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

Summary

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.

Abstract

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.

Introduction

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 exist^4,5, there is a need to develop a validated assessment method to determine if these hearts are usable and to potentially predict their post-transplant performance^6,7. 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.

Protocol

The institutional animal care and use committee of the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (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' experimental goals.

1. Animal preparation and anesthetic induction

1. 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.
2. 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.
3. After confirming anesthetic adequacy, perform an orotracheal intubation using a 6.5-8 mm endotracheal tube as described in ¹⁰.
4. 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.
5. 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.
6. 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.
7. Place the cautery electrode pad on the animal's back. Place the animal in Trendelenburg position to facilitate central venous access.
8. Locate the right jugular vein using an ultrasound. Once located, using a needle, enter the skin at a 45° angle until blood is seen and the needle is seen in the vein on the ultrasound.
9. Place the guidewire in the needle and then insert a 7 Fr percutaneous central venous sheath introducer over the guidewire.
10. 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.
11. Using the same technique, insert a central venous catheter (e.g., double lumen) into the left jugular vein.
12. 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.
13. 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.

2. Establishing surgical access

1. 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.
2. 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.
3. 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.
4. Carefully cut the pericardium open with the cautery. Insert fingers under the pericardium while cutting to avoid injury to the heart.
5. Administer 300 units/kg of heparin IV to achieve systemic anticoagulation. If the test is available, ensure activated clotting time (ACT) ˃ 300 s.

3. Withdrawal of life-sustaining therapies and declaration of death

1. Administer a bolus of 3 mg/kg propofol IV, and then stop mechanical ventilation and disconnect the endotracheal tube.
   NOTE: This step corresponds to "Withdrawal of Life Support" to launch the Normothermic Regional Perfusion. For euthanasia conditions, refer to section 8.
2. Monitor arterial pressure and peripheral O₂ saturation. Start functional warm ischemia time when systolic pressure < 50 mmHg.
3. 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.

4. Placement of thoracoabdominal normothermic regional perfusion

1. 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.
2. Dissect and ligate the supra-aortic vessels to avoid and exclude cerebral perfusion. Alternatively, apply a large cross-clamp over the vessels.
3. 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.
4. 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.
5. 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.
6. Setup and prime the NRP system according to the investigators' 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's perfusionists at the division of cardiac surgery.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. Continuously infuse 10 µg/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.

5. Maintenance of reperfusion and targets for weaning off

1. 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.
2. 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's objectives.
3. Evaluate NRP weaning off criteria and ensure that all criteria are met as detailed in Table 1.

6. Cardiac recovery assessment

1. Generate pressure-volume (PV) loop analyses to measure cardiac contractility. The conductance catheter allows for continuous measurements of LV-PV relations.
   1. 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.
2. 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.
3. Analyze cardiac function through echocardiography using a standard transesophageal probe and a transthoracic probe placed directly on the heart.
4. 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.
5. 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's objectives.

7. NRP removal and cardiac evaluation

1. 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.
2. Evaluate cardiac function every 30 min for 2 h after NRP has stopped as described in steps 6.1. to 6.4.

8. Experiment termination

1. After 2 h, place a 16 G or 18 G cardioplegia catheter in the ascending aorta. Connect the cannula to the NRP circuit.
2. After that, decompress the right cardiac cavities by incising the IVC and the left cavities by incising the left atrium using the cautery.
3. Clamp the ascending aorta after the insertion of the cannula and administer 1.5 L of Del Nido cardioplegia solution at 4 °C to the heart. Ensure that the perfusion pressure is 50 mmHg.
4. 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.
5. 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.

Figure 1: Schematic summary of the experimental protocol. Abbreviations: NRP = Normothermic Regional Perfusion. Please click here to view a larger version of this figure.

	Definition/Criteria
Functional Warm Ischemia Period	Period that starts when systolic pressure < 50 mmHg and ends when NRP is initiated. In this protocol, this period is 15 min
Circulatory Arrest	It is established when the heart is in asystole or in ventricular fibrillation
Stand-Off Period	Period that starts with the circulatory arrest and ends with the declaration of death. In this protocol, this period is 5 min
Successful NRP weaning off	Minimal use of inotropes and vasopressors
	CI > 2.2 L/min/m2
	MAP > 55 mmHg
	RAP < 15 mmHg
	PCWP < 15 mmHg
	Normal LV and RV function

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.

Representative Results

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.

Figure 2: Cardiac during NRP. Cardiac index (median ± 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. Please click here to view a larger version of this figure.

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 Please click here to view a larger version of this figure.

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 ± 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. Please click here to view a larger version of this figure.

Discussion

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 models^{11,12,13,14,15,16}. Modifications to this protocol can be adapted to the investigator'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.

Materials

Name	Company	Catalog Number	Comments
Amiodarone			As available in the institution
Angiocath 20G	BD	381704
Atropine 0.4 mg/mL			As available in the institution
Biomedicus Centrifugal Pump	Medtronic
Cardioplegia Solution (Del Nido)	in-house made		Another solution can be used at the discretion of the researcher
Cautery Pencil	Covidien	E2515H
Central Venous Catheter double-lumen	Cook Medical	C-UDLM-501J-LSC
Central Venous Sheath Introducer 7 Fr
Conductance Catheter
CPB pack	Medtronic
DLP Aortic Root Cannula	Medtronic	12218
DLP double-stage venous cannula (29 or 37 F)	Medtronic
Dobutamine			As available in the institution
Dopamine			As available in the institution
Electrode Polyhesive	Covidien	E7507
EOPA Arterial Cannula (17 or 21 F)
Epinephrine			As available in the institution
O2 Face Mask			As available in the institution
Gloves, Nitrile, Medium	Fischer	27-058-52
Heparin 1000 IU/mL			As available in the institution
Inhaled Isofurane			Provided by the institution's animal facility
Jelco 16 or 18 G catheter
Ketamine inj. 50 mL vial (100 mg/mL)	Health Canada		Health Canada approval is required
Lidocaine/Xylocaine 1%			As available in the institution
Magnesium Sulfate 5 g/10 mL			As available in the institution
Midazolam inj. 10 mL vial (5 mg/mL)	Health Canada		Health Canada approval is required
MPS Quest delivery disposable pack	Quest Medical	5001102-AS
Norepinephrine			As available in the institution
Normal Saline (NaCl 0.9%) 1L bag	Baxter	JB1324
Pipette Tips 1 mL	Fisherbrand	02-707-405
Propofol 1 mg/mL			As available in the institution
Rocuronium			As available in the institution
Set Admin Prim NF PB W/ Checkvalve	Smith Medical	21-0442-25
Sodium Bicarbonate (NaOH) 8.4%			As available in the institution
Sofsil 0 wax coated	Covidien	S316
Solumedrol 500 mg/5 mL			As available in the institution
Suction Tip	Covidien	8888501023
Suction Tubing 1/4'' x 120''	Med-Rx	70-8120
Suture 3.0 Prolene Blu M SH	Ethicon	8523H
Suture 5.0 Prolene BB	Ethicon	8580H
Suture Prolene Blum 4-0 SH 36	Ethicon	8521H
Suture BB 4.0 Prolene	Ethicon	8881H
Tracheal Tube, 6.5 mm	Mallinckrodt	86449
Vasopressin			As available in the institution