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 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.
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.
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
2. Establishing surgical access
3. Withdrawal of life-sustaining therapies and declaration of death
4. Placement of thoracoabdominal normothermic regional perfusion
5. Maintenance of reperfusion and targets for weaning off
6. Cardiac recovery assessment
7. NRP removal and cardiac evaluation
8. Experiment termination
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.
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.
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'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.
The authors have nothing to disclose.
We would like to thank Melanie Borie, Caroline Landry, Henry Aceros and Ahmed Menaouar for their precious help and support.
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 |