline and following 2 and 3 h post-reperfusion of the donor heart in the recipient. This can be modified by the investigators according the experimental design.</li>
	</ol>
	</li>
	<li>Metabolic assessment:
	<ol>
		<li>For metabolic assessments, collect arterial and venous (alternatively: mixed venous) blood samples and store the plasma for subsequent analyses. Real-time blood gas analyses and lactate levels should also be obtained.</li>
		<li>Collect these samples at baseline in the donor, before procurement in the donor, at baseline in the recipient, and at 15, 30 and 60 min of reperfusion of the donor heart (after removing cross-clamp). This can be modified according to the experimental design.</li>
	</ol>
	</li>
	<li>Experiment termination and euthanasia:
	<ol>
		<li>Once all assessments are finished, exsanguinate the recipient animal into the venous reservoir of the CPB circuit by opening the venous line clamp. Alternatively, exsangination can be achieved by harvesting the cardiac alograft to collect samples (i.e. myocardial biopsies).</li>
	</ol>
	</li>
</ol>

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G., G&#243;mez Bueno, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+graft+failure+after+heart+transplantation:+characteristics+in+a+contemporary+cohort+and+performance+of+the+RADIAL+risk+score.">Primary graft failure after heart transplantation: characteristics in a contemporary cohort and performance of the RADIAL risk score.</a> The Journal of Heart and Lung Transplantation. 32 (12), 1187-1195 (2013).</li><li>Fedak, P. W. 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D., Boylen, P., Cohen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+blood+perfusion+improves+myocardial+recovery+after+heart+transplantation.">Donor blood perfusion improves myocardial recovery after heart transplantation.</a> Journal of Heart and Lung Transplantation. 16 (6), 667-673 (1997).</li><li>Wicomb, W. N., Cooper, D. K., Barnard, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-four-hour+preservation+of+the+pig+heart+by+a+portable+hypothermic+perfusion+system.">Twenty-four-hour preservation of the pig heart by a portable hypothermic perfusion system.</a> Transplantation. 34 (5), 246-250 (1982).</li><li>Badiwala, M. 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(102), e52907 (2015).</li><li>Fukunaga, N., Bissoondath, V., Rao, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submandibular+Gland-preserving+Technique+for+Heterotopic+Cervical+Heart+Transplantation+in+Mice.">Submandibular Gland-preserving Technique for Heterotopic Cervical Heart Transplantation in Mice.</a> Transplantation. 1, (2018).</li><li>Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Heterotopic+Abdominal+Heart+Transplant+Model.">Mouse Heterotopic Abdominal Heart Transplant Model.</a> Rodent Transplant Medicine. , 107-118 (2014).</li><li>Robinson, N., Souslian, L., Gallegos, R. P., Rivard, A. L., Dalmasso, A. P., Bianco, R. 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The authors have nothing to disclose.

This manuscript describes a large-animal pre-clinical model of orthotopic heart transplantation. Various small animal models of heterotopic heart transplantation have been successfully used to study the effects of novel treatments to improve organ preservation and decrease ischemia-reperfusion injury11,12,13. Additionally, small animal models permit survival models and long-term follow-up to investigate the development of reject...

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure1. Although ischemic times of up to four hours are tolerated adequately, an ischemic time of greater than six hours is associated with inferior outcomes2. Primary graft dysfunction remains the principal cause of early morbidity and mortality following transplantation2,3. The causes of primary graft dysfunction are multifactorial and include the use of marginal organs, recipient pulmonary vascular disease, hyperacute rejection, and ischemia-reperfusion injury sustained at the time of transplantation3.
Multiple studies have investigated novel methods for donor heart preservation to reduce the incidence of primary graft dysfunction4,5,6,7. It is common practice to assess new techniques and treatments in murine models of ischemia-reperfusion injury or heterotopic heart transplantation. Additionally, small animal models permit survival models and long-term follow-up to investigate the development of rejection and cardiac allograft vasculopathy11,12,13. However, most of these strategies fail initial clinical pilot trials or never reach this stage. It is of paramount importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings.
The porcine heart is often considered the most anatomically similar to the human heart when using large-animal models. As such, it is an ideal platform to perform cardiac surgical research. However, there are several important factors to consider when using a porcine model. First, the tissue is typically described as fragile and friable, especially in the right atrium and the pulmonary artery, being prone to tears14. Additionally, the pig heart is considered sensitive to manipulation and prone to arrhythmias, which is why one should routinely administer an anti-arrythmetic to each animal at the beginning of the experiment. An important anatomical difference between the porcine model and clinical heart transplantation is the left hemiazygous vein in the swine which drains directly into the coronary sinus. This has to be ligated during the recipient procedure to avoid continuous bleeding. Finally, the porcine model is very sensitive to ischemia, but it is still appropriate for acute studies in heart transplantation15.
This manuscript describes a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies5,6,8,9.

<table><tbody><tr><td>Amiodarone</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Angiocath 20G</td><td>BD</td><td>381704</td><td></td></tr><tr><td>Calcium Chloride 1g/10ml</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Cardioplegia solution</td><td></td><td></td><td>This should be chosen at the investigators discretion.&amp;nbsp;</td></tr><tr><td>Cautery Pencil</td><td>Covidien</td><td>E2515H</td><td></td></tr><tr><td>Central Venous Catheter double-lumen</td><td>Cook Medical</td><td>C-UDLM-501J-LSC</td><td></td></tr><tr><td>CPB pack</td><td>Medtronic</td><td></td><td>Custom-made cardiopulmonary bypass perfusion circuit.</td></tr><tr><td>D5W 5% 250ml</td><td>Baxter</td><td>JB1064</td><td></td></tr><tr><td>DLP Aortic Root Cannula/stabber</td><td>Medtronic</td><td>12218</td><td></td></tr><tr><td>DLP single-lumen venous cannula (24F or 28F)</td><td></td><td></td><td>This should be chosen at the investigators discretion.&amp;nbsp;</td></tr><tr><td>Dobutamine</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Electrode Polyhesive</td><td>Covidien</td><td>E7507</td><td></td></tr><tr><td>EOPA arterial cannula (17F or 21F)</td><td></td><td></td><td>This should be chosen at the investigators discretion.&amp;nbsp;</td></tr><tr><td>Epinephrine</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Eppendorf Tubes, 1.5 mL</td><td>Sarstedt&amp;nbsp;</td><td>72.690.001</td><td></td></tr><tr><td>Gloves, nitrile, medium</td><td>Fischer</td><td>27-058-52</td><td></td></tr><tr><td>Heparin 1000 IU/ml</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Ketalean (Ketamine) inj. 100mg/ml, 50ml/vial</td><td>Health Canada</td><td></td><td>Requires health canada approval</td></tr><tr><td>Lidocaine/Xylocaine 1%</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Magnesium Sulfate 5g/10ml</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Midazolam inj. USP 5mg/ml vial/10ml</td><td>Health Canada</td><td></td><td>Requires Health canada approval</td></tr><tr><td>MPS Quest delivery disposable pack</td><td>Quest medical</td><td>5001102-AS</td><td></td></tr><tr><td>NACL 0.9% 1L</td><td>Baxter</td><td>JB1324</td><td></td></tr><tr><td>Organ Bag</td><td>CardioMed</td><td>2990</td><td></td></tr><tr><td>Pipette Tips, 1 mL</td><td>Fisherbrand</td><td>02-707-405</td><td></td></tr><tr><td>Propofol 1mg/ml</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Rocuronium</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Set Admin Prim NF PB W/Checkvalve</td><td>Smith Medical</td><td>21-0442-25</td><td>Intravenous infusion pump line. Researchers should choose infusion lines compatible with the infusion pump available at their facilities</td></tr><tr><td>Set Intro Sheath 8.5FRx 10CM</td><td>Arrow</td><td>SI-09880</td><td></td></tr><tr><td>Sofsilk 0 wax coated</td><td>Covidien</td><td>S316</td><td></td></tr><tr><td>Solumedrol 500mg/5ml</td><td></td><td></td><td>Purchased from institutional pharmacy</td></tr><tr><td>Suction tip</td><td>Covidien</td><td>8888501023</td><td></td></tr><tr><td>Suction Tubing 1/4&quot; x 120&quot;</td><td>Med-Rx</td><td>70-8120</td><td></td></tr><tr><td>Suture 5.0 Prolene BB</td><td>Ethicon</td><td>8580H</td><td></td></tr><tr><td>Suture Prolene Blum 4-0 SH 36</td><td>Ethicon</td><td>8521H</td><td></td></tr><tr><td>Sutures 2.0 Prolene Blu M SH</td><td>Ethicon</td><td>8523H</td><td></td></tr><tr><td>Sutures BB 4.0 Prolene&amp;nbsp;</td><td>Ethicon</td><td>8881H</td><td></td></tr><tr><td>Tracheal Tube, 6.5mm</td><td>Mallinckrodt</td><td>86449</td><td></td></tr></tbody></table>

a pre-clinical porcine model of orthotopic heart transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

This pre-clinical model has been used successfully since 19945,6,8,9. Table 1 demonstrates representative results from pressure-volume relationships and echocardiographic parameters taken at baseline, and 3 h post-transplantation in a set of 5 experiments. Although we see a decrease in myocadial contractility following transplantation, this was not statistically significant.</p...

Watch this Scientific Journal Video about A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation at JoVE.com

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

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JoVE Journal

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9.5K Views.  University Health Network. Het is belangrijk om nieuwe strategieën voor het behoud van het hart te evalueren met behulp van klinisch relevant grootdiermodel voor een betrouwbare klinische vertaling. Het porcine hart model voor onderzoek in hartchirurgie is een zeer nuttig, omdat de menselijke en varkensharten delen veel gelijkenissen in termen van anatomie, fysiologie, zelfs grootte, dus het is een bijna ideaal platform voor onderzoek. Laat een assistent na het openen van het hartzakje van een verdoofd donorvarken de ...

Video: A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Immunology and Infection

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

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 ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Porcine Heart Excision: A Protocol to Extract Porcine Heart for Cardiac Transplant

Source: Hatami, S., et al. <a href="https://www.jove.com/v/58430">Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism.</a> J. Vis. Exp. (2019).
In this video, we describe a step-by-step protocol to extract the heart from a porcine model for cardiac transplantation.

Source: Hatami, S., et al. Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism. J. Vis. Exp. (2019).
In ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

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