We would like to thank Melanie Borie, Caroline Landry, Henry Aceros and Ahmed Menaouar for their precious help and support.

The institutional animal care and use committee of the Centre de Recherche du Centre Hospitalier de l&#39;Universit&#233; de Montr&#233;al (CRCHUM) approved all experimental protocols, and animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals. For this protocol, 3-4-month-old Large White male or female pigs weighing 50-60 kg was used. Animal size can vary according to the investigators&#39; experimental goals.
1. Animal preparation and anesthetic induction
<ol>
	<li>Premedicate the animal using an intramuscular injection of atropine (0.04 mg/kg), midazolam (0.3 mg/kg), and ketamine (20 mg/kg). Administer isoflurane at an end tidal concentration of 1%-3% in 3 L/min O2, using a face mask for anesthesia induction and maintenance.</li>
	<li>Confirm adequate anesthesia by testing for a relaxed jaw tone, and absence of pain using toe pinch response. Ensure that the animal is placed in dorsal decubitus position and maintained in normothermia using a warming blanket.</li>
	<li>After confirming anesthetic adequacy, perform an orotracheal intubation using a 6.5-8 mm endotracheal tube as described in 10.</li>
	<li>Place the oxygen saturation monitor on the ear or bottom lip of the animal for continuous monitoring. Adjust ventilation to a positive end-expiratory pressure (PEEP) of 5 cm H2O, a peak inspiratory pressure (PIP) of 15 cm H2O, a fraction of inspired oxygen (FIO2) of 0.5, and a respiratory rate (RR) of 15 respirations/min reaching a tidal volume (TV) of 6-8 mL/kg, and normal partial pressure of O2 and CO2. Adjust the RR to maintain an end-tidal CO2 level of 35-45 mmHg.</li>
	<li>Gain a peripheral intravenous access via the ear vein using a 20 G catheter. Start a maintenance infusion of normal saline solution (0.9% NaCl) and administer 2 g of magnesium sulfate (MgSO4) as an intravenous bolus to prevent arrhythmias.</li>
	<li>Prepare the surgical sites by adequately shaving and scrubbing the thorax and groins. Disinfect groins and neck using chlorhexidine and drape appropriately while providing venous and arterial access.</li>
	<li>Place the cautery electrode pad on the animal&#39;s back. Place the animal in Trendelenburg position to facilitate central venous access.</li>
	<li>Locate the right jugular vein using an ultrasound. Once located, using a needle, enter the skin at a 45&#176; angle until blood is seen and the needle is seen in the vein on the ultrasound.</li>
	<li>Place the guidewire in the needle and then insert a 7 Fr percutaneous central venous sheath introducer over the guidewire.</li>
	<li>Remove the guidewire while keeping the sheath introducer in place, then insert a Swans-Ganz catheter to ensure cardiac output measurements and right-heart catheterization. 
	NOTE: The left jugular vein can be used instead of the right one. If access is not possible, insert the sheath directly in the internal jugular vein (left or right) after midline sternotomy. In that case, make sure to dissect the vein.</li>
	<li>Using the same technique, insert a central venous catheter (e.g., double lumen) into the left jugular vein.</li>
	<li>Insert another 7 Fr percutaneous central venous sheath introducer into the right femoral vein using the Seldinger technique as described in steps 1.7. to 1.10., for the introduction of a conductance catheter into the right ventricle (RV). This can be done percutaneously with echo-guidance, or after midline sternotomy.</li>
	<li>Allow a period of stability for 10 min post-intubation and manipulation, then measure baseline hemodynamic and cardiac function parameters including pulmonary and systemic pressures, cardiac output, RV and LV pressure-volume loops, and transthoracic echocardiography.</li>
</ol>
2. Establishing surgical access
<ol>
	<li>Using a cautery pen, perform a midline incision from the mid-cervical region to the xiphoid. After that, divide the subcutaneous fat, layer by layer, along the midline using the electrocautery to reach the peri-sternum.</li>
	<li>Cut around the sternal notch and use a finger to retract and sweep soft tissues from under the sternum. Open the sternum with a bone saw and ensure the sternum is completely divided while being careful with the vein beneath the upper part of the sternal bone. Cauterize the sternum and/or apply bone wax to ensure adequate hemostasis.</li>
	<li>Using the electrocautery, dissect and remove the thymus by lifting it from the pericardium. Cauterize the vessels that supply the thymus from the aorta and the superior vena cava (SVC) to prevent bleeding.</li>
	<li>Carefully cut the pericardium open with the cautery. Insert fingers under the pericardium while cutting to avoid injury to the heart.</li>
	<li>Administer 300 units/kg of heparin IV to achieve systemic anticoagulation. If the test is available, ensure activated clotting time (ACT) &#707; 300 s.</li>
</ol>
3. Withdrawal of life-sustaining therapies and declaration of death
<ol>
	<li>Administer a bolus of 3 mg/kg propofol IV, and then stop mechanical ventilation and disconnect the endotracheal tube. 
	NOTE:&#160;This step corresponds to &#34;Withdrawal of Life Support&#34; to launch the Normothermic Regional Perfusion. For euthanasia conditions, refer to section 8.</li>
	<li>Monitor arterial pressure and peripheral O2 saturation. Start functional warm ischemia time when systolic pressure &#60; 50 mmHg.</li>
	<li>Establish circulatory arrest when there is asystole (or ventricular fibrillation) and an absence of arterial pulsatility. Initiate a minimum observation phase of 5 min after circulatory arrest is established (stand-off period) and subsequently declare animal death.</li>
</ol>
4. Placement of thoracoabdominal normothermic regional perfusion
<ol>
	<li>Using the cautery, carefully dissect the aortopulmonary space. Retract the RV outflow tract inferiorly, the pulmonary artery to the left, and the aorta to the right to avoid direct lesions to the latter. Take an assistants help if needed during this step.</li>
	<li>Dissect and ligate the supra-aortic vessels to avoid and exclude cerebral perfusion. Alternatively, apply a large cross-clamp over the vessels.</li>
	<li>Insert two 18 G catheters in the carotids bilaterally distally to the occlusion to evaluate potential blood flow to the brain. Collect and measure blood throughout the experiment and reinfuse via the NRP circuit.</li>
	<li>Using the metzenbaum and right-angle forceps, dissect delicately between the SVC and the innominate artery, and between the IVC and pericardium. Encircle the SVC and IVC using an umbilical tape or a simple 0-silk suture and secure them with a tourniquet.</li>
	<li>Using a 3-0 suture, place two concentric purse-string sutures on the distal ascending aorta adventitia; avoid full thickness sutures. Place a purse-string suture on the right atrium (RA). Secure all sutures with a tourniquet.</li>
	<li>Setup and prime the NRP system according to the investigators&#39; and experimental needs. For this protocol, use an ECMO circuit with a centrifugal pump, primed with 2 L of crystalloid solution containing 500 mg of Solumedrol, 200 mL of 8.4% sodium bicarbonate (NaOH), 300 units/kg of heparin, and 2 g of MgSO4. 
	NOTE: The setup used here is similar to the one used by the institution&#39;s perfusionists at the division of cardiac surgery.</li>
	<li>Using a 17 to 21 F arterial cannula, cannulate the aorta, then tighten the tourniquet holding the purse-string suture to secure the cannula in place. 
	NOTE: Alternatively, insert an extracorporeal membrane oxygenator (ECMO) cannula using the Seldinger technique to minimize blood loss. Another method consists of using a standard bypass arterial cannula. The aortic cannula is then connected to the arterial line of the NRP circuit using a 3/8-3/8 connector; ensure complete deairing to avoid air embolism.</li>
	<li>Create a 5 mm incision at the center of the purse-string on the RA, and then dilate it using a small-angled instrument like a right-angle or snap. Cover the incision with a finger to avoid excessive bleeding.</li>
	<li>Use a double-stage venous cannula to cannulate the RA then tighten the purse-string sutures using a tourniquet to secure the cannula in place.</li>
	<li>Connect the cannulas to the venous line of the NRP circuit using a 1/2-3/8 connector and ensure complete deairing to avoid an airlock in the system.</li>
	<li>Ensure that a minimum of 15 min has passed between the declaration of death and the initiation of NRP to encompass for the time needed in clinical practice preparation, draping, and having access to the heart.</li>
	<li>Initiate NRP 15 min after the start of functional warm ischemia time. Maintain normothermia throughout the procedure. Adjust flowrates progressively to reach a perfusion index of 2.5 L/min/m2. Restart mechanical ventilation with an FIO2 of 50% and a TV of 6 mL/kg. 
	NOTE: Modify these settings according to the experimental design.</li>
	<li>Continuously infuse 10 &#181;g/kg/min of dopamine and 4 units/min of vasopressin IV. Use epinephrine and norepinephrine during initial perfusion and then titrate them as needed to aid in pressure regulation while maintaining arterial pressure above 50 mmHg.</li>
</ol>
5. Maintenance of reperfusion and targets for weaning off
<ol>
	<li>Reperfuse the heart for 30 min before attempting to wean for NRP. If weaning is unsuccessful, perform an additional 15 min of reperfusion to support with NRP before attempting to wean again for a total maximum reperfusion time of 180 min.</li>
	<li>Proceed to cardiac assessment after successful weaning. If weaning is not achieved after this time, stop the experiment, and report the issue. 
	NOTE: This should be adapted to each investigator&#39;s objectives.</li>
	<li>Evaluate NRP weaning off criteria and ensure that all criteria are met as detailed in Table 1.</li>
</ol>
6. Cardiac recovery assessment
<ol>
	<li>Generate pressure-volume (PV) loop analyses to measure cardiac contractility. The conductance catheter allows for continuous measurements of LV-PV relations.
	<ol>
		<li>Obtain steady-state recordings to generate volume-dependent parameters, stroke work, and developed pressure, and occlude the IVC with an umbilical tape to generate volume-independent parameters through occlusion, namely preload recruitable stroke work.</li>
	</ol>
	</li>
	<li>Measure central venous pressure, pulmonary artery pressure, cardiac output, RV pressure, and pulmonary capillary wedge pressure using the Swans-Ganz catheter introduced in the beginning of the experiment.</li>
	<li>Analyze cardiac function through echocardiography using a standard transesophageal probe and a transthoracic probe placed directly on the heart.</li>
	<li>Perform metabolic assessments by collecting arterial and venous blood samples for analyses. Draw blood directly from the coronary sinus to assess heart metabolic function specifically. Analyze blood gases and lactate levels.</li>
	<li>Additionally, collect myocardial biopsies from the RV and LV if needed. Obtain samples from other transplantable organs of interest (e.g., lungs, liver, kidneys), depending on the investigator&#39;s objectives.</li>
</ol>
7. NRP removal and cardiac evaluation
<ol>
	<li>When weaning off criteria are met, stop NRP. Remove the cannula from the RA and quickly tighten the purse-string suture to minimize blood loss. Secure the suture with knots and follow the same procedure to remove the aortic cannula.</li>
	<li>Evaluate cardiac function every 30 min for 2 h after NRP has stopped as described in steps 6.1. to 6.4.</li>
</ol>
8. Experiment termination
<ol>
	<li>After 2 h, place a 16 G or 18 G cardioplegia catheter in the ascending aorta. Connect the cannula to the NRP circuit.</li>
	<li>After that, decompress the right cardiac cavities by incising the IVC and the left cavities by incising the left atrium using the cautery.</li>
	<li>Clamp the ascending aorta after the insertion of the cannula and administer 1.5 L of Del Nido cardioplegia solution at 4 &#176;C to the heart. Ensure that the perfusion pressure is 50 mmHg.</li>
	<li>Stop the mechanical ventilation and place ice slush (0.9% NaCl) in the thoracic cavity after the initiation of cardioplegia for cooling. Proceed with cardiectomy in a traditional manner once the cardioplegia infusion is complete.</li>
	<li>Use the collected heart to obtain extensive myocardial biopsies for assessment and evaluation. Perform other biopsies from other organs (e.g., lungs, liver, kidneys) depending on the experimental design and objective of the investigators 
	NOTE: Figure 1 provides a summary of the protocol steps shown and Table 1 provides definition of the parameters and criteria used in this study.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64009/64009fig01.jpg" /> 
Figure 1: Schematic summary of the experimental protocol. Abbreviations: NRP = Normothermic Regional Perfusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64009/64009fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Definition/Criteria</td>
		</tr>
		<tr>
			<td>Functional Warm Ischemia Period</td>
			<td>Period that starts when systolic pressure &#60; 50 mmHg and ends when NRP is initiated. In this protocol, this period is 15 min</td>
		</tr>
		<tr>
			<td>Circulatory Arrest</td>
			<td>It is established when the heart is in asystole or in ventricular fibrillation</td>
		</tr>
		<tr>
			<td>Stand-Off Period</td>
			<td>Period that starts with the circulatory arrest and ends with the declaration of death. In this protocol, this period is 5 min</td>
		</tr>
		<tr>
			<td rowspan="6">Successful NRP weaning off</td>
			<td>Minimal use of inotropes and vasopressors</td>
		</tr>
		<tr>
			<td>CI &#62; 2.2 L/min/m2</td>
		</tr>
		<tr>
			<td>MAP &#62; 55 mmHg</td>
		</tr>
		<tr>
			<td>RAP &#60; 15 mmHg</td>
		</tr>
		<tr>
			<td>PCWP &#60; 15 mmHg</td>
		</tr>
		<tr>
			<td>Normal LV and RV function</td>
		</tr>
	</tbody>
</table>
Table 1: Representative definition of parameters and criteria used in this protocol. Abbreviations: CI = Cardiac Index; MAP = Mean Arterial Pressure; NRP = Normothermic Regional Perfusion; PCWP = Pulmonary Capillary Wedge Pressure; RAP = Right Atrial Pressure.

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The authors have nothing to disclose.

This manuscript describes a large-animal model donation after circulatory death (DCD) followed by thoracoabdominal normothermic regional perfusion. In this experiment, the heart is reperfused for a minimum of 30 min and a maximum of 3 h before it is weaned off from the ECMO circuit. The heart is then functioning on its own for 2 h which allows for valuable cardiac assessment on the short term. Therefore, the major limitation of this protocol is the short-term follow-up; however, a long-term assessment would be resource-intensive and costly. This procedure focuses on acute ischemia-reperfusion injury, and can be a reliable method to assess the cardiac function of a donor heart within the donor and establish if the organ can be transplanted. It can also be an adequate preclinical model that can be used to investigate novel pharmacologic and non-pharmacologic interventions that might improve cardiac functional recovery and have been previously validated only in small-animal models11,12,13,14,15,16. Modifications to this protocol can be adapted to the investigator&#39;s objectives and in case of troubleshooting. These modifications can include but are not limited to the cardioplegia, the priming solutions, the ischemic times, and the addition of pharmacological interventions to study.
When using large-animal models, the porcine heart is one of the preferred platforms to perform cardiac surgical research considering its anatomical similarity to the human heart. However, some important factors in porcine hearts should be considered when using this model. For example, porcine cardiac tissue is very fragile and friable and is prone to tears, especially in the pulmonary artery and the right atrium9. Cannulation and heart manipulation should be done with caution. Another important factor to consider is that the porcine heart is very sensitive to ischemia and prone to arrhythmias, which is why antiarrhythmics like the magnesium sulfate used in this experimental protocol should be administered routinely to every animal before the experiment. Nevertheless, this model is still considered appropriate for the study of acute ischemia in heart transplantation9. Although this protocol only describes NRP for cardiac functional assessment, the same procedure, when optimized, can and should preferably be used to evaluate other organs as well thus maximizing the potential of information gained from the experimental animal.
This experimental procedure requires at least one trained surgeon, and in order to optimize the protocol within each research group, around three to five experiments should be performed. One member of the team should be assigned to perform the anesthesia and blood sampling for analysis, and another team member should be in charge of the thoracoabdominal normothermic regional perfusion and management of the heart to include administration of antiarrhythmics, vasopressors, and other medications if needed. Several steps in this protocol are critical for the adequate continuation of the experiment, such as the induction of anesthesia and intubation, cardiac manipulation, cannulation, and decannulation, and should preferably be performed by an experienced surgeon. It is through repetition and consistency that this model can be optimized and be reliable for various future uses.

Over 300,000 individuals die in North America each year of heart failure (HF); cardiac transplantation remains the only treatment option for some of these patients with end stage disease1. Historically, the exclusive source for heart transplantation was donor hearts obtained after neurological determination of death (NDD), but even then, only around 40% were adequate for transplantation2. Between 15% to 20% of patients die while waiting for a heart donation, with shortage of donor hearts being one of the reasons that creates a discrepancy between the hearts available and the hearts needed2. In order to increase the organ donor pool, one important consideration is the use of hearts donated after circulatory death (DCD)3. There is reluctance to use DCD hearts because these organs are invariably submitted to a period of unprotected (warm) ischemia after cessation of blood circulation and may sustain irreversible damage. Although reports for successful DCD heart transplantation with excellent early outcomes do exist4,5, there is a need to develop a validated assessment method to determine if these hearts are usable and to potentially predict their post-transplant performance6,7. To limit ischemic periods of DCD hearts and to continuously monitor them during storage and transportation, ex situ heart perfusion systems were developed8. However, this technology relies on complex machines with perfusion equipment, and has a high upfront cost without any guarantee that the procured organ will be suitable for transplantation. A novel protocol for DCD heart transplantation based on normothermic regional perfusion (NRP) was proposed by Messer et al3. This technique involves restoring myocardial perfusion while the heart is still in the donor and excluding cerebral circulation. It allows a functional assessment in situ before procurement3.
When using large-animal models, the porcine heart is one of the preferred platforms to perform cardiac surgical research considering its anatomical similarity to the human heart. However, some important factors in porcine hearts should be considered when using this model. For example, porcine cardiac tissue is very fragile and friable and is prone to tears, especially in the pulmonary artery and the right atrium9. Another important factor to consider is that the porcine heart is very sensitive to ischemia and prone to arrhythmias, which is why antiarrhythmics should be administered routinely to every animal before the experiment; nevertheless, it is still considered an appropriate model for the study of acute ischemia in heart transplantation9.
This manuscript 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 novel therapeutic studies and strategies for translational research.

<table><tbody><tr><td>Amiodarone</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Angiocath 20G</td><td>BD</td><td>381704</td><td></td></tr><tr><td>Atropine 0.4 mg/mL</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Biomedicus Centrifugal Pump</td><td>Medtronic</td><td></td><td></td></tr><tr><td>Cardioplegia Solution (Del Nido)</td><td>in-house made</td><td></td><td>Another solution can be used at the discretion of the researcher</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>Central Venous Sheath Introducer 7 Fr</td><td></td><td></td><td></td></tr><tr><td>Conductance Catheter</td><td></td><td></td><td></td></tr><tr><td>CPB pack</td><td>Medtronic</td><td></td><td></td></tr><tr><td>DLP Aortic Root Cannula</td><td>Medtronic</td><td>12218</td><td></td></tr><tr><td>DLP double-stage venous cannula (29 or 37 F)</td><td>Medtronic</td><td></td><td></td></tr><tr><td>Dobutamine</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Dopamine</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Electrode Polyhesive</td><td>Covidien</td><td>E7507</td><td></td></tr><tr><td>EOPA Arterial Cannula (17 or 21 F)</td><td></td><td></td><td></td></tr><tr><td>Epinephrine</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>O2 Face Mask</td><td></td><td></td><td>As available in the institution</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>As available in the institution</td></tr><tr><td>Inhaled Isofurane</td><td></td><td></td><td>Provided by the institution&#039;s animal facility</td></tr><tr><td>Jelco 16 or 18 G catheter</td><td></td><td></td><td></td></tr><tr><td>Ketamine inj. 50 mL vial (100 mg/mL)</td><td>Health Canada</td><td></td><td>Health Canada approval is required</td></tr><tr><td>Lidocaine/Xylocaine 1%</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Magnesium Sulfate 5 g/10 mL</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Midazolam inj. 10 mL vial (5 mg/mL)</td><td>Health Canada</td><td></td><td>Health Canada approval is required</td></tr><tr><td>MPS Quest delivery disposable pack</td><td>Quest Medical</td><td>5001102-AS</td><td></td></tr><tr><td>Norepinephrine</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Normal Saline (NaCl 0.9%) 1L bag</td><td>Baxter</td><td>JB1324</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 1 mg/mL</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Rocuronium</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Set Admin Prim NF PB W/ Checkvalve</td><td>Smith Medical</td><td>21-0442-25</td><td></td></tr><tr><td>Sodium Bicarbonate (NaOH) 8.4%</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Sofsil 0 wax coated</td><td>Covidien</td><td>S316</td><td></td></tr><tr><td>Solumedrol 500 mg/5 mL</td><td></td><td></td><td>As available in the institution</td></tr><tr><td>Suction Tip</td><td>Covidien</td><td>8888501023</td><td></td></tr><tr><td>Suction Tubing 1/4&#039;&#039; x 120&#039;&#039;</td><td>Med-Rx</td><td>70-8120</td><td></td></tr><tr><td>Suture 3.0 Prolene Blu M SH</td><td>Ethicon</td><td>8523H</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>Suture BB 4.0 Prolene</td><td>Ethicon</td><td>8881H</td><td></td></tr><tr><td>Tracheal Tube, 6.5 mm</td><td>Mallinckrodt</td><td>86449</td><td></td></tr><tr><td>Vasopressin</td><td></td><td></td><td>As available in the institution</td></tr></tbody></table>

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.

This preclinical model has been successfully used in our institution for multiple experiments. First, we demonstrated that DCD hearts, initially reperfused with NRP, demonstrated similar functional recovery following transplantation when compared to conventional beating heart donation preserved with cold storage. Further, we have used this protocol to show that cardiac functional assessment following NRP was predictive of post-transplantation recovery. Finally, we have also studied the effects of NRP on cerebral perfusion and lung preservation following circulatory arrest.
Figure 2 demonstrates representative results of cardiac function at baseline and following DCD and NRP measured using a pulmonary artery catheter. Here, we see that there is a significant decline in heart function following DCD; however, these organs demonstrated similar functional recovery post-transplantation when compared to conventionally transplanted hearts (beating heart donation followed by cold storage). Figure 3 shows measurements of cerebral oximetry during NRP with the supra-aortic vessels clamped; the measurements confirm the absence of adequate cerebral perfusion. Finally, Figure 4 shows measurements of lung compliance during NRP and after weaning from support. It showed no significant chang from baseline during NRP.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64009/64009fig02.jpg" /> 
Figure 2: Cardiac during NRP. Cardiac index (median &#177; interquartile range) was measured at baseline, during NRP, and following transplant using pulmonary artery catheter. DCD hearts (in HTK storage solution) showed similar functional recovery compared to control hearts (standard-beating donations) after transplant (n = 5/group). Abbreviations: DCD = donation after circulatory death; HTK = Histidine-Tryptophan-Ketoglutarate storage solution; NRP = Normothermic Regional Perfusion; Tx = Treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64009/64009fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64009/64009fig03.jpg" /> 
Figure 3: Cerebral oximetry measurement. Cerebral oximetry measurements throughout the experiment showed progressive decline during withdrawal of life sustaining measures and remained at minimal levels during NRP. Abbreviations: NRP = Normothermic Regional Perfusion; WLST = Withdrawal of Life-Sustaining Treatment <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64009/64009fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64009/64009fig04.jpg" /> 
Figure 4: Lung compliance measurement during NRP and after weaning. Lung compliance, measured by the ratio between tidal volume and mean pressure as seen on the ventilator (median &#177; interquartile range), showed no significant changes from baseline during NRP (n = 5). Static compliance (Static) is measured during an end-respiratory hold. Dynamic compliance (Dyn) is measured throughout the respiratory cycle. Abbreviations: NRP = Normothermic Regional Perfusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64009/64009fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

large-animal-model-donation-after-circulatory-death-normothermic

Nanyang Technological University

Research

JoVE Journal

Medicine

1.6K Views.  Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM). Dit protocol beschrijft een groot diermodel van donatie na circulatoire sterfte gevolgd door thoracoabdominale normotherme regionale perfusie die de klinische harttransplantatie nauwkeurig nabootst. Dit kan worden gebruikt om potentiële strategieën in translationeel onderzoek te evalueren. Het belangrijkste voordeel van dit protocol is dat het een geschikt preklinisch model biedt voor harttransplantatie gezien de anatomische en chirurgische technieken overeenkomsten tussen de zwijnen en de ...

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

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.

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 ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

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 ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

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

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